FIELD OF INVENTION

The present invention relates to new microorganisms having improved capability of utilizing monomeric sugars found in renewable resources, in particular lignocel- lulose. More specifically, the invention relates to genetically modified Ralstonia and Cupriavidus hosts and methods of using them.

BACKGROUND

Lignocellulose is the main renewable resource with several applications, including feedstock for bulk chemicals. The main components of lignocellulose are cellulose, hemicellulose and lignin. The cellulose fraction comprises polymers of the hexose sugar, glucose. The hemicellulose fraction is mainly comprised of polymers of the pentose sugars xylose, and arabinose, and the hexose sugars mannose, glucose and galactose. These are abundant in the lignocellulosic biomass of soft woods, whereas xylose occurs in both soft and hard woods.

There are numerous examples for efforts to utilize lignocelluloses more effectively by microorganisms and to extend the substrate utilization range of microorganisms towards low-cost and abundant carbon sources. For example, attempts have been made to broaden the utilization of unfermentable pentoses like xylose and arabi- nose occurring in lignocellulosic biomass in many microorganisms. Many of these investigations aimed at the extension of carbohydrate metabolism in ethanol producers, such as Zymomonas mobilis and Saccharomyces cerevisiae for ethanol fermentation bioprocess. WO 2009/081941 , for example, describes genetical modification of ethanol producing bacterium belonging to the genus Zymomonas to uti- lize glucose, mannose, and xylose.

Bacteria belonging to genus Ralstonia and Cupriavidus are chemolithoautotrophs which are capable of accumulating large amounts of polyhydroxyalkanoates (PHAs) from renewable resources or from carbon dioxide. Strains of the genus Ralstonia and Cupriavidus, in particular, R. eutropha (new name Cupriavidus ne- cator) have been shown to possess large biotechnological potential as these strains can be cultivated to high cell densities in large scale (Schlegel et al. 1961 ) and is suitable for large-scale industrial bioprocesses. Unfortunately, Cupriavidus and Ralstonia have a very narrow carbohydrate substrate range limited to fructose, N-acetylglucosamine and gluconate. Attempts to broaden the substrate utili- zation range of R. euthropha H16 have been described in the prior art (Pries et al. 1990, Schlegel and Gottschalk, 1965). A spontaneous glucose-utilizing mutant of R. euthropha H16 which presumably transported this sugar into the cell by a passive transporter has been reported to be isolated (Schlegel and Gottschalk 1965, Konig, et al. 1969). Furthermore, Pries et al. 1990 have reported of the introduc- tion of /3-galactosidase gene and the ga/ operon from E. coli into R. eutropha strain H16 and utilization of lactose and galactose by this bacterium.

Further, Buchholz et al. 1994 have described the construction of a genomic library of Pseudomonas saccharophila and its transfer to Alcaligenes eutrophus (Ralstonia eutropha). An insert containing xyl genes encoding xylose isomerase and xylulokinase made the host A. eutrophus (pGN3) to grow on xylose.

In spite of the previous attempts to extend the substrate utilization range of certain microorganisms towards low-cost and abundant carbon sources, there is still a need for new methods and microorganisms for more efficient utilization of renewable resources, in particular lignocelluloses. SUMMARY

One object of the present invention is to provide a process for effectively utilizing lignocelluloses, in particular monomeric sugars found in lignocelluloses.

Another object of the present invention is to provide new microorganisms capable of utilizing lignocelluloses, in particular monomeric sugars found in lignocellulose. To achieve these objects the invention is characterized by the features that are enlisted in the independent claims. Other claims represent the preferred embodiments of the invention. The invention described herein provides, in one aspect, a new microorganism, which is genetically modified to express genes broadening its capability of using different monomeric sugars which are present in lignocellulose.

The present invention provides, in one aspect, a microorganism of the genus Cu- priavidus or Ralstonia, which is capable of using mannose as carbon source.

In another aspect the invention provides a microorganism of the genus Cupriavidus or Ralstonia, which is capable of using xylose as carbon source.

In one embodiment the microorganism is genetically modified to express phos- phomannose isomerase (EC 5.3.1 .8) and facilitated diffusion protein for mannose uptake (EC 1 .3.1 .74) and/or xylose proton symporter E, and optionally mannofruc- tokinase (EC 2.7.1 .4 ). Typically, microorganisms of the genus Cupriavidus or Ralstonia are incapable of utilizing mannose without said modification, whereas the modified microorganisms are capable of utilizing mannose. The genes used for said genetic modification may be exogenous or endogenous, typically they are ex- ogenous. In various embodiments, when the microorganism is genetically modified to express a gene encoding facilitated diffusion protein for mannose uptake (EC 1 .3.1 .74), in particular gene glf, the microorganism host is surprisingly capable of utilizing also glucose.

In one embodiment the microorganism is genetically modified to express xylose isomerase (EC 5.3.1 .5), xylulokinase (EC 2.7.1 .17 ) and a high affinity ABC- transporter (Xyl G, EC 3.6.3.17 , Xyl F, and/or Xyl H) and/or xylose proton symporter E. Typically, microorganisms of the genus Cupriavidus or Ralstonia are incapable of utilizing xylose without said modification, whereas the modified microorganisms are capable of utilizing xylose. The genes used for said genetic modifi- cation may be exogenous or endogenous, typically they are exogenous. Typically, said microorganisms are incapable of utilizing arabinose without said genes, whereas in various embodiments the modified microorganisms are surprisingly capable of utilizing arabinose as the carbon source. Furthermore, when the microorganisms are genetically modified to express high affinity ABC-transporter (in par- ticular genes Xyl G, (EC 3.6.3.17), Xyl F and Xyl H), the microorganism hosts are surprisingly capable of utilizing glucose and galactose. In one further embodiment of the invention the microorganism hosts are genetically modified to express glucokinase (EC 2.7.1 .2). This modification makes the genetically modified micoorganism to be also capable of utilizing glucose.

The microorganisms are thus capable of utilizing glucose as carbon source in addition to mannose, or in addition to mannose, xylose and arabinose.

In a preferred embodiment of the invention the microorganisms are capable of uti- lizing mannose, glucose, galactose, xylose and arabinose as carbon source.

In one further embodiment the microorganisms are genetically modified to express transaldolase (EC 2.2.1 .2) and/or transketolase (EC 2.2.1 .1 ). The genes encoding transaldolase and/or transketolase improve the fastness of growth of the modified microorganism in particular on pentoses, such as xylose and arabinose.

The microorganism may be any species or strains belonging to the genus Cu- priavidus or Ralstonia. Preferably, the microorganism belongs to C. necator (earlier R. eutropha or Alkaligenes eutrophus) species.

In one aspect the invention provides a vector comprising a gene encoding phos- phomannose isomerase (EC 5.3.1 .8 ), a gene encoding facilitated diffusion protein for mannose uptake (EC 1 .3.1 .74), a gene encoding xylose proton symporter E and/or a gene encoding mannofructokinase (EC 2.7.1 .4), and regulatory elements for regulating expression of said genes in Cupriavidus or Ralstonia. Said genes may be in the same or separate vectors.

In another aspect the vector may comprise a gene encoding xylose isomerase (EC 5.3.1 .5 ), xylulokinase (EC 2.7.1 .17 ), a high affinity ABC- transporter (Xyl G, EC 3.6.3.17, Xyl F, and/or Xyl H) and/or xylose proton symporter E, and regulatory elements for regulating expression of said genes in Cupriavidus or Ralstonia. Said genes may be in the same or separate vectors. In one embodiment the vector may comprise a gene encoding transaldolase (EC 2.2.1 .2) and/or transketolase (EC 2.2.1 .1 ). Said genes may be in the same or separate vectors. In one further embodiment the vector may comprise a gene encoding glucokinase (EC 2.7.1 .2). Said gene may be in the same or separate vectors as the above listed genes.

In one still further embodiment a vector comprise any one or any combination of the above listed genes.

Any promoter that can be expressed in strains of the genera Ralstonia or Cu- priavidus can be used in the vector.

Suitable promoters comprise for example /ac-promoter, neo/ anamyc/n-promoter , glyceraldehyde-3-phophate dehydrogenase promoter, P2, hydrogenase (SH) promoter and P|_ .

In one preferred embodiment the vector is a broad host range plasmid which can be used to extend the substrate utilization range of various different host strains of Cupriavidus or Ralstonia.

In one aspect the invention provides a method for modifying the microorganisms of the genera Ralstonia or Cupriavidus. The method comprises the hosts are genet- ically modified to express one or more nucleotide sequences selected from the group comprising a nucleotide sequence or sequences encoding phosphomannose isomerase (EC5.3.1 .8) and facilitated diffusion protein for mannose uptake (EC1 .3.1 .74) and/or xylose proton symporter E, and optionally mannofructokinase (EC2.7.1 .4), wherein said microorganism is capable of growing on mannose and optionally also on glucose as the carbon source,

and/or

a nucleotide sequence or sequences encoding xylose isomerase (EC 5.3.1 .5), xylulokinase (E 2.7.1 .17) and a high affinity ABC- transporter and/or xylose proton symporter E, wherein said microorganism is capable of growing on xylose, or xylose and arabinose, and optionally also on glucose and galactose as the carbon source. In one embodiment the method may further comprise that the microorganism is genetically modified to express transaldolase (EC2.2.1 .2) and/or transketolase (EC2.2.1 .1 ). In one embodiment the method may further comprise that the microorganism is genetically modified to express glucokinase (EC 2.7.1 .2).

In one still further aspect the invention provides a method for cultivating the modified microorganism in a cultivation medium comprising mannose, glucose, galac- tose, xylose or arabinose, any of them, any combination of them, or all of them, as carbon source.

In one embodiment the cultivation medium may comprise mannose as carbon source and optionally glucose.

In another embodiment the cultivation medium may comprise either or both xylose and arabinose as carbon source, and optionally also glucose and/or galactose.

In one further emdodiment the cultivation medium may comprise mannose and ei- ther or both xylose and arabinose as carbon source, and optionally also glucose and/or galactose.

In one further aspect the method comprises that the microorganism is allowed to synthesize bulk chemicals, such as lipids, for example polyhydroxyalkanoates, fat- ty acids or fatty acid derivatives, and the chemical is recovered from the cells, or from the cultivation medium.

Considerable advantages are obtained by means of the present invention. By means of the invention new microorganisms belonging to genus Cupriavidus or Ralstonia are obtained, said microorganisms having an extended substrate utilization range. The modified microorganisms as described herein can utilize effectively low-cost carbohydrates, in particular lignocellulose comprising monomeric sugars. Xylose, mannose and arabinose are wood sugars present in a polymerized form in the hemicelluloses of plant cell walls. The microorganisms of the present invention are also able to use monomeric glucose, which is found in particular in sucrose and starch and also in cellulose and hemicellulose fractions in lignocellulosic materials. The modified microorganisms of the present invention are suitable for pro- ducing many bulk chemicals.

DESCRIPTION OF THE FIGURES

Figure 1 . Pathways of mannose and glucose utilization established in R. eutropha strain H16. GLF, glucose-facilitated diffusion transporter; KDPG, Entner-Doudoroff pathway; MAK, mannofructokinase; PMI, phosphomannose isomerase, GLK, glucokinase; PGI, phosphoglucose isomerase.

Figure 2. Physical maps of the constructed plasmids pBBRI MCS-S./g/f, pBBR1 MCS-3::glf::mak, pBBR1 MCS-3: :glf::pmi, pBBR1 MCS-3: :glf::mak::pmi, pBBRI MCS^ ^g/f and pBBR1 MCS-2::P _n/( ::g'// ::g'/f. The genes glf, glk, mak and pmi, respectively, were excised from hybrid plasmids of pJET1 .2 or pCR2.1 after subcloning using the indicated restriction sites, and were then ligated in various arrays to the linearized expression vectors pBBR1 MCS-2 and pBBR1 MCS-3. Relevant cleavage sites and structural genes are indicated (Km ^R , kanamycin resistance cassette, Tc ^R , tetracycline resistance cassette; mob, mobilization site; rep, origin of replication; glf, gene encoding the glucose-facilitated diffusion transporter (GLF) from Z. mobilis; glk, gene encoding the glucokinase (GLK) from E. coli; mak, gene encoding a mannofructokinase (MAK) from E. coli; pmi, gene encoding a phosphomannose isomerase (PMI) from E. coli).

Figure 3. Transcription analysis of glf, mak and pmi in R. eutropha strain H16 har- boring pBBR1 MCS-3::g/f::ma/ ;;pm/ ^' and in the negative control harboring only the vector pBBR1 MCS-3. Expression of glf, mak and pmi was analyzed by RT-PCR in samples containing total RNA isolated from cells of the exponential growth phase of R. eutropha strain H16 harboring pBBR1 MCS-3::g/f::ma/ ;;pm/ ^' and of R. eutropha strain H16 harboring the vector pBBR1 MCS-3. Cells were grown in MSM containing 1 % (wt/vol) sodium gluconate as sole carbon source. The resulting PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide. Lanes 1 , 3 and 5 represent the RT-PCR assay for glf, mak and pmi, respectively, for R. eutropha strain H16 harboring pBBRI MCS- 3::glf::mak::pmi, whereas lanes 2, 4 and 6 represent the negative controls to detect whether there is any expression of glf, mak and pmi in the strain harboring only the vector pBBR1 MCS-3. A k/Pst\ DNA Marker (lanes M, Fermentas, Germany) served for size comparison.

Figure 4. Crude extracts and solubilized membrane proteins of recombinant R. eutropha strain H16 harboring pBBR1 MCS-2 or pBBR1 MCS-2::P„*::gr/fc:gr//: Cells were grown for 48h in MSM medium containing 1 % (wt vol) sodium glucose as carbon source. Proteins were separated in 12.5% (wt/vol) SDS-polyacrylamide gels and stained with Coomassie brilliant blue. A, crude extracts. Lane 1 : R. eutropha strain H16 pBBR1 MCS-2, lane 2: R. eutropha strain H16 pBBR1 MCS-2::P„/ ₍ ::g'// ::g'/f; B, solubilized membrane proteins. Lane 3: R. eutropha strain H16 pBBR1 MCS-2, lane 4: R. eutropha strain H16 pBBR1 MCS-2::P„/ _f ::g// ::g/ ^' . St, molecular weight standard. Arrows indicate detect- ed recombinant proteins glucokinase (Glk) and glucose transporter (Glf).

Figure 5. Growth of recombinant R. eutropha strain H16. Recombinant strains of R. eutropha harboring different plasmids. Cells were cultivated in liquid MSM containing 1 % (wt/vol) glucose as sole carbon source. □ pBBR1 MCS-3::g/f; • pBBR1 MCS-2::P _n/( ::g/f; Δ pBBR1 MCS-2::P _n/( ::g// ::g/f; A pBBR1 MCS-3.\-g/f.\777a/ .\ m/ ^' ; ♦ control R. eutropha H16 strain G ⁺ 1 . Cultivations were done in Erlenmeyer flasks incubated on a horizontal rotary shaker at 1 10 rpm at 30 °C. The optical density was measured at 600 nm in a Klett photometer.

Figure 6. Recombinant strains of R. eutropha harboring plasmid pBBW MCS-3::glf::mak::pmi. Cells were cultivated in liquid MSM containing tetra- cycline (12.5 pg/ml) and Δ 0.5% (wt/vol) sodium gluconate, ▲ 0.5% (wt/vol) man- nose,□ 1 % (wt/vol) mannose,♦ 2% (wt/vol) mannose, or o 4% (wt/vol) mannose as sole carbon source. Cultivations were done in Erlenmeyer flasks incubated on a horizontal rotary shaker at 130 rpm and at 30 °C. The optical density was measured at 600 nm in a Klett photometer. Figure 7. The utilization pathway of D-xylose in bacteria (XI, xylose isomerase; XK, xylulokinase; PPP, pentose phosphate pathway) Figure 8. Physical maps of the constructed plasmids pBBR1 MCS-3: :xy//4B, pBBR1 MCS-3: :xy//4BE and pBBR1 MCS-3 ::xy//4BFGH. The genes xylA, xylB, xylE and xylFGH, respectively, were excised from hybrid plasmids of pJET1 .2 after subcloning using the indicated restriction sites, and were then ligated to the linear- ized expression vector pBBR1 MCS-3. Relevant cleavage sites and structural genes are indicated (Tc ^R , tetracycline resistance cassette; mob, mobilization site; rep, origin of replication; xy/A, gene encoding the xylose-isomerase (XylA); xy/B, gene encoding the xylulokinase (XylB); xylE, gene encoding the low affinity transporter (XylE); and xylFGH, genes encoding the ABC-transporter (XylFGH) from E. coli, respectively).

Figure 9. Growth of the recombinant plasmid pBBR1 MCS-3: :xy//4BFGH harboring strain of R. eutropha strain H16, and of R. eutropha strain H16, which were cultivated in fluid MSM containing different concentrations (0.5%, 1 %, 2% [wt/vol]) of D-xylose, 1 % (wt/vol) glucose and with 1 % (wt/vol) Na-gluconate as sole carbon source, respectively. □ pBBR1 MCS-3: :xy//4BFGH, 0.5% (wt/vol) D-xylose; ■ pBBR1 MCS-3: :xy//4BFGH, 1 .0% (wt/vol) D-xylose; Δ pBBR1 MCS-3: xylABFGH, 2.0% (wt/vol) D-xylose; A pBBR1 MCS-3: xylABFGH, 4.0% (wt/vol) D-xylose; 0 pBBR1 MCS-3: \xylABFGH, 1 .0% (wt/vol) Na-gluconate; ♦ pBBRI MCS- 3::xylABFGH, 1 .0% (wt/vol) glucose; + control R. eutropha H16, 1 .0% (wt/vol) Na- gluconate. Cultivations were done in Erlenmeyer flasks incubated on a horizontal rotary shaker at 1 10 rpm at 30 °C. The optical density was measured at 600 nm in a Klett photometer.

DETAILED DESCRIPTION OF THE INVENTION

"A genetically modified microorganism" refers here to a microorganism whose ge- netic material has been altered using genetic engineering techniques. These techniques involve the use of nucleic acid molecules from different sources, for example from another microorganism or they may be at least partly synthetic. Nucleic acid molecules from different sources may be combined to one or more molecules. These constructions may be transferred into a microorganism, giving it modified or novel genetic constructions. "A gene" refers here typically to a nucleotide sequence encoding a specific product, here usually an enzyme. The coding region is typically operationally linked into regulatory regions, in particular into a promoter, which is able to function in the host cell. Into a host cell is here typically introduced a genetic construct, which consist essentially of a coding region of a gene and a regulatory region, in particular a promoter, functional in the host cell. A suitable vector may be used to introduce the desired genetic construct into a host cell. "A gene" may refer to one or more genes.

Genetical modification may mean that one or more exogenous genes are intro- duced into and expressed in a microorganism host, or that one or more of the expressed genes are endogenous and one or more are exogenous, or that the expression of one or more endogenous genes is increased in a host compared to the parent host.

Microorganism hosts in various embodiments of the invention are preferably bac- teria belonging to the genus Cupriavidus or Ralstonia.

A microorganism refers here typically to an isolated microorganism, i.e. a microorganism is used as a pure culture.

In one embodiment the species may be any Ralstonia species including, but not limited to Ralstonia insidiosa, R. mannitolytica, R. pickettii, R. solanacearum and R. syzygii. In another embodiment the species may be any Cupriavidus species including, but not limited to Cupriavidus basilensis, C. campinensis, C. gilardii, C. laharis, C. metallidurans, C. necator, C. oxalaticus, C. pauculus, C. pinatubonen- sis, C. respiraculi and C. taiwanensis (Bacterial Nomenclature up-to-date (approved lists, validation list), May 2010, compiled by DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH). Preferred species are R. eutropha and C. metallidurans. Most preferred species is R. eutropha (present name C. necator, earlier names Alcaligenes eutrophus and Hydrogenomonas eutropha). Cupriavidus and Ralstonia strains are availably to the public from microbial culture collections, such as ATCC, DSMZ and NCIB. The invention has been exemplified by using R. eutropha strain H 16 (present name C. necator). This strain is a wild type strain, which is available from Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ, under accession number DSM428, from American Type Culture Collection under accession number ATCC17699, and from National Collection of Industrial and Marine Bacteria, NCIBM, under Accession number NCIB10442. All these strains are available to the public.

Deposited R. eutropha H16 strains, which can be used in the invention are

Ralstonia eutropha H16, DSMZ Accession No.: DSM428, DSM 15443 (mutant of DSM 428)

Taxonomy ID: 381666; Inherited blast name: b-proteobacteria

Other names:

synonyms: Cupriavidus necator H16, Alcaligenes eutropha H16, Wautersia eutropha H16, Cupriavidus necator ATCC 17699, Ralstonia eutropha ATCC 17699; equivalentic names: Ralstonia eutropha strain H16, Ralstonia eutropha str. H16

Other R. eutropha strains which can be used in the invention are:

Ralstonia eutropha H850

DSMZ Accession No.: DSM 13439

Taxonomy ID: 53482; Inherited blast name: b-proteobacteria

Other names:

synonyms: Wautersia eutropha H850, Cupriavidus necator H850, Alcaligenes eut- rophus H850

Ralstonia eutropha JMP134

DSMZ Accession No.: DSM 4058

Taxonomy ID: 264198; Inherited blast name: b-proteobacteria

Other names:

synonyms: Cupriavidus pinatubonensis JMP134, Cupriavidus necator JMP134, Wautersia eutropha JMP134, Ralstonia sp. JMP134;

equivalent names: Ralstonia eutropha str. JMP134 and Ralstonia eutropha strain JMP134

In addition, also the following other Ralstonia eutropha strains can be used in the invention:

Ralstonia eutropha 335 (DSMZ Accession No.: DSM 531 , ATCC 17697)

Ralstonia eutropha N9A (DSMZ Accession No.: DSM 518;

Ralstonia eutropha B19 (DSMZ Accession No.: DSM 515): Ralstonia eutropha G27 (DSMZ Accession No.: DSM 516):

Ralstonia eutropha G29 (DSMZ Accession No.: DSM 517):

Ralstonia eutropha 336 (DSMZ Accession No.: DSM 529, ATCC 17698)

Ralstonia eutropha 338 (DSMZ Accession No.: DSM 530, ATCC 17700);

Ralstonia eutropha TA06 (DSMZ Accession No.: DSM 4182);

Ralstonia eutropha H850 (DSMZ Accession No.: DSM 5536; NRRL B-15940) Ralstonia eutropha KTO ₂ (DSMZ Accession No.: DSM 6519);

Ralstonia eutropha Tfa17 (DSMZ Accession No.: DSM 1 1098)

Ralstonia eutropha N-1 (DSMZ Accession No.: DSM 13513;ATCC 43291 )

Ralstonia eutropha NRRL B-2804 (DSMZ Accession No.: DSM 30029,

ATCC 25207, NRRL B-2804);

"Lignocellulosic biomass" refers here to plant biomass that comprises mainly cellulose, hemicelluloses and lignin. Lignocellulosic biomass is for example agricul- tural residues (e.g. wheat, barley or rice straw, husk (chaff), corn stover, sugar cane bagasse), energy crops, wood or plant materials or residues and municipal waste, e.g. paper waste.

"Bulk chemical" refers to any useful chemicals, which may be produced from inexpensive raw materials in high amounts. Examples of useful bulk chemicals are li- pids, for example polyhydroxyalkanoates, in particular polyhydroxybutyrate (PHB), or other lipids, such as fatty acids and/or fatty acid derivatives, for example acyl- glyserols.

The term "lipid" refers to a fatty substance, whose molecule generally contains, as a part, an aliphatic hydrocarbon chain, which dissolves in nonpolar organic sol- vents but is poorly soluble in water. Lipids are an essential group of large molecules in living cells. Lipids are, for example, fats, oils, waxes, wax esters, sterols, terpenoids, isoprenoids, carotenoids, polyhydroxyalkanoates, nucleic acids, fatty acids, fatty alcohols, fatty acid esters, phospholipids, glycolipids, sphingolipids and acylglycerols. The terms lipids and oils are used in this description synonymously. The term "acyglycerol" refers to an ester of glycerol and fatty acids. Acylglycerols occur naturally as fats and fatty oils. Examples of acylglycerols include triacylglyc- erols (TAGs, triglycerides) diacylglycerols (diglycerides) and monoacylglycerols (monoglycerides). "Raw materials" or "starting materials" are materials which form or are included to the cultivation medium of a Cupriavidus or Ralstonia host and which the microorganism can use for its growth and/or production of desired chemicals. Advantageously the materials are inexpensive and available in large amounts. Suitable starting materials are for example lignocellulosic biomass (e.g. wood or plant materials, their fractions or residues) and agricultural waste(s) or residue(s), micro- or macroalgae and any materials, which comprise carbohydrates, such as glucose, galactose, mannose, xylose, or arabinose or combinations thereof. Some starting materials may comprise mainly xylose (hemicelluloses), some may comprise mainly glucose (cellulose or starch), and some may comprise mainly mannose (galactoglucomannan based hemicelluloses).

Sugars, mannose, glucose, xylose, arabinose, and galactose need to be provided to microorganisms as monomeric sugars. The raw materials containing these sugars in polymeric form, e.g. lignocelluloses, can be treated with any known method to hydrolyse the sugar polymers into monomeric sugars that are utilizable by microorganisms according to the present invention.

In the cultivation of Cupriavidus or Ralstonia can be used any cultivation medium comprising components suitable for Cupriavidus or Ralstonia growth. In industrial applications the cultivation medium typically comprises monomeric sugars orinating from lignocellulosic materials as carbon source, and in addition usually nutrients and salts or components thereof as micro- and macronutrients, and water. A cultivation medium may comprise mannose, glucose, galactose, xylose, or arabinose or any combination thereof as carbon source, typically as the main carbon source, in some embodiments as the sole carbon source.

In the cultivation of Cupriavidus or Ralstonia can be used cultivation conditions suitable for cultivation of Cupriavidus or Ralstonia. Such cultivation conditions are well known to a person skilled in the art. In the cultivation can be used liquid or sol- id media. Liquid cultures may be agitated, suitable agitation 50 - 1000 rpm, typically 100 - 600 rpm. The cultivation temperature may be 15 to 45 °C, preferably 25 to 37 °C, typically 30 °C. The incubation time may be 1 day to 14 days, typically 2 to 7 days, preferably 24 to 72 hours. The cultivation may be a batch, fed-batch or continued cultivation.

The term "endogenous gene" refers here to a gene which is natural to the Cu- priavidus or Ralstonia host. The term "exogenous gene" refers here to a gene which is not natural to the Cu- priavidus or Ralstonia host.

The genes introduced into the Ralstonia or Cupriavidus host may be endogenous or exogenous, typically they are exogenous, if/since the host does not possess the genes naturally. In some embodiments the endogenous genes naturally produced by the host may be overexpressed in the host for example by expressing the coding region under a strong promoter.

A gene encoding "phosphomannose isomerase" may be any gene which encodes an enzyme having phosphomannose isomerase activity. According to Enzyme Classification the enzyme has EC number 5.3.1 .8. In a specific embodiment of the invention the gene may be a gene encoding phosphomannose isomerase (PMI) or mannose-6-phosphate isomerase from Escherichia coli strain K12 (EC 5.3.1 .8) (ACCESSION No. P00946) (gene name: pmi, origin: Escherichia coli strain K12) (SEQ ID NO:1 ).

A gene encoding "facilitated diffusion protein for mannose uptake" may be any gene which encodes facilitated diffusion protein for mannose uptake activity (EC 1 .3.1 .74 ) or a gene encoding xylose proton symporter E. In a specific embodiment of the invention the gene may be a gene encoding glucose-facilitated diffusion transporter (GLF) from Zymomonas mobilis (EC 1 .3.1 .74) (ACCESSION No.

P21906) (gene name: glf, origin: Zymomonas mobilis) (SEQ ID NO:2). Surprising- ly, a Ralstonia or Cupriavidus host introduced to express a gene encoding facilitated diffusion protein for mannose uptake activity, in particular glf, can use glucose as carbon source.

A gene encoding "mannofructokinase" may be any gene which encodes man- nofructokinase (EC 2.7.1 .4) activity. In a specific embodiment of the invention the gene may be a gene encoding mannofructokinase (MAK) (EC 2.7.1 .4) from Esch- erichia coli, in particular from strain K12 (ACCESSION No. P23917) (gene name: mak, origin: Escherichia coli strain K12) (SEQ ID NO:3).

A gene encoding "xylose isomerase" may be any gene, which encodes xylose isomerase (EC 5.3.1 .5) activity. In a specific embodiment of the invention the gene may be a gene encoding D-xylose isomerase (XylA) from Escherichia coli strain K12 (ACCESSION No. P00944) (EC 5.3.1 .5) (gene name: xylA, origin:

Escherichia coli strain K12) (SEQ ID NO:4).

A gene encoding "xylulokinase" may be any gene, which encodes xylulokinase activity (EC 2.7.1 .17). In a specific embodiment of the invention the gene may be a gene encoding xylulose kinase (XylB) from Escherichia coli strain K12

(ACCESSION No. P09099) (EC 2.7.1 .17) (gene name: xylB, origin: Escherichia coli strain K12) (SEQ ID NO:5).

A gene encoding "xylose proton symporter E" may be any gene, which encodes xylose proton symporter E . In a specific embodiment of the invention the gene may be a gene encoding D-xylose-proton symporter (XylE) from Escherichia coli strain K12 (gene name: xylE, origin: Escherichia coli strain K12) (ACCESSION NO.P0AGF4) (SEQ ID NO:6).

A gene encoding "high affinity ABC- transporter" may be any gene, which encodes high affinity ABC- transporter. In a specific embodiment of the invention the gene may be a gene encoding D-xylose-binding periplasmic protein (XylF) from Escherichia coli strain K12 (gene name: xylF, origin: Escherichia coli strain K12) (ACCESSION No. P37387) (SEQ ID NO:7), or a gene encoding xylose import ATP-binding protein (XylG) from Escherichia coli strain K12 (gene name: xylG, origin: Escherichia coli strain K12) (ACCESSION No. P37388) (EC 3.6.3.17) (SEQ ID NO:8), or a gene encoding xylose transport system permease protein (XylH) from Escherichia coli strain K12 (gene name: xylH, origin: Escherichia coli strain K12) (ACCESSION No. P0AGI4) (SEQ ID NO:9). Surprisingly, a Ralstonia or Cu- priavidus host introduced to express genes, which encode high affinity ABC- transporter, in particular genes encoding Xyl F, Xyl G and Xyl H, can use glucose and galactose as carbon source. A gene encoding "transaldolase " may be any gene, which encodes transaldolase (EC 2.2.1 .2). In a specific embodiment of the invention the gene may be a gene encoding transaldolase B (TalB) from Escherichia coli strain K12 (gene name: talB, origin: Escherichia coli strain K12) (ACCESSION No. BAB96586) (EC

2.2.1 .2) (SEQ ID NO:10).

A gene encoding "ketolase " may be any gene, which encodes transketolase (EC

2.2.1 .1 ) . In a specific embodiment of the invention the gene may be a gene encoding transketolase 1 (TktA) from Escherichia coli strain K12 (gene name: tktA, origin: Escherichia coli strain K12) (ACCESSION No. P27302) (EC 2.2.1 .1 ) (SEQ ID NO: 1 1 ).

A gene encoding "glucokinase" may be any gene, which encodes glucokinase (EC

2.7.1 .2) . In a specific embodiment of the invention the gene may be a gene encoding glucokinase (glk) from Escherichia coli strain K12 (gene name: glk, origin: Escherichia coli strain K12) (ACCESSION No. P0A6V8.1 ) (EC 2.7.1 .2)

GLK_ECOLI RecName: Full=Glucokinase; AltName: Full=Glucose kinase [Escherichia coli K-12] (SEQ ID NO: 12).

A Ralstonia or Cupriavidus host introduced to express glucokinase is capable of utilizing glucose. Within the scope of the present invention is any gene, the gene product of which is known to catalyze the reaction of interest. Within the scope of the present invention are also genes, which encode the same or equivalentic function and are highly homologous to the genes of interest. This means, for example, that the proteins encoded by the genes have high identity % when compared to each other. For example the following genes may be used:

The respective genes from different Escherichia strains (e.g. Escherichia albertii [phosphomannose isomerase ZP_02903372.1 , mannose-6-phosphate isomerase, class I [Escherichia albertii TW07627] (SEQ ID NO:13); mannofructokinase ZP_02902328.1 , manno kinase [Escherichia albertii TW07627] (SEQ ID NO:14); Escherichia fergusonii [phosphomannose isomerase YP_002382576.1 , mannose- 6-phosphate isomerase [Escherichia fergusonii ATCC 35469] (SEQ ID NO:15); xy- lose isomerase YP_002384641 .1 ]) xylose isomerase [Escherichia fergusonii ATCC 35469] (SEQ ID NO:16); Shigella strains (e.g. Shigella flexneri [phosphomannose isomerase NP_837299.1], mannose-6-phosphate isomerase [Shigella flexneri 2a str. 2457T] (SEQ ID NO:M),;Shigella sonnei [phosphomannose iso- merase YP_310481 .1 , mannose-6-phosphate isomerase [Shigella sonnei Ss046] (SEQ ID NO:18); xylose isomerase YP_312597.1 ] , xylose isomerase [Shigella sonnei Ss046] (SEQ ID NO:19); Shigella dysenteriae [phosphomannose isomerase ZP_03064125.1 , mannose-6-phosphate isomerase, class I [Shigella dysenteriae 1012] (SEQ ID NO:20); mannofructokinase ZP_03064822.1 , man- no(fructo)kinase [Shigella dysenteriae 1012] (SEQ ID NO:21 ),;xylose isomerase ZP_03063610.1 ]), xylose isomerase [Shigella dysenteriae 1012] (SEQ ID NO:22); Citrobacter strains (e.g. Citrobacter koseri [phosphomannose isomerase

P_001453192.1 , hypothetical protein CKO_01624 [Citrobacter koseri ATCC BAA- 895] (SEQ ID NO:23); xylose isomerase YP_001456500.1 ], xylose isomerase [Citrobacter koseri ATCC BAA-895] (SEQ ID NO:24); Citrobacter rodentium

[phosphomannose isomerase YP_003365034, mannose-6-phosphate isomerase [Citrobacter rodentium ICC168] . (SEQ ID NO:25); mannofructokinase

YP_003364074.1 , probable manno(fructo)kinase [Citrobacter rodentium ICC168] (SEQ ID NO:26); xylose isomerase YP_003367655.1 ] , D-xylose isomerase

[Citrobacter rodentium ICC168] (SEQ ID NO:27); Citrobacter youngae [phosphomannose isomerase ZP_06352970.2, mannose-6-phosphate isomerase, class I [Citrobacter youngae ATCC 29220] (SEQ ID NO:28); xylose isomerase

ZP_06571492.1 ]), xylose isomerase [Citrobacter youngae ATCC 29220] (SEQ ID NO:29); Salmonella strains (e.g. Salmonella enterica [phosphomannose isomer- ase YP_002146578.1 , mannose-6-phosphate isomerase, class I [Salmonella enterica subsp. enterica serovar Agona str. SL483] (SEQ ID NO:30; xylose isomerase ZP_02342859.1 ], xylose isomerase [Salmonella enterica subsp. enterica serovar Saintpaul str. SARA29] (SEQ ID NO:31 ); Salmonella typhimurium [phosphomannose isomerase CAA40399.1 ]), phosphomannose isomerase [Salmonella typhimurium] (SEQ ID NO:32); Enterobacter strains (e.g. Enterobacter cloacae [phosphomannose isomerase ABF71065.1 ,mannose-6-phosphate isomerase [Enterobacter cloacae](SEQ ID NO:33); xylose isomerase YP_003610718.1 ], xylose isomerase [Enterobacter cloacae subsp. cloacae ATCC 13047] (SEQ ID NO:34); Enterobacter cancerogenus [phosphomannose isomerase ZP_05967481 .1 ]), mannose-6-phosphate isomerase, class I [Enterobacter cancerogenus ATCC 35316] (SEQ ID NO:35); Cronobacter strains (e.g. Cronobacter turicensis [phosphomannose isomerase YP_003210382.1 ] , mannose-6-phosphate isomerase [Cronobacter turicensis z3032] (SEQ ID NO:36); Cronobacter sakazakii [phosphomannose isomerase YP_001438057.1]) , hypothetical protein ESA_01968 [Cronobacter sakazakii ATCC BAA-894] (SEQ ID NO:37); Klebsiella strains (e.g. Klebsiella pneumoniae [phosphomannose isomerase YP 002238770.1 , mannose- 6-phosphate isomerase, class I [Klebsiella pneumoniae 342] (SEQ ID NO:38); mannofructokinase YP_002240149.1 ]), manno(fructo)kinase [Klebsiella pneumoniae 342] (SEQ ID NO:39); Serratia strains (e.g. Serratia odorifera [phosphomannose isomerase ZP_06640499.1 ]), mannose-6-phosphate isomerase [Serratia odorifera DSM 4582] (SEQ ID NO:40).

Furthermore, the following genes may be used:

D-xylose transporter XylE [Escherichia fergusonii ATCC 35469] YP_002385136.1 (SEQ ID NO: 41 );

D-xylose transporter XylE [Shigella flexneri 2a str. 301] NP_709888.1 (SEQ ID NO: 42);

D-xylose-proton symporter (D-xylose transporter) [Salmonella enterica subsp. en- terica serovar Schwarzengrund str. SL480] , Xyl E, ZP_02664392.1 (SEQ ID NO: 43);

sugar transporter family protein [Citrobacter youngae ATCC 29220] , Xyl E, ZP_06356420.1 (SEQ ID NO: 44);

D-xylose: proton symporter [Acidobacterium capsulatum ATCC 51 196] , Xyl E, YP_002753901 .1 (SEQ ID NO: 45):

xylulokinase [Shigella flexneri 2a str. 301 ] NP_709351 .1 (SEQ ID NO: 46);

xylulose kinase [Citrobacter rodentium ICC168] YP_003367656.1 (SEQ ID NO: 47);

D-xylose transporter subunit XylF [Shigella flexneri 5 str. 8401 ] YP_691285.1 (SEQ ID NO: 48);

D-xylose transporter subunit XylF [Citrobacter koseri ATCC BAA-895]

YP_001456501 .1 (SEQ ID NO: 49);

D-xylose ABC transporter, ATP-binding protein [Shigella boydii CDC 3083-94] YP_001882237.1 , Xyl G, (SEQ ID NO: 50);

D-xylose ABC transporter, periplasmic D-xylose-binding protein [Klebsiella pneumoniae 342] , Xyl G, YP_002236058.1 (SEQ ID NO: 51 );

xylose transporter ATP-binding subunit [Citrobacter koseri ATCC BAA-895] YP_001456502.1 , Xyl G, (SEQ ID NO: 52); D-xylose ABC transporter, permease protein [Shigella dysenteriae 1012] ZP_03063638.1 , Xyl H, (SEQ ID NO: 53);

inner-membrane translocator [Enterobacter cloacae subsp. cloacae ATCC 13047]YP_003610715.1 , Xyl H, (SEQ ID NO: 54);

glucokinase [Shigella dysenteriae 1012] ZP_03065183.1 (SEQ ID NO: 55); glucokinase [Escherichia fergusonii ATCC 35469]YP_002381954.1 (SEQ ID NO: 56);

transaldolase [Shigella dysenteriae 1012] ZP_03066451 .1 (SEQ ID NO: 57); transaldolase B [Escherichia fergusonii ATCC 35469] YP_002381239.1 (SEQ ID NO: 58);

transketolase [Shigella dysenteriae 1012] ZP_03063878.1 (SEQ ID NO: 59); transketolase [Escherichia fergusonii ATCC 35469] YP_002383968.1 (SEQ ID NO: 60)

can be used in the invention. Alternative sources for various genes encoding the desired enzyme activities are for example :

Alternative sources for glucokinases: glucokinase [Shigella boydii Sb227]YP_408803.1

glucokinase [Escherichia albertii TW07627]ZP_02901664.1

glucokinase [Salmonella enterica subsp. enterica serovar Typhimurium str. LT2] NP_461344.1

glucokinase [Citrobacter youngae ATCC 29220]ZP_06351950.1

Glucokinase [Citrobacter rodentium ICC168]YP_003366335.1

glucokinase [Enterobacter cloacae subsp. cloacae NCTC 9394] CBK871 15.1 glucokinase [Citrobacter koseri ATCC BAA-895] YP_001452005.1

glucokinase [Cronobacter sakazakii ATCC BAA-894]YP_001436959.1 glucokinase [Enterobacter cancerogenus ATCC 35316]ZP_05968594.1 glucokinase [Klebsiella pneumoniae subsp. pneumoniae MGH 78578]

YP_001336384.1

glucokinase [Serratia odorifera DSM 4582] ZP_06639362.1

Glucokinase [Erwinia billingiae Eb661 ] CAX60782.1

Glucokinase [Erwinia amylovora CFBP1430] YP_003531826.1

Alternative sources for mannofructokinases are for example: fructokinase [Shigella sonnei Ss046] YP_309383.2

hypothetical protein CKO_02777 [Citrobacter koseri ATCC BAA-895]

YP_001454319.1

fructokinase [Enterobacter sp. 638] YP_001 175599.1

fructokinase [Escherichia fergusonii ATCC 35469] YP_002383736.1

fructokinase [Enterobacter cloacae subsp. cloacae ATCC 13047]

YP 00361 1661 .1 Alternative sources for facilitated diffusion transporter, GLF and XylE are for example:

D-xylose transporter XylE [Shigella sonnei Ss046] YP_312943.1

xylose-proton symport [Citrobacter sp. 30_2] ZP_04558597.1

sugar transporter family protein [Citrobacter youngae ATCC 29220]

ZP_06356420.1

D-xylose transporter XylE [Salmonella enterica subsp. arizonae serovar

62:z4,z23:-] YP_001572426.1

D-xylose-proton symporter (D-xylose transporter) [Citrobacter rodentium ICC168] YP_003367154.1

D-xylose transporter XylE [Mannheimia succiniciproducens MBEL55E]

YP_089566.1

D-xylose transporter XylE [Actinobacillus succinogenes 130Z] YP_001343806.1 D-xylose transporter XylE [Bacillus cereus ATCC 10987] NP_978526.1 xylose permease [Bacillus megaterium DSM319] YP_003597063.1

Due to high homology between facilitated diffusion protein for mannose uptake (EC1 .3.1 .74) , in particular GLF, and xylose proton symporter E, xyl E, genes en- coding xylose proton symporter E, xyl E, may be used instead of genes encoding facilitated diffusion protein for mannose uptake, and vice versa.

Alternative sources for xylulokinases are for example: xylulokinase [Shigella sp. D9] ZP_05434777.1

xylulokinase [Escherichia fergusonii ATCC 35469]

YP_002384640.1

xylulokinase [Citrobacter sp. 30_2] ZP_04559359.1

xylulokinase [Shigella boydii Sb227] YP_409882.1

xylulose kinase [Salmonella enterica subsp. enterica serovar Paratyphi C strain RKS4594] YP_002639258.1

hypothetical protein CKO_05020 [Citrobacter koseri ATCC BAA-895]

YP_001456499.1

xylulokinase [Shigella sonnei Ss046] YP_312598.1

xylulokinase [Citrobacter youngae ATCC 29220] ZP_06355683.1

xylulokinase [Klebsiella sp. 1_1_55] ZP_06551221 .1

hypothetical protein ECL_00202 [Enterobacter cloacae subsp. cloacae ATCC 13047] YP_003610719.1

xylulokinase [Klebsiella pneumoniae 342] YP_002236061 .1

xylulokinase [Klebsiella variicola At-22] YP_0034371 18.1

Xylulose kinase [Cronobacter turicensis z3032] YP_003212435.1

Xylulose kinase [Yersinia intermedia ATCC 29909] ZP_04636833.1

Xylulokinase [Erwinia billingiae Eb661 ] CAX60830.1

xylulokinase [Serratia odorifera DSM 4582] ZP_06638097.1 Alternative sources for XylF are for example:

D-xylose transporter subunit XylF [Shigella boydii Sb227] YP_409884.1

D-xylose transporter subunit XylF [Enterobacter sp. 638] YP_001 174896.1

D-xylose transporter subunit XylF [Cronobacter sakazakii ATCC BAA-894] YP_001440178.1

D-xylose-binding periplasmic protein [Cronobacter turicensis z3032]

YP_003212438.1

D-xylose transporter subunit XylF [Enterobacter cloacae subsp. cloacae ATCC 13047] YP_003610717.1

hypothetical protein ENTCAN_08293 [Enterobacter cancerogenus ATCC 35316] ZP_05969667.2

D-xylose ABC transporter, periplasmic substrate-binding protein [Erwinia billingiae Eb661 ] CAX60828.1

xylose ABC superfamily ATP binding cassette transporter, binding protein [Serratia odorifera DSM 4582] ZP_06638100.1

D-xylose transporter subunit XylF [Actinobacillus succinogenes 130Z]

YP_001343809.1 Alternative sources for XylG are for example: xylose transporter ATP-binding subunit [Shigella flexneri 5 str. 8401 ] YP_691284.1 D-xylose ABC transporter, ATP-binding protein [Shigella dysenteriae 1012] ZP_03063359.1

xylose transporter ATP-binding subunit [Enterobacter cloacae subsp. cloacae ATCC 13047] YP_003610716.1

D-xylose ABC transporter, ATP-binding protein [Klebsiella pneumoniae 342] YP_002236058.1

hypothetical protein ENTCAN_08292 [Enterobacter cancerogenus ATCC 35316] ZP_05969666.1

xylose transporter ATP-binding subunit [Cronobacter sakazakii ATCC BAA-894] YP_001440177.1

Xylose import ATP-binding protein xylG [Cronobacter turicensis z3032]

YP_003212439.1

D-xylose ABC transporter, ATPase subunit [Erwinia billingiae Eb661 ] CAX60827.1 xylose ABC superfamily ATP binding cassette transporter, ABC protein [Serratia odorifera DSM 4582] ZP_06638101 .1

COG1 129: ABC-type sugar transport system, ATPase component [Actinobacillus pleuropneumoniae serovar 1 str. 4074] ZP_00134918.2

Alternative sources for XylH are for example: hypothetical protein CKO_05024 [Citrobacter koseri ATCC BAA- 895]YP_001456503.1

xylose ABC transporter membrane protein [Enterobacter cloacae subsp. cloacae NCTC 9394] CBK87790.1

hypothetical protein ENTCAN_08291 [Enterobacter cancerogenus ATCC 35316] ZP_05969665.1

inner-membrane translocator [Enterobacter sp. 638] YP_001 174894.1 hypothetical protein ESA_04159 [Cronobacter sakazakii ATCC BAA- 894]YP_001440176.1

D-xylose ABC transporter, permease protein [Klebsiella pneumoniae 342] YP_002236057.1

Xylose transport system permease protein xylH [Cronobacter turicensis z3032] YP_003212440.1

sugar transport system permease protein [Yersinia enterocolitica subsp.

enterocolitica 8081 ] YP_001008391 .1

D-xylose ABC transporter, permease protein [Erwinia billingiae Eb661 ]

CAX60826.1

monosaccharide-transporting ATPase [Serratia proteamaculans 568]

YP_001476339.1 xylose ABC superfamily ATP binding cassette transporter, membrane protein [Serratia odorifera DSM 4582] ZP_06638102.1

monosaccharide-transporting ATPase [Actinobacillus succinogenes 130Z] YP_001343807.1

Alternative sources for transaldolase are for example transaldolase B [Shigella boydii Sb227] YP_406573.1

transaldolase [Citrobacter youngae ATCC 29220] ZP_06354813.1

transaldolase B [Citrobacter koseri ATCC BAA-895] YP_001454897.1

transaldolase B [Salmonella enterica subsp. arizonae serovar 62:z4,z23:~ YP_001571972.1

transaldolase B [Citrobacter rodentium ICC168] YP_003363651 .1

transaldolase B [Enterobacter sp. 638] YP_001 175308.1

hypothetical protein ENTCAN_05194 [Enterobacter cancerogenus ATCC 35316] ZP_05966848.1

transaldolase B [Citrobacter sp. 30_2] ZP_04559788.1

transaldolase [Enterobacter cloacae subsp. cloacae NCTC 9394] CBK87626.1 transaldolase B [Klebsiella pneumoniae 342] YP_002240539.1

transaldolase B [Enterobacter cloacae subsp. cloacae ATCC

13047]YP_00361 1333.1

transaldolase B [Cronobacter sakazakii ATCC BAA-894] YP_001439387.1 transaldolase B [Cronobacter turicensis z3032] YP_003208998.1

transaldolase [Escherichia albertii TW07627] ZP_02902842.1

transaldolase [Yersinia kristensenii ATCC 33638] ZP_04625574.1

transaldolase A [Serratia odorifera DSM 4582] ZP_06637249.1

transaldolase B [Erwinia amylovora CFBP1430] YP_003532302.1

transaldolase B [Erwinia amylovora ATCC 49946] YP_003537736.1

transaldolase B [Erwinia pyrifoliae DSM 12163] CAY73102.1

Alternative transketolases are for example: transketolase [Shigella flexneri 2a str. 2457T] NP_838419.1

transketolase [Shigella boydii CDC 3083-94] YP_001881708.1

transketolase [Escherichia albertii TW07627] ZP_02900973.1

transketolase [Citrobacter koseri ATCC BAA-895] YP_001455802.1 transketolase [Citrobacter sp. 30_2] ZP_04560349.1

transketolase [Citrobacter youngae ATCC 29220] ZP_06355044.1

transketolase [Enterobacter cancerogenus ATCC 35316] ZP_05970189.1 transketolase [Enterobacter cloacae subsp. cloacae NCTC 9394] CBK86553.1 transketolase [Klebsiella pneumoniae subsp. pneumoniae MGH 78578]

YP_001336984.1

transketolase 1 [Citrobacter rodentium ICC168] YP_003368408.1 transketolase [Klebsiella variicola At-22] YP_003440166.1

transketolase [Klebsiella pneumoniae 342] YP_002239249.1

Transketolase 1 [Cronobacter turicensis z3032] YP_00321 1835.1

transketolase [Cronobacter sakazakii ATCC BAA-894] YP_001436540.1

transketolase [Enterobacter cloacae subsp. cloacae ATCC 13047]

YP_003614730.1

transketolase [Erwinia tasmaniensis Et1/99] YP_001908733.1

transketolase [Serratia odorifera DSM 4582] ZP_06641778.1

The accession numbers are derived from NCBI database (National Center for Biotechnology Information, U.S. National Library of Medicine, 8600 Rockville Pike, Bethesda MD, 20894 USA). The date of providing the accession numbers was 2nd June 2010.

Within the scope of the present invention are proteins encoded by any of the above listed genes, in particular those comprising the amino acid sequences SEQ ID NO: 1 to 60, preferably sequences SEQ ID NO: 1 to 12.

It is evident that small variations in the nucleotide sequence of a gene do not sig- nificantly change the catalytic properties of the encoded protein. For example many changes in the nucleotide sequence do not change the amino acid sequence of the encoded protein. Also an amino acid sequence may have variations, which do not change the functional properties of a protein, in particular they do not prevent an enzyme from carrying out its catalytic function. As it is known to a per- son skilled in the art, variations can exist or can be made to regions which are outside of the region critical to the function of the enzyme and still result in an active polypeptide. Such variations in the nucleotide sequence or DNA molecules or in an amino acid sequence are known as "functional equivalents", because they do not significantly change the function of the gene to encode a protein with a particu- lar function, e.g. catalyzing a particular reaction or, respectively, change the particular function of the protein. Within the scope of the present invention are func- tional equivalents, including fragments or derivatives, or closest homologues of the listed proteins, in particular functional equivalents of sequences SEQ ID NO: 1 to 60, preferably sequences SEQ ID NO: 1 to 12.

Within the scope of the present invention are genes encoding amino acid se- quences showing at least 50%, preferably at least 60 % identity, preferably at least 65 %, preferably at least 70 %, preferably at least 75 %, preferably at least 80 %, preferably at least 85 %, more preferably at least 90 %, still more preferably at least 95 %, more and more preferably at least 98 % identity to any of the amino acid sequences of the above listed proteins, in particular sequences SEQ ID NO: 1 to 60, preferably sequences SEQ ID NO: 1 to 12.

Within the scope of the present invention are genes encoding proteins comprising any of the amino acid sequences SEQ ID NO:1 to 60, preferably sequences SEQ ID NO: 1 to 12.

Within the scope of the present invention are also genes encoding amino acid se- quences showing at least 50%, preferably at least 60 % identity, preferably at least 65 %, preferably at least 70 %, preferably at least 75 %, preferably at least 80 %, preferably at least 85 %, more preferably at least 90 %, still more preferably at least 95 %, more and more preferably at least 98 % identity to any of the amino acid sequences SEQ ID NO:1 to 60, preferably sequences SEQ ID NO: 1 to 12. The term "identity" refers to the identity between two nucleic acid or amino acid sequences, respectively compared to each other from the first nucleic acid to the last nucleic acid or from the first amino acid encoded by the corresponding gene to the last amino acid. The identity of the full-length sequences can be measured by using BLAST program (Altschul, S. F., T.L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller und D. J. Lipman 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389- 3402). In the comparison is preferably used the mature sequences of the proteins.

Bacteria belonging to genus Cupriavidus or Ralstonia and capable of expressing genes encoding phosphomannose isomerase and facilitated diffusion protein for mannose uptake or xylose proton symporter E and optionally mannofructokinase are capable of growing on mannose as the carbon source. A Cupriavidus host introduced to express a gene encoding facilitated diffusion protein for mannose uptake activity, in particular glf, can use also glucose as carbon source.

Mannose can be found in wood and plant materials, especially in hemicelluloses of soft woods (e.g. pine or spruce) consisting of galactoglucomannane polymer, in some fruits or berries (e.g. cranberry), and in cell structures of various microorganisms such as fungi (yeasts and filamentous fungi) and microalgae (e.g. Chlorella, Nannochloropsis and Phaeodacylum).

Bacteria belonging to genus Cupriavidus or Ralstonia and capable of expressing genes encoding xylose isomerase and xylulokinase and a high affinity ABC- transporter or xylose proton symporter E are capable of growing on xylose or xylose and arabinose as the carbon source. A Cupriavidus host introduced to express genes, which encode high affinity ABC- transporter, in particular genes encoding Xyl F, Xyl G and Xyl H, are able to use also glucose and galactose as carbon source.

In addition the microorganism may be genetically modified to express transal- dolase and/or transketolase making the growth of the microorganism faster.

As is described herein a microorganism introduced to express a gene encoding facilitated diffusion protein for mannose uptake is capable of using also glucose. This is achieved in particular by gene glf.

The microorganism host is capable of utilizing glucose also, if to the host is introduced to express a gene encoding glucokinase EC 2.7.1 .2).

In one embodiment a microorganism according to this disclosure is capable of uti- lizing one or more of mannose, glucose, galactose, xylose and/or arabinose as carbon source.

In one embodiment a microorganism according to this disclosure is capable of utilizing mannose and optionally glucose as carbon source. In one further embodiment a microorganism according to this disclosure is capable of utilizing xylose, or xylose and arabinose, and optionally glucose and/or galactose as carbon source. A microorganism introduced to express xylose isomerase, xylulokinase and a high affinity ABC- transporter or xylose protein symporter E, is capable of utilizing xylose. Surprisingly, the microorganism host is capable of utilizing also arabinose. To the best knowledge of the present inventors, this is the first time a Ralstonia or Cupriavidus host has obtained the capability to use arabinose. In the prior art Buchholz et al. (1994) transferred an Alcaligenes eutrophus host by a 28-kb insert, 16.4 of which comprised the minimal information required for xylose utilization by A. eutrophus. Buchholz et al. assumed that the xyl genes encoding xylose isomerase and xylulokinase were located within this region, since they were induced during growth on xylose. Buchholz et al. obtained three transconjugants which grew on xylose as sole source of carbon and energy, but ribose or arabinose- utilizing strains were not obtained. As herein disclosed the microorganism host has been introduced to express inserts which consist essentially of nucleotide sequences needed for xylose utilization. Surprisingly the constructions make the host capable of utilizing also arabinose. A microorganism introduced to express a high affinity ABC- transporter, in particular genes encoding Xyl F, Xyl G and Xyl H, is surprisingly capable of using also glucose and galactose as carbon source.

The activity of MAK (mannofructokinase), PMI (phosphomannose isomerase), GLK (glucokinase) can be assayed by methods well known in the art. Soluble pro- tein fractions of recombinant host strain can be applied for MAK, PMI and GLK activity measurements at 30°C using a Nicolet Evolution 100 UV/VIS spectrophotometer (Thermo Electron Corporation, Cambridge, UK). Activity of MAK can be assayed with an NADH-coupled system as described by Sebastian and Asensio (1972) with some modifications as described in the examples. PMI activity can be measured with an NADP-coupled assay (Kang and Markovitz 1967) with some modifications as described in the examples. GLK activity can be measured with an NADPH-coupled system according to the method of Gottschalk (Gottschalk et al. 1964) with modifications as described in the examples.

XylA and XylB activity can be assayed by methods well known in the art. Soluble protein fractions of recombinant host strain can be applied for XylA and XylB activity measurements at 30 °C using a Nicolet Evolution 100 UVA/IS spectrophotometer (Thermo Electron Corporation, Cambridge, UK). Activity of XylA (Gao et al. 2002, Mejnen et al. 2008) or XylB (Shamanna and Sangerson 1979, Eliasson et al. 2000) can be assayed with an NADH-coupled system with some modifica- tions, using sorbitol dehydrogenase or pyruvate kinase and lactate dehydrogenase, respectively, as auxiliary enzymes as described in the examples

"Regulatory elements" refer here to regulatory elements which can regulate the expression of a foreign gene introduced into a host cell, here in particular into Cupriavidus or Ralstonia. Regulatory elements include promoters, terminators, enhancers and signal sequences.

"Vector" refers here to a nucleic acid molecule, typically a DNA molecule, which can be used to transfer foreign genetic material into a host cell. A vector may be a plasmid, bacteriophage, virus or cosmid, here it is typically a plasmid. Preferably the vector is a broad host range vector. In one embodiment the vector may be an episomal vector not incorporated to the genome of the host cell. In another embodiment the vector may be integrated into the genome of the host cell.

Broad-host-range plasmids permit replication in diverse bacterial species and are not restricted to bacteria of a certain genus or species.

In one embodiment of the invention a vector may comprise a gene encoding phosphomannose isomerase, a gene encoding facilitated diffusion protein for mannose uptake, a gene encoding xylose proton symporter E and/or a gene encoding mannofructokinase. The genes may be in the same or in separate vectors. In another embodiment of the invention a vector may comprise a gene encoding xylose isomerase, xylulokinase, xylose proton symporter E, and/or a high affinity ABC- transporter A. The genes may be in the same or in separate vectors. In one embodiment of the invention the vector may be a broad host range vector. This provides the advantage that the extension of the substrate range can be achieved with the broad host range vectors in any species or strains of Cupriavi- dus or Ralstonia. Preferably the vector is a high copy number vector.

Furthermore, the vector comprises regulatory elements, in particular a promoter, for regulating expression of said genes in Cupriavidus or Ralstonia. Preferably the vector comprises a strong promoter for expression of the desired genes in Cu- priavidus or Ralstonia.

In one embodiment regulatory elements in the vector may comprise neokanamy- c/ ^' n-promoter, /ac-promoter, or glyceraldehyde-3-phophate dehydrogenase promoter. In addition, the following promoters can be used: promoter P2, typically from Pseudomonas syringae (Solaiman and Swingle 2010), in particular of plas- mid pBS29-P2 (Solaiman et al. 2010), the hydrogenase (SH) promoter, typically from R. eutropha (Porthun et al. 2002, Schwartz et al. 1998), and the promoter P _L .typically from the ebb operon (Jeffke et al. 1999, Schaferjohann et al. 1996, Windhovel and Bowien 1990). Preferably the promoters are constitutive.

Suitable promoters are here in particular a constitutive /ac-promoter (P _/ac ) (Siedow et al. 1999), neo/ anamyc/ ^' n-promoter (P _n k), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter (PGAPDH) ■

Vectors, suitable for use in the invention are for example pBBRI MCS and its derivatives pBBR1 MCS-2, pBBR1 MCS-3, pBBR1 MCS-4 and pBBR1 MCS-5 (Kovach et al. 1995). Furthermore, the shuttle plasmid pBHR1 can be used (MoBiTec GmbH, Gottingen, Germany) (Aneja et al. 2009) in the invention.

Plasmid pKT230 (Park et al. 1995), which is derived from the broad host range plasmid RSF1010 (Franklin et al. 1981 ) can also be used in the invention.

Also plasmid pBS29 (Swingle et al. 2008) or pBS29-P2 (Solaiman et al. 2010) , which are derived from plasmid pUCP26 capable of replicating in E. coli and Pseudomonas species (West et al. 1994), can be used in this invention.

Within the scope of the present invention is also the production of desired chemicals by using the modified microorganisms according to the invention. The desired chemicals may be products naturally produced by the host, such as polyhydrolyal- kanoates (Reinecke and Steinbuchel 2009 and Verlinden et al. 2007) or the host may be introduced to express the desired chemicals, for example lipids. Genes which can be used to genetically modify microorganism hosts to express various lipids has been disclosed in WO 2007136762. The capability of a Cupriavidus or Ralstonia host to produce lipids was exemplfied by introducing a Cupriavidus or Ralstonia host to express fatty acid ethyl esters (FAEE). This was carried out by expressing in a polyhydroxy butyrate (PHB) -negative mutant of R. eutropha H16 host genes encoding wax ester synthase/diacylglycerol acyltransferase {atfA), pyruvate decarboxylase (pdc) and alcohol dehydrogenase B (adhB).

The benefit of this invention is an extended substrate utilization range of Cupriavi- dus or Ralstonia towards abundant and low-cost carbohydrates. The extension of substrate range was achieved by using broad host range plasmids, which make it possible to extend the substrate range in any species or strain of the Cupriavidus or Ralstonia host. By chemical methods it is possible to introduce a mutation only in a specific strain. Furthermore, in chemically induced mutants there is the risk of additional mutations that might affect the productiviness and/or may lead to unfavourable side effects. The present invention was exemplified by constructing two different recombinant strains of Ralstonia, more specifically two R. eutropha H16 strains. The strains were developed to be capable of utilizing mannose/glucose, and xylose/arabinose respectively, by metabolic engineering. The induction of different gene in R. eu- tropha, necessary for either glucose/mannose uptake and utilization of mannose, or for xylose uptake and utilization, repectively, complemented the lacking parts of either glucose/mannose or xylose/arabinose catabolism, respectively. The recombinant strains of R. eutropha with the extended substate utilization range synthesized poly(3-hydroxybutyrate) PHB from glucose, mannose, xylose and arabinose in similar amounts than from gluconate.

In one embodiment of the invention in Cupriavidus or Ralstonia may be expressed genes, which are responsive of sugar uptake and conversion. Hexokinases from other prokaryotes, such as Leuconostoc mesenteroides or Streptomyces sp., which also exhibit an activity for mannose (Anderson and Sapico 1975, Coulombel et al. 1982), may be expressed in Cupriavidus or Ralstonia to improve sugar conversion in the host. An overexpression of the chromosomally encoded hexokinase with its side activity for mannose may be used to enhance mannose utilization in Ralstonia. The pBBR plasmids used in the examples can be replaced for example with high copy number vectors and/or with stronger promoters (Kovach et al. 1995, Siedow et al. 1999), or other vectors more suitable for expression of the desired genes for mannose utilization. The /ac-promoter may be replaced with a stronger constitutive promoter, for example the neo/ anamyc/n-promoter as described herein and which give elevated expression levels. The desired genes may also be integrated into the genome of Cupriavidus or Ralstonia giving advantages, such as stability. Furthermore, mutants containing marker-free integrations in their genomes are not subjected to any selection, which save costs especially for large scale cultivations.

According to one further embodiment the present invention provides a method, which comprises that the microorganism is allowed to synthesize bulk chemicals, such as lipids, for example polyhydroxyalkanoates, fatty acids or fatty acid derivatives, and the chemical is recovered from the cultivation medium or from the cells. In the cultivation of Cupriavidus or Ralstonia can be used any cultivation medium comprising components suitable for Cupriavidus or Ralstonia growth. Typically they include a carbon and/or a nutrient source, salts, typically mineral salts, and water. Selection markers, such as antibiotic resistance genes may be needed to confirm the presence of desired genes in the host cells.

The substrate utilization of Ralstonia has been here exemplified by cultivating Cupriavidus or Ralstonia in a mineral salts medium (MSM) described by Schlegel et al. (1961 ). In industrial applications the cultivation medium typically comprises monomeric sugars orinating from lignocellulosic materials, as carbon source, and in addition possibly nutrients and salts or components thereof as micro- and mac- ronutrients, and water.

In the cultivation of Cupriavidus or Ralstonia can be used cultivation conditions suitable for cultivation of Cupriavidus or Ralstonia. Such cultivation conditions are well known to a person skilled in the art. In the cultivation can be used liquid or solid media. Liquid cultures may be agitated, suitable agitation 50 - 1000 rpm, typically 100 - 600 rpm. The cultivation temperature is typically 15 to 45, preferably 25 to 37 °C, typically 30 °C. The incubation time may be 1 day to 14 days, typically 2 to 7 days, preferably 24 to 72 hours. The cultivation may be a batch, fed-batch or continued cultivation.

Escherichia coli and Zymomonas mobilis were cultivated by methods well known to a person skilled in the art and described in the examples.

As herein described recombinant Ralstonia strains and wild type strains were cultivated on different carbon sources. Cells were incubated on MSM agar plates containing 1 % (wt/vol) of the respective carbon source for 5 days at 30 °C. Visible colony growth was phenotypically examined. Characters: (++) growth after an in- cubation time of 24-72 h, (+) growth after more than 72 h of incubation, (-) no visible growth detectable. The use of the following carbon sources were examined: L- Arabinose, D- fructose, D-Galactose, D- Gluconate, D-Glucose, D-glycerol, D- maltose- mannitol, D-mannose, N-acetyl-D-glucosamine, pyruvate, D, ribose, D- trehalose and D-xylose.

The activity of MAK (mannofructokinase), PMI (phosphomannose isomerase), GLK (glucokinase) can be assayed by methods well known in the art. Soluble pro- tein fractions of recombinant host strain can be applied for MAK, PMI and GLK activity measurements at 30°C using a Nicolet Evolution 100 UV/VIS spectrophotometer (Thermo Electron Corporation, Cambridge, UK). Activity of MAK can be assayed with an NADH-coupled system as described by Sebastian and Asensio (1972) with some modifications as described in the examples. PMI activity can be measured with an NADP-coupled assay (Kang and Markovitz 1967) with some modifications as described in the examples.

GLK activity can be measured with an NADPH-coupled system according to the method of Gottschalk (Gottschalk et al. 1964) with modifications as described in the examples. The present invention has been exemplified by generating recombinants of R. eu- tropha strain H16 which, in contrast to the wild type, were able to utilize mannose and/or glucose as sole carbon sources. Up to four genes (glf, glk, mak, pmi) were introduced episomally in R. eutropha strain H16 under control of a constitutive ne- okanamycin- or /ac-promoter, enabling uptake and conversion of glucose and mannose into intermediates of the KDPG pathway.

Bacteria belonging to the genera Ralstonia and Cupriviadus typically have a very narrow substrate utilization spectrum and are only limited amount of sugars. E.g. the capability of Ralstonia eutropha H 16 to use carbohydrates and related compounds is limited to fructose, N-acetylglucosamine and Gluconate. To broaden the substrate utilization range of Ralstonia and Cupriviadus strains, the strains were engineered to use mannose as sole carbon source for growth. The genes for a facilitated diffucion protein (glf) from Zymomomas mobilis and for mannofructokinase (mak), phosphomannose isomerase (pmi) and glucokinase (g// ) from E. coli either alone or in combination constitutively expressed in R. eutropha under control of the lac-promoter, using an episomal broad host range vector. Recombinant strains harboring pBBR1 MCS-3::g// r.makwpmi or pBBRMCS-3: glfv.pmi grew on mannose, whereas pBB^ MCS-3::glf::mak and pBBRI MCS-S!!g/f did not confer the ability to utilize mannose as carbon source to R. eutropha. The recombinant strains harboring pBBR1 MCS-3::g/f: :/77a/ ::p/77/ ^' exhibited faster growth than pBBRI MCS- 3::glf::pmi on mannose. These data indicated that phosphomannose isomerase is required to convert mannose-6-phosphate into fructose-6-phophate for subsequent catabolism via Entner-Doudoroff pathway. In addition, surprisingly all plas- mids conferred to Ralstonia also the ability to grow in the presence of glucose.

Furthermore, expression of the respective enzymes was demonstrated at the tran- scriptional level and by measuring the activities of mannofructokinase (0.14 U mg - 1 protein), and phosphomannose isomerase (0.25 U mg -1 protein).

To identify genes in the genome of R. eutropha H16 coding for functional enzymes required for uptake and catabolism of D-xylose, a protein-protein-BLAST-analysis (Altschul et al. 1990) using an amino acid sequence database of R. eutropha H16 (Pohlmann et al. 2006) was performed. No genes encoding a transporter specific for D-xylose could be identified (Pohlmann et al. 2006). Also no genes encoding enzymes catabolizing D-xylose, e.g. a xylose isomerase or xylulose kinase, were identified. Indeed, R. eutropha H16 possesses genes for a fructokinase and a glu- cokinase (Pohlmann et al. 2006). All other genes, encoding enzymes of the non- oxidative pentose phosphate pathway for further catabolism of D-xylulose-5- phosphate are present in R. eutropha strain H16.

The construction of Ralstonia hosts to use xylose as sole carbon source for growth was exemplified by using R. eutropha strains. The genes for a low-affinity D- xylose-proton-symporter (xylE), a high-affinity ABC-transporter (xylFGH), a xylose isomerase (xylA), and a xylulokinase (xylB), from E. coli were in combination con- stitutively expressed in Ralstonia hosts under control of a lac-promoter and glycer- aldehyde-3-phosphate dehydrogenase (GADPH) promoter, respectively, using an episomal broad host range vector. Recombinant strains harboring pBBRI MCS- 3::xylABFGH grew on xylose conferring the ability to utilize xylose as carbon source to Ralstonia. In addition, surprisingly, plasmids conferring xylose- utilization to Ralstonia conferred also the ability to grow in the presence of arabi- nose. To exemplify the ability of recombinant R. eutropha strains to produce PHB from mannose or glucose, Ralstonia cells were cultivated under conditions permissive for PHB accumulation in MSM containing 1 % (wt/vol) mannose, glucose or sodium gluconate as sole carbon source containing tetracycline or kanamycin, respectively, for selection and 0.05% (wt vol) NH CI. The PHB contents of the cells were analyzed gas chromatographically as described in the materials and methods section. Cultures containing gluconate as a sole carbon source served as a reference for the recombinant strain, and samples were taken in the early stationary growth phase. GC analysis of the cells revealed a PHB content of 63% (wt/wt) of cell dry mass. Cells grown in presence of 1 % (wt/vol) glucose or mannose were also examined in the stationary growth phase, and PHB contents of 65 and 67% (wt/wt) of cell dry mass, respectively, were determined.

To exemplify the ability of recombinant R. eutropha strains to produce PHB from xylose and arabinose, cells were cultivated under conditions permissive for PHB accumulation in MSM containing 1 % (wt/vol) D-xylose, L-arabinose or sodium gluconate as sole carbon source containing tetracycline, respectively, for selection and 0.05% (wt/vol) NH CI. The PHB contents of the cells were analyzed gas chromatographically as described in the Examples in the materials and methods section. Cultures containing gluconate as a sole carbon source served as a reference for the recombinant strain, and samples were taken in the early stationary growth phase. GC analysis of the cells revealed that the cells were able to produce PHB. Cells grown in presence of 1 % (wt/vol) L-arabinose or D-xylose were also examined in the stationary growth phase, and production of PHB was shown. The recombinant strains of R. eutropha with the extended substrate utilization range were thus able to synthesize poly(3-hydroxybutyrate) PHB from glucose, mannose, xylose and arabinose in similar amounts than from gluconate. Examples

Example 1

Materials and methods

Bacterial strains, plasmids, oligonucleotides and cultivation conditions. All bacteria, plasmids and primers used in this application are listed in Table 1 . Cells of R. eutropha strain H16 were cultivated in mineral salts medium (MSM) as described by Schlegel et al. (1961 ). Carbon sources were added to liquid MSM as indicated in the text. Liquid cultures in Erlenmeyer flasks were incubated on a horizontal rotary shaker at an agitation of 1 10 rpm. Solid media were prepared by ad- dition of 1 .5% (wt vol) agar-agar. Cells of Escherichia coli were cultivated at 37 °C in Lysogeny Broth (LB) [Sambrook et al.1989]. Cells of Zymomonas mobilis were cultivated in a medium containing 10 g/L Bacto peptone, 10 g/L yeast extract, and 20 g/L glucose. Antibiotics were applied according to Sambrook et al. (1989) and as indicated in the text.

Table 1 . Bacterial strains, plasmids and oligonucleotides

Strain, plasmid Source or refer¬

Relevant characteristics

or primer ence

Strains

E. coli K-12 Wild type DSM 426

E. coli TOP 10 F ^" mcrA, A(mrr-hsdRMS-mcrBC) f80lacZ AM15, Invitrogen

AlacX74, deoR, recAl, araD139, A(ara- leu)7697, gall), galK, rpsL, endAl, nupG

E. coli S17-1 recAl, thil, hsdR17(r _k -, m _k +), proA, ira-genes Simon et al. 1983 of RP4 plasmid chromosomally integrated

(Mobilization strain)

E. coli J E5511 Hfr, manA4, lpp-1, pps-6 E. coli Genetic

Stock Center (Hirota et al. 1977)

R. eutropha Wild type DSM 428 strain H16

Z. mobilis subsp. Wild type DSM 424 ATCC mobilis 10988

Z. mobilis subsp. Wild type DSM3580 ATCC mobilis 9191

Table 1 continues

Plasmids pJET1.2/blunt Ap ^r Fermentas pCR2.1 Ap ^r , Km ^r , LacZ- Invitrogen pJET1.2::g// with g//as blunt end PCR product in the examples pJET1.2::mofc with mak as blunt end PCR product in the examples pJET1.2::p/T7/ ^' with pmi as blunt end PCR product in the examples pCR2.1::P _nk with neokanamycin promoter as Xho\/Nde\ in the examfragment ples pCR2.1::P _nk ::g/fc with neokanamycin promoter and glk as in the examXho\/EcoR\ fragment ples pBBRlMCS-3 Tc ^r , LacZ- , mob, rep Kovach et al.

1995 pBBRlMCS-2:: P _nk ::glf with neokanamycin promoter and g//as in the examXho\/Sac\ fragment ples pBBRlMCS- with neokanamycin promoter, glk and g//as in the exam2::P _nk ::glk::glf Xho\/Sac\ fragment ples pBBRlMCS-3::g// with g//as Kpn\/Pst\ fragment in the examples pBBRlMCS-3::g//::/77afc with g//and mak as Kpn\/Xba\ fragment in the examples pBBRlMCS-3::g//::pm; with g//as Kpn\/Pst\ fragment and pmi in the examXba\/Sac\ fragment ples pBBRlMCS-3 with glf, mak and pmi as Kpn\/Sac\ fragment in the exam::glf::mak::pmi ples Table 1 continues

Oligonucleotides

Pglf_Kpn\J 5 ^' GGTACCAAGGAAGGACTGATCATG AGTTCTGAAAGTAGTCAGGGTCTA GTC3 ^' (SEQ

ID NO:61)

Pglf_Pst\_r 5 CTGCAGCTACTTCTGGGAGCGCCACATC3 ^' (SEQ ID NO: 62)

Pmak_Pst\J 5 ^' CTGCAGAAGGAAGGACTGATC GTGCGTATAGGTATCGATTTAGGCG3 ^' (SEQ ID NO:

63)

Pmak_Xba\_r 5 ^' TCTAGATTACTCTTGTGGCCATAACCACGC3 ^' (SEQ ID NO: 64)

Ppmi_Xba\J 5 ^' TCTAGAAAGGAAGGTCGACTC ATGCAAAAACTCATTAACTCAGTG CAAAAC3 ^' (SEQ

ID NO:65)

Ppmi_Sac\_r 5 ^' GAGCTCTTACAGCTTGTTGTAAACACGCGCTAAAC3 ^' (SEQ ID NO: 66)

g//_RT_f 5 GTTCTATCGATTGGGTTAATGCCAGTGG3 ^' (SEQ ID NO: 67)

g//_RT_r 5 ^' GGAACATCTGCGGTGCATAATACAGC3 ^' (SEQ ID NO: 68)

mak_Rl_f 5 GCGAGGTTGCAGCGGGAAGTG3 ^' (SEQ ID NO: 69)

moir_RT_r 5 ^' AATTTC ACTG CCTTTCAG CG C ATGTCC3 ^' (SEQ ID NO: 70)

pm/ ^' _RT_f 5 CGCTGACGCCTTTCCTTGCGAT3 ^' (SEQ ID NO: 71)

pm/_RT_r 5 GCGGTGTTTCAGCGAACAGGAACA3 ^' (SEQ ID NO: 72)

pJET1.2 forward se5'CGACTCACTATAGGGAGAGCGGC3' (SEQ ID NO: 73) Fermen- quencing primer, 23- tas mer

pJET1.2 reverse se5'AAGAACATCGATTTTCCATGGCAG3' (SEQ ID NO: 74) Fermen- quencing primer, 24- tas mer

M13/pUC-forward, 5 ^' GTAAAACGACGGCCAGT3 ^' (SEQ ID NO: 75) Jena 17-mer

Bioscience

F_Pnk_Xftol 5 CTCGAGCCGGAATTGCCAGCTGGGG3 ^' (SEQ ID NO: 76)

R_Pnk_Wdel 5 ^' CATATGAAACGATCCTCATCCTGTCTCTTG3 ^' (SEQ ID NO: 77)

f_glk_Nde\ 5 CATATGACAAAGTATGCATTAGTCGGTGATGTGGG3 ^' (SEQ ID NO: 78)

R_glk_Sal\ 5 GTCGACTTACAGAATGTGACCTAAGGTCTGGCGTAAATGTGC3 ^' (SEQ ID NO: 79)

F jl/JomHI 5 GGATCCATGAGGATCGTTTCGCATGAGTTCTGAAAGTAGTCAGGGTCTAGTC3 ^' (SEQ ID

NO: 80)

R_glf_Sac\ 5 ^' GAGCTCCTACTTCTGGGAGCGCCACATCTCCTCG3 ^' (SEQ ID NO: 81) Table 2. Growth of recombinant R. eutropha strain H16 and of the wild type (negative control) on different carbon sources. Cells were incubated on MSM agar plates containing 1 % (wt/vol) of the respective carbon source for 5 days at 30 °C. Visible colony growth was phenotypically examined. Characters: (++) growth after an incubation time of 24-72 h, (+) growth after more than 72 h of incubation, (-) no visible growth detectable.

Carbon R. eutropha strain R. eutropha harboring R. eutropha harbor- source H16 wild type pBBRI MCS- ing pBBRI MCS-

3 : glf::mak::pmi 2 : : Pnt _< ::glk::glf

L-Arabinose

D-Fructose ++ ++ ++

D-Galactose

D-Gluconate ++ ++ ++

D-Glucose - + ++

D-Glycerol + + +

D-Maltose

D-Mannitol

D-Mannose - +

/V-Acetyl-D- ++ ++ ++ glucosamine

Pyruvate ++ ++ ++

D-Ribose

D-Trehalose

D-Xylose Isolation, analysis and modification of DNA. Plasmid DNA was prepared from crude lysates by the alkaline extraction method (Birnboim and Doly 1979). Total DNA of Z. mobilis DSM 424 or DSM3580 and of E. coli strain K-12 was prepared using the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) accord- ing to the manufacturer's protocol. DNA was restricted with restriction endonucle- ases (Gibco/BRL, Gaithersburg, USA) under conditions recommended by the manufacturer. All other genetic procedures and manipulations were conducted as described by Sambrook et al. (1989).

Constructions of plasmids and its transfer into R. eutropha strain H16. The coding regions of gif from Z. mobilis mobilis DSM424 and DSM3580, and of glk, mak and pmi from E. coli, respectively, were amplified by PCR using oligonucleotides Pglf_Kpn\ , Pglf_Pst\_r, F_glf_BamH\ and R_glf_Sac\ for amplification of gif, F_Pnk_Xftol and R_Pnk_A/c/el for P _n/( , F_glk_Nde\ and R_glk_Sal\ for glk, Pmak_Pst\ and Pmak_Xba\_r for amplification of mak, or Ppmi_Xba\ and Ppmi_Sac\_r for amplification of pmi, respectively (Table 1 ). For PCR, KOD Hot Start DNA Polymerase (Merck, Darmstadt, Germany) was used according to the manufacturer's instructions. PCR products were then cloned into the pJET1 .2/blunt cloning vector (Fermentas, Germany) using T4 DNA ligase (Gibco BRL, Gaithersburg, USA) or into cloning vector pCR2.1 (Invitrogen, Karlsbad, Germany) and transferred into E. coli strain TOP10. Plasmids were isolated from ampicillin resistant clones, and the cloned fragments were excised by restriction with respective suitable restriction enzymes for further cloning, extracted from gel after separation using the E.Z.N.A gel extraction kit (Omega Bio-tec, Bangalore, India). For expression experiments in R. eutropha strain H16, the broad host range vectors pBBR1 MCS-2 and pBBR1 MCS-3 were used for cloning of gif, glk, mak and pmi. Vector pBBR1 MCS-2 conferred kanamycin resistance, whereas pBBR1 MCS-3 conferred tetracycline resistance for selection to E. coli and R. eutropha strain H16. The coding region of gif was excised by restriction with Kpn\ and Pst\ and ligated to Kpn\/Pst\ linearized plasmid pBBR1 MCS-3, yielding plas- mids pBBR1 MCS-3::g/f (Fig.2). The coding sequence of the neokanamycin- promoter P _nk was amplified using primers F_Pnk_X/?ol and R_Pnk_A/c/el using isolated vector pBBR1 MCS-2 and further ligated into the vector pCR2.1 (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions yielding pCR2.1 \ \ P _nk . The glk-gene was amplified by tailored PCR from total DNA of E. coli K12 using oligonucleotides F_glk_Nde\ and R_glk_Sal\ as primers. The resulting PCR product was cloned as Nde\/Sal\ fragment into pCR2.1 ::P„/ ₍ , yielding pCR2.1 vP _nk v.glk. The glf-gene was amplified by tailored PCR from total DNA of Z. mobilis DSM3580 using oligonucleotides F_glf_BamH\ and R_glf_Sac\ as primers. The resulting PCR product was cloned as BamH\/Sac\ fragment into pBBR1 MCS-2, yielding pBBRI MCS^g/f. To obtain plasmid pBBR1 MCS-2::P _n/( ::g'// ::g'/f, the coding region of Pnk'-'-glk was excised by restriction with Xho\ and EcoRI from plasmid pCR2.1 vP _nk \\glk and ligated into Xho\/EcoR\ linearized pBBR1 MCS-2::g/f. The coding sequence of glk was further excised by restriction with Nde\/Sal\ from plasmid pBBR1 MCS-2::P„/ ₍ ::g'// ::g'/f, the linearized plasmid was blunted using T4 Polymerase (Gibco BRL, Gaithersburg, USA) and religated using T4 DNA ligase (Gibco BRL, Gaithersburg, USA) yielding plasmid pBBRI MCS^ ^g/f. To ob- tain plasmids pBBR1 MCS-3: :g/f::ma/ and pBBR1 MCS-3::g/f::pm/ ^' , the coding regions of mak and pmi were excised by restriction with Pst\ and Xba\ or Xba\ and Sac\, respectively, and ligated to Pst\/Xba\ and Xba\/Sac\ linearized plasmid pBBR1 MCS-3: :g/f, respectively (Fig. 2). The excised coding regions of mak and pmi were also ligated together to Pst\/Sac\ linearized plasmid pBBR1 MCS-3::g/f yielding pBBR^ MCS-3 ^{^} . ^{^} .glf ^{^} . ^{^} .mak ^■ . ^■ .pmi (Fig. 2). All plasmids were transferred to E. coli strain S17-1 by transformation (Hanahan 1983).

Transfer of DNA by conjugation. Transfer of plasmids pBBR1 MCS-2, pBBR1 MCS-2::P _n/( ::g/f, pBBR1 MCS-2::P _n/( ::g// ::g/f, pBBR1 MCS-3, pBBRI MCS- 3v.gif, pBBR1 MCS-3: :glf::mak, pBBR1 MCS-3 v.glfv.pmi and pBBRI MCS- 3:.glf:.mak:.pmi (Table 1 ) was performed by conjugation applying a previously described protocol (Friedrich et al. 1981 ), using E. coli S17-1 as donor and R. eu- tropha strain H16 as recipient.

DNA sequence analysis. DNA was sequenced in a 48-capillary 3730 DNA Analyzer electrophoresis system (Applied Biosystems, Foster City, CA). For sequenc- ing of inserts in the pJET1 .2/blunt cloning vector (Fermentas, Germany) the pJET1 .2 forward sequencing primer, 23-mer and pJET1 .2 reverse sequencing primer, 24-mer were used. The universal M13/pUC-forward primer and the M13/pUC-reverse primer were used for sequencing inserts in plasmids pBBR1 MCS-2 and pBBR1 MCS-3 (Kovach et al. 1995).

RT-PCR analysis of total RNA isolated from R. eutropha strain H16. DNA-free total RNA of R. eutropha strain H16 was prepared using the RNeasy RNA purifica- tion kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. For identification of glf-, mak- and pm/ ^' -derived mRNA, RT-PCR was applied using oligonucleotides g _RT_f, g _RT_r, makJRJ , mak_RT_r, pmi_RT and pmi_RT_r (Table 1 ). RT-PCR was carried out using a Qiagen OneStep RT-PCR kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol and 0.5 ng RNA as template. To exclude any DNA contamination that could serve as template for PCR, template RNA was added in a control experiment, after inactivation of reverse transcriptase for 15 min at 95 °C in presence of Tag-polymerase. Absence of PCR products indicated that the RT-PCR products were not derived from contaminating DNA. Preparation of soluble protein fractions from R. eutropha strain H16. Cells of recombinant strains of R. eutropha strain H16 were cultivated as described above in presence of 1 % (wt/vol) sodium gluconate as sole carbon source. Cells were harvested by centrifugation for 15 min at 4 °C and 3,500 x g, washed with 100 mM MOPS buffer (pH 7.0) and resuspended in two volumes of the same buffer. Dis- ruption was done by sonication employing a Sonifer 250 (Branson Sonic Power Company) with an amplitude of 16 μιτι (1 min/ml; 50% output control). During ul- trasonication samples were cooled with an ice-NaCI mixture. Soluble membrane- free protein fractions were prepared by 60 min ultracentrifugation of the crude extracts at 100,000 x g and 4 °C. Solubilisation of membrane proteins. Recombinant cells of R. eutropha H16 were cultivated as described above in presence of 1 % (wt vol) glucose as sole carbon source. After 48 h cells were harvested by 15 min centrifugation at 3,500 x g and 4 °C, washed once with 0.9% (wt/vol) NaCI and resuspended in one volume of 100 mM Tris/HCI buffer (pH 7.0). Cell disruption was carried out by a threefold passage through a precooled French Pressure Cell at 1 .000 MPa. The obtained lysates were centrifugated as before in order to remove residual cells. The membrane fraction was prepared by 1 h centrifugation of the supernatant at 100,000 x g and 4 °C , resuspended in 2 ml_ of 20 mM Tris/HCI buffer (pH 7.4) containing 200 mM NaCI and 2% (vol/vol) TritonX-1 14 and incubated for 12 h on ice on a rotary shaker (W3, VWR international GmbH, Darmstadt, Germany). The lysate was centrifugated for 20 min at 3,500 x g and 4 °C. The supernatant was incubated for 5 min at 37 °C and centrifugated for 20 min at 3,500 x g and 37 °C to remove Tri- tonX-1 14 according to Bordier (Bordier, 1981 ). The supernatant was further diluted with 2 volumes of the same buffer without TritonX-1 14 to decrease the detergent concentration.

One-dimensional PAGE. Protein samples were resuspended in gel loading buffer (0.6% (wt/vol) SDS; 1 .25% (vol/vol) /3-mercaptoethanol; 0.25 mM EDTA; 10% (vol/vol) glycerol; 0.001 % (wt/vol) bromophenol blue; 12.5 mM Tris/HCI, pH 6.8) and were separated in 12.5% (wt/vol) SDS-polyacrylamide gels as described by Laemmli (1970). The proteins were stained with Coomassie brilliant blue R-250 (Weber and Osborn, 1969). Samples of crude extracts and solubilized membrane proteins were analyzed by this method.

MAK, PMI and GLK activity assays. Soluble protein fractions of recombinant R. eutropha strain H16 were applied for MAK, PMI and GLK activity measurements at 30°C using a Nicolet Evolution 100 UV/VIS spectrophotometer (Thermo Electron Corporation, Cambridge, UK). Activity of MAK was assayed with an NADH-coupled system as described by Sebastian and Asensio (1972) with some modifications, using pyruvate kinase and lactate dehydrogenase as auxiliary enzymes. The buffered reaction mixture (100 mM MOPS, pH 7.0) contained 2 mM MgCI ₂ , 2 mM ATP, 0.2 NADH, 5 mM mannose, 0.2 mM PEP, 5 U ml ^"1 pyruvate kinase, 5 U ml ^"1 lactate dehydrogenase and 5 to 100 μΙ of soluble extract. PMI activi- ty was measured with an NADP-coupled assay (Kang and Markovitz 1967) with some modifications, using phosphoglucose isomerase and glucose-6-phosphate dehydrogenase as auxiliary enzymes. The buffered reaction mixture (100 mM MOPS, pH 7.0) contained 5 mM MgCI ₂ , 1 mM NADP, 3 mM mannose-6- phosphate, 1 U ml ^"1 phosphoglucose isomerase, 1 U ml ^"1 glucose-6-phosphate dehydrogenase and 10 to 100 μΙ of soluble extract.

GLK activity was measured with an NADPH-coupled system according to the method of Gottschalk (Gottschalk et al. 1964) with modifications concerning the buffer. The buffer mixture (100 mM Tris/HCI, pH 7.6) contained 7 mM MgCI ₂ , 0.9 mM NADP, 460 mM D-glucose, 0.7 mM ATP, 0.35 U ml ^"1 glucose-6-phosphate- dehydrogenase and 10 to 50 μΙ of enzyme preparation.

Analysis of PHB content of recombinant R. eutropha cells by GC. Lyophilized cell material was subjected to methanolysis in presence of methanol and sulfuric acid (85%, vol/vol, MeOH and 15%, vol/vol, H ₂ SO ₄ ) for 4 h at 100 °C, and the resulting methylesters of the PHA constituents were characterized by gas chromatography using an Agilent 6850 GC (Agilent Technologies, Waldbronn, Germany) equipped with a BP21 capillary column (50 m by 0.22 mm; film thickness, 250 nm; SGE, Darmstadt, Germany) and a flame ionization detector (Agilent Technologies). A 2 μΙ portion of the organic phase was analyzed after split injection (split ratio, 1 :5), and a constant hydrogen flow of 0.6 ml min ^"1 was used as carrier gas. The temperatures of the injector were 250 °C and 220 °C, respectively. The following temperature program was applied: 120 °C for 5 min, increase of 3 °C min ^"1 to 180 °C and increase of 10 °C min ^"1 to 220 °C and 220 °C for 31 min. Substances were identified by comparison of their retention times to those of standard fatty acid methyl ester.

The Z. mobilis glf gene plus the E. coli mak and pmi genes were episomally introduced and expressed in R. eutropha and characterized the recombinant strains. The engineered pathways of glucose and mannose catabolism in the recombinant R. eutropha strain are depicted in Fig. 1 .

Construction of different pBBR1 expression vectors for R. eutropha strain H16. The coding regions of the Z. mobilis glf gene (1 ,449 bp) and the E. coli genes mak (936 bp), pmi (1 ,203 bp) and glk (966 bp) were amplified by PCR from ge- nomic gDNA of Z. mobilis DSM 424 and E. coli strain K-12, respectively. The four PCR products comprised beside the coding regions also suitable ribosome binding sites for R. eutropha upstream of the respective start codon to enable expression in this host. These fragments were cloned via the pJET1 .2/blunt or pCR2.1 vector into the broad-host range vectors pBBR1 MCS-2 and pBBR1 MCS-3 under the con- trol of the neokanamycin- or /ac-promoter, respectively, which allow constitutive expression of the cloned genes in R. eutropha H16, yielding plasmids pBBR1 MCS-2::P _n *::gr/f, pBBR1 MCS-2::P _nk ::glk::glf, pBBR1 MCS-3::gr/f, pBBR1 MCS-3: :g/f::ma/ , pBBR1 MCS-3 wglfwpmi and pBBR1 MCS-3: :g/f::ma/ ::pm/ ^' (Fig. 2). Accurate construction of these plasmids was confirmed by sequencing. Additionally, the functionality of the pmi gene in pBBR1 MCS-3: :glf::pmi and pBBW MCS-3::glf::mak::pmi was confirmed by complementation of the pmi (ma- nA) deficient E. coli mutant JE551 1 (Hirota et al. 1977). The recombinant E. coli manA mutants exhibited good growth on solid M9 medium containing 0.5% (wt/vol) mannose as a sole carbon source after two days of incubation at 37 °C. In contrast, the pBBR1 MCS-3 vector did not confer growth to the manA mutant.

Transfer of plasmids to R. eutropha strain H16 and establishment of glucose and mannose utilization. Both plasmids pBBRI MCS^ ^g/f and pBBRI MCS- 2v. Pnk. glkv.gif were transferred via conjugation from E. coli S17-1 to R. eutropha strain H16. Transconjugants were selected on MSM agar plates containing 1 % (wt/vol) sodium gluconate and kanamycin (300 pg/ml), and were from there transferred to MSM plates containing 1 % (wt/vol) glucose as sole carbon source. Only recombinant strains harboring either pBBRI MCS^ ^g/f or pBBRI MCS- 2vPnkV.glkv.gif exhibited significant growth after 5 days of incubation.

To investigate if all three genes are necessary for mannose utilization, all constructed plasmids and plasmid pBBR1 MCS-3 were mobilized from E. coli strain S17-1 to R. eutropha strain H16. The transconjugants were selected on MSM agar plates containing 1 % (wt/vol) sodium gluconate as a sole carbon source and tetracycline (12.5 Mg/ml). They were then transferred to MSM agar plates containing mannose as a sole carbon source at various concentrations (0.2, 0.5, 1 .0, 2.0, 4.0% [wt/vol]). Only recombinant strains harboring pBBR1 MCS-3: :glf::pmi or pBBW MCS-3::glf::mak::pmi exhibited significant growth in presence of any tested mannose concentration. Whereas cells harboring plasmid pBBR1 MCS-3::g ^f /f::ma/ ;;pm/ ^' exhibited distinct growth on mannose after 3-4 days of incubation, cells with pBBR1 MCS-3: :glf::pmi only showed growth after an incubation time of 5-6 days. In contrast, recombinant strains harboring plasmid pBBRI MCS-S.-.g/f or pBBW MCS-3: :glf::mak, respectively, exhibited barely no growth even after an incubation period of 30 days. Only after prior exposure of the latter recombinant strains on MSM plates containing fructose instead of gluconate, slight growth occurred after a prolonged time of incubation. Control of expression of glf, glk, mak and pmi. To confirm the presence of glf, mak, and pmi derived mRNA, transcriptional analyses of the recombinant strains were performed. For this, cells of the recombinant strains of R. eutropha harboring pBBR1 MCS-3.:g'/f:.77?a/ .:pm/ ^' or pBBR1 MCS-3 were cultivated in MSM containing 1 % (wt/vol) sodium gluconate as a sole carbon source. Since P _/ac is a constitutive promoter in R. eutropha strain H16 (Siedow et al. 1999), all three genes should be expressed constitutively without further induction. Transcriptional analyses were done by one-step RT-PCR. Total RNA from cells of late logarithmic growth phase served for RT-PCR using primers g _RT_f, g _RT_r, mak_RT , mak_RT_r, p/7i/_RT_f and pm/_RT_r specific for glf, mak and pmi (Table 1 ). PCR products of the expected size of 351 bp for glf, mak and pmi, respectively, were obtained if RNA isolated from recombinant pBBR1 MCS-3::g ^f /f::ma/ ;;pm/ ^' harboring cells of strain H16 was analyzed. This clearly demonstrated that glf, mak and pmi were only transcribed in cells of the recombinant strain of R. eutropha strain H16 har- boring pBBR1 MCS-3::g ^f /f::ma/ ;;pm/ ^' but not in the negative control harboring only the vector pBBR1 MCS-3. The absence of PCR products in the control without any activity of reverse transcriptase indicated that the RT-PCR product was not derived from contaminating DNA (Fig. 3).

To further confirm the transcription of glk and glf in recombinant strains harboring plasmid pBBR1 MCS-2::P _n/( ::g'// ::g'/f at the protein level, an analysis by SDS-PAGE was performed. As Glf is a transmembrane protein (Parker et al. 1995), membrane proteins of both R. eutropha strain H16 harboring vector pBBR1 MCS-2 and R. eutropha strain H16 harboring plasmid pBBR1 MCS-2::P _n/( ::g'// ::g'/f were solubilized and separated by PAGE. The electropherogram showed a distinct protein band with an apparent mass of 50 kDa representing Glf, that was absent in the control. Similarly, analysis of the soluble cell fraction showed a distinct protein band at 35 kDa, representing Glk (Fig. 4).

The presence of functional active MAK, PMI and GLK in the recombinant strains was confirmed by enzymatic analyses. Coupled enzyme assays employing the soluble protein fractions obtained from cells of the recombinant strain of H16 harboring pBBRI MCS-S.-.g/f.-.ma/ /.pm/, or pBBRM CS-2 P _nk ::glk::glf cultivated in MSM containing 1 % (wt/vol) sodium gluconate as sole carbon source, demon- strated the presence of active PMI (0.25 U mg ^"1 ), MAK (0.14 U mg ^"1 ) and also GLK (0.52 U mg ^"1 ), respectively, whereas these activities except GLK (0.02 U mg ^"1 ) were absent in the soluble protein fractions of the negative control of strain H16 harboring plasmids pBBR1 MCS-2 or pBBR1 MCS-3, respectively, thus indicating the absence of PMI and MAK in the negative control strain.

Investigations on utilization of several carbon sources by the recombinant strains of R. eutropha H16. In comparison to the other recombinant strains, R. eutropha strain H16 harboring plasmids pBBR1 MCS-2::P _n / _f ::g// ::g/f or pBBW MCS-3::glf::mak::pmi exhibited the fastest growth on glucose or mannose of all tested substrates, respectively, as a sole carbon source. These strains were therefore further investigated with regard to the utilization of several other carbon sources employing MSM agar plates each containing 1 % (wt/vol) of L-arabinose, D-fructose, D-galactose, D-gluconate, D-glucose, glycerin, maltose, D-mannitol, D- mannose, /V-acetyl-D-glucosamine, pyruvate, D-ribose, trehalose, or D-xylose as sole carbon source. The wild type of R. eutropha served as negative control. The results are summarized in Table 2. Both, the wild type strain and the recombinant strains, were able to grow in presence of D-fructose, D-gluconate, glycerol, N- acetylglucosamine, and pyruvate (Table 2). All other carbon sources mentioned above were not utilized for growth, except mannose by the recombinant harboring pBBR1 MCS-3::g ^f /f::ma/ ;;pm/ ^' and glucose by the recombinant strains harboring pBBR1 MCS-2::P _n *::gr/fr::gr/f or pBBR1 MCS-3;;g/ :;mafcvpm/ ^' (Table 2).

Growth of the recombinant R. eutropha with glucose as sole carbon source.

Recombinant strains of R. eutropha strain H16 were cultivated in liquid MSM containing 1 % (wt/vol) glucose. Although all strains exhibited growth on glucose, re- combinant strain harboring pBBR1 MCS-2::P„ _/( ::g'// ::g'/f showed fastest growth (μ = 0.32 h ^"1 ) in comparison to the recombinant strain harboring pBBR1 MCS-3.:g/f.:ma/ .:pm/ ^' (μ = 0.12 h ^"1 ) (Fig. 5). This was in the same order as the positive control strain G ⁺ 1 (μ = 0.30 h ^"1 ). Interestingly, recombinant cells harboring plasmids carrying only the glf gene either under the control of the neokan- amycin- or /ac-promoter exhibited a strongly diminished growth (μ = 0.085 and 0.073, respectively) when compared to recombinant cells harboring plasmids with also the other genes (Fig. 5). Growth of the recombinant R. eutropha with mannose or gluconate as sole carbon source. Recombinant cells of R. eutropha strain H16 harboring plasmid pBBW MCS-3..glf..mak::pmi were cultivated in liquid MSM containing different concentrations of mannose as sole carbon source to evaluate optimal concentra- tion for maximal growth. Furthermore, it was examined if the introduced genes are affecting growth of the recombinant strain in comparison to wild type strain H16 in MSM containing 0.5% (wt vol) sodium gluconate as sole carbon source. According to our expectations, the recombinant strain H16 harboring pBBR1 MCS-3::g/ ;:ma/ .:p/7i/ ^' exhibited faster growth on gluconate (μ = 0.33 h ^"1 ) than on mannose (μ = 0.078 h ^"1 ). Maximal growth on mannose occurred at 1 % (wt/vol) mannose, whereas lower and higher concentrations of this sugar yielded slightly slower growth (Fig. 6). With gluconate as sole carbon source growth of the recombinant strain was only slightly diminished in comparison to the wild type.

Example 2 Bacterial strains, plasmids, oligonucleotides and cultivation conditions. All bacteria, plasmids and primers used in this study are listed in Table 3 and 4, respectively. Cells of R. eutropha strain H16 were cultivated in nutrient broth, or mineral salts medium (MSM) as described by Schlegel et al. (1961 ). Carbon sources were added to liquid MSM as indicated in the text. Liquid cultures in Er- lenmeyer flasks were incubated on a horizontal rotary shaker at an agitation of 1 10 rpm. Solid media were prepared by addition of 1 .5% (wt/vol) agar-agar. Cells of Escherichia coli were cultivated at 37 °C in Lysogeny Broth (LB, [Sambrook et al. 1989]), or M9 mineral salts medium (Sambrook et al. 1989) with D-xylose as sole carbon source. Antibiotics were applied according to Sambrook et al. (1989) and as indicated in the text.

Isolation, analysis and modification of DNA. Plasmid DNA was prepared from crude lysates by the alkaline extraction method (Birnboim and Doly 1979). Total DNA of E. coli strain K-12 was prepared using the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. DNA was restricted with restriction endonucleases (Gibco/BRL, Gaithersburg, USA) under conditions recommended by the manufacturer. All other genetic procedures and manipulations were conducted as described by Sambrook et al. (1989). Table 3. Bacterial strains, plasmids and oligonucleotides used in this study

Source or refer¬

Strain, plasmid or primer Relevant characteristics

ence

Strains

E. coli K-12 Wild type DSM 426

E. coli TOP10 F mcrA, A(mrr-hsdRMS-mcrBC) f80lacZ Invitrogen

ΔΜ15, AlacX74, deoR, recAl, araD139,

A(ara-leu)7697, galU, galK, rpsL, endAl,

nupG

E. coli S17-1 recAl, thil, ftsdR17(r _k -, m _k +), proA, tra- Simon et al. 1983 genes of RP4 plasmid chromosomally

integrated (Mobilization strain)

E. coli H B 101 hsdS, recA, proA, lacy, 9a/K, xyl-5, mtl E. coli Genetic

Stock Center (Hirota et al. 1977)

E. coli JWL255 xyl91pR E. coli Genetic

Stock Center (Hirota et al. 1977) f. co// LR2-168 thi, arg, met, his, gal, xyl-7 E. coli Genetic

Stock Center (Hirota et al. 1977)

/?. eutropha strain H 16 Wild type DSM 428 Table 3 continued

Source or refer¬

Strain, plasmid or primer Relevant characteristics

ence

Plasmids

pJET1.2/blunt Ap ^r Fermentas pJET1.2::xy/A with xylA as blunt end PCR product in the examples pJET1.2::xy/B with xylB as blunt end PCR product in the examples pJET1.2::xy/E with xylE as blunt end PCR product in the examples pBBRlMCS-3 Tc ^r , LacZ-a, mob, rep Kovach et al.

1995 pBBRlMCS-3:: xy/FGH with xylFGH as Spe\/Sac\\ fragment in the examples pBBRlMCS-3:: xyA48 with xylAB as Apa\/Spe\ fragment in the examples pBBRlMCS-3:: xyA48£ with xylABE as Apa\/Sac\ \ fragment in the examples pBBRlMCS-3:: xylABFGH with xylABE as Apa\/Sac\ \ fragment in the examples

Table 4. PCR and sequencing primers used in this study

Oligonucleotides Sequence Function

P_xylA_Apa\J ATT AGG GCC CAA GGA 5 ^' -primer for the amplification of the codGGT TAC AGC ATG CAA ing region of xylA. The primer contains GCC TAT TTT GAC CAG the restriction site for Apa\ (underlined).

(SEQ ID NO: 82)

P_xy/A_Smal_r ATT GCC CGG GTT ATT TGT 3 ^' -primer for the amplification of the codCGA ACA GAT AAT G ing region of xylA. The primer contains the restriction site for Sma\ (underlined).

(SEQ ID NO: 83)

P_xylB_Sma\J ATT GCC CGG GAA GGA 5 ^' -primer for the amplification of the codGGT TAC AGC ATG TAT ATC ing region of xy/B. The primer contains GGG ATA GAT CTT the restriction site for Sma\ (underlined).

(SEQ ID NO: 84)

P_xy/B_Spel_r CCC GAC TAG TTT ACG CCA 3 ^' - primer for the amplification of the

TTA ATG GCA GAA G coding region of xy/B. The primer contains the restriction site for Spel (under¬

(SEQ ID NO: 85) lined).

P_xylE_Spe\ CCC GAC TAG TAA GGA 5 ^' -primer for the amplification of the cod¬

GGT TAC AGC ATG AAT ACC ing region of xy/E. The primer contains CAG TAT AAT TC the restriction site for Spel (underlined).

(SEQ ID NO: 86)

P_xy/E_Sacll_r ATT GCC GCG GTT ACA 3 ^' - primer for the amplification of the

GCG TAG CAG TTT GTT G coding region of xy/E. The primer contains the restriction site for Sacll (under¬

(SEQ ID NO: 87) lined).

P_xy/FGH_Spel_ CCC GAC TAG TAA GGA 5 ^' -primer for the amplification of the codf GGC TAC AGC ATG AAA ATA ing region of xy/FGH: The primer conAAG AAC ATT CTA C tains the restriction site for Spel (underlined).

(SEQ ID NO: 88) P- ATT GCC GCG GTC AAG 3 ^' - primer for the amplification of the xy/FGH_Sacll_r AAC GGC GTT TGG TTG coding region xy/FGH. The primer contains the restriction site for Sacll (under¬

(SEQ ID NO: 89) lined).

AAAACCGCGGCGGCTCGGA 5 ^' -primer for the amplification of the AGTCG promoter P _GAPDH region: The primer contains the restriction site for Sacll (under¬

(SEQ ID NO: 90) lined).

pJET1.2 forCG ACTCACTATAG G GAGAG Fermentas (sequencing primer, 23-mer) ward CGGC (SEQ ID NO: 91 )

pJET-l .2 reAAGAACATCGATTTTCCATG Fermentas (sequencing primer, 24- verse GCAG (SEQ ID NO: 92)

M13/pUC- GTAAAACGACGGCCAGT Jena Bioscience (17-mer)

forward (SEQ ID NO: 93)

Constructions of plasmids and its transfer into R. eutropha strain H16. For amplification of the coding regions of xylA, xylB, xylE, xylFGH, talB and tktA from E. coli strain K12 by PCR, oligonucleotides listed in Table 4 were used. The oligo- nucleotides P_xy/A_>4pal_f and P_xy/A_Smal_r were used for amplification of xy- IA, P_xylB_Sma\ and P_xy/B_Spel_r for xylB, P_xylE_Spe\ and P_xy7E_Sacl l_r for xylE, and P_xy/FGH_Spel_f and P-xy/FGH_Sacll_r for xylFGH, respectively. Primers contained a ribosomal binding site in order to permit RNA translation in the target bacterium R. eutropha H16. KOD Hot Start DNA Polymer- ase (Merck, Darmstadt, Germany) was used for PCR amplification according to the manufacturer's instructions. PCR products were ligated to the pJET1.2/blunt cloning vector (Fermentas, Germany) using T4 DNA ligase (Gibco BRL, Gaithersburg, USA) or to plasmid pBBR1 MCS-3 (Kovach et al. 1995), and transferred into E. coli strain TOP10. Plasmids were isolated from ampicillin or tetracy- dine resistant clones, respectively, and the cloned fragments were excised by restriction with the respective suitable restriction enzymes for further cloning, extracted from gel after separation using the E.Z.N.A gel extraction kit (Omega Bio- tec, Bangalore, India). For expression experiments in R. eutropha strain H16, the broad host range vector pBBR1 MCS-3 was used for cloning of xylA, xylB, xylE, and xylFGH. Vector pBBR1 MCS-3 conferred tetracycline resistance for selection in E. coli and R. eutropha strain H16. The coding regions of xylA or xylB were ex- cised by restriction with Apa\ and Sma\ or Sma\ and Spe\, respectively, ligated to Apa\ and Spe\ linearized plasmid pBBR1 MCS-3, yielding plasmids pBBRI MCS- 3::xylAB (Fig.8). To obtain plasmids pBBR1 MCS-3..xylABE and pBBRI MCS- 3::xylABFGH, the coding regions of xylE and xylFGH were excised by restriction with Spe\ and Sacll, respectively, and ligated to Spe\ and Sacll linearized plasmid pBBR1 MCS-3::xy//¾B yielding plasmids pBBR1 MCS-3: xylABE and pBBRI MCS- 3::xylABFGH, respectively (Fig. 8). All plasmids were transferred to E. coli strain S17-1 by transformation (Hanahan 1983).

Transfer of DNA by conjugation. Transfer of plasmids pBBR1 MCS-3, pBBR1 MCS-3::xy//ABE, pBBR1 MCS-3::xy//ABFGH (Table 3) was performed by conjugation applying a previously described protocol (Friedrich et al. 1981 ), using E. coli S17-1 as donor and R. eutropha strain H16 as recipient.

DNA sequence analysis. DNA was sequenced in a 48-capillary 3730 DNA Analyzer electrophoresis system (Applied Biosystems, Foster City, CA). For sequencing of inserts in the pJET1 .2/blunt cloning vector (Fermentas, Germany) the pJET1 .2 forward sequencing primer, 23-mer and the pJET1 .2 reverse sequencing primer, 24-mer were used. The universal M13/pUC-forward primer and the M13/pUC-reverse primer were used for sequencing inserts in plasmid pBBR1 MCS-3 (Kovach et al. 1995).

Preparation of soluble protein fractions from R. eutropha strain H16. Cells of recombinant strains of R. eutropha strain H16 were cultivated in nutrient broth medium. Cells were harvested by centrifugation for 15 min at 4 °C and 3,500 x g, washed with 50 mM Tris/HCI buffer (pH 7.5) and resuspended in two volumes of the same buffer. Disruption was done by sonication employing a Sonifer 250 (Branson Sonic Power Company) with an amplitude of 16 m (1 min/ml; 50% out- put control). During ultrasonication samples were cooled with an ice-NaCI mixture. Soluble membrane-free protein fractions were prepared by 60 min ultracentrifuga- tion of the crude extracts at 100,000 x g and 4 °C. XylA and XylB activity assays. Soluble protein fractions of recombinant R. eutropha strain H16 were applied for XylA and XylB activity measurements at 30 °C using a Nicolet Evolution 100 UVA/IS spectrophotometer (Thermo Electron Corporation, Cambridge, UK). Activity of XylA (Gao et al. 2002, Mejnen et al. 2008) or XylB (Shamanna and Sangerson 1979, Eliasson et al. 2000) was assayed with an NADH-coupled system with some modifications, using sorbitol dehydrogenase or pyruvate kinase and lactate dehydrogenase, respectively, as auxiliary enzymes. The buffered reaction mixture (50 mM Tris/HCI, pH 7.5) for XylA activity measurement contained 10 mM MgSO , 1 mM triethanolamine, 0.2 mM NADH, 50 mM D-xylose, 0.5 U ml ^"1 sorbitol dehydrogenase, and 5 to 100 μΙ of soluble extract. The buffered reaction mixture (50 mM Tris/HCI, pH 7.5) for XylB activity measurement contained 2 mM MgCI ₂ , 2 mM ATP, 0.2 mM NADH, 10 mM xylulose, 0.2 mM PEP, 10 U ml ^"1 pyruvate kinase, 10 U ml ^"1 lactate dehydrogenase and 5 to 100 μΙ of soluble extract. D-xylose utilization in R. eutropha. After D-xylose is taken up by the cell, the xylA gene encoding the enzyme D-xylose isomerase (Xyll or XylA; EC 5.3.1 .5) catalyzes the reversible conversion of the aldose D-xylose into its keto-form D- xylulose. Subsequently, the D-xylulose is phosphorylated to D-xylulose-5- phosphate, utilizing ATP as energy and phosphate source. This step is catalyzed by a xylulose kinase (XylK or XylB; EC 2.7.1 .17). D-Xylulose-5-phosphate can be utilized further as an intermediate of the non-oxidative pentose phosphate pathway. As E. coli strain K12 is one of the most extensively studied organisms utilizing D-xylose, the genes xylA, encoding xylose-isomerase, xylB encoding xyluloki- nase, xylE encoding the low affinity transporter and xylFGH encoding the ABC- transporter were amplified using genomic DNA of E. coli K12, episomally introduced and expressed in R. eutropha H16. The engineered pathway of xylose ca- tabolism in the recombinant R. eutropha strain is depicted in Fig. 7.

Construction of different pBBR1 expression vectors for R. eutropha strain H16. The coding regions of the genes xylA (1 ,323 bp), xylB (1 ,455 bp), xylE (1 ,476 bp) and xylFGH (3,771 bp) were amplified by PCR from genomic gDNA of E. coli strain K12. The three PCR products comprised beside the coding regions also suitable ribosome binding sites for R. eutropha upstream of the respective start codon to enable expression in this host. These fragments were cloned via the pJET1 .2/blunt vector into the broad-host range vector pBBR1 MCS-3 under the control of the /ac-promoter, which allows constitutive expression of the cloned genes in R. eutropha H16 (Siedow et al. 1999), yielding plasmids pBBRI MCS- 3::xylAB, pBBR1 MCS-3..xylABE and pBBR1 MCS-3..xylABFGH (Fig. 8). Accurate construction of these plasmids was confirmed by sequencing. Additionally, the functionality of the xylA or xylB genes in pBBR1 MCS-3::xy//¾B, pBBRI MCS- 3:.xylABE and pBBR1 MCS-3::xy//4BFGH was confirmed by complementation of the xylA deficient E. coli mutant HB101 , xylB deficient E. coli mutant JWL255, or xylAB deficient E. coli mutant LR2-168, respectively (Hirota et al. 1977). The recombinant E. coli mutants HB101 , JWL255 or LR2-168harboring pBBRI MCS- 3::xylAB, pBBR1 MCS-3..xylABE or pBBR1 MCS-3::xy//4BFGH, respectively, exhibited good growth on solid M9 medium containing 0.5% (wt/vol) xylose as a sole carbon source after two days of incubation at 37 °C. In contrast, vector pBBR1 MCS-3 did not confer growth to the mutants HB101 , JWL255 or LR2-168, respectively.

Transfer of plasmids to R. eutropha strain H16 and establishment of D- xylose utilization. Plasmids pBBR1 MCS-3::xy//¾B, pBBR1 MCS-3..xylABE and pBBR1 MCS-3::xy//4BFGH were transferred via conjugation from E. coli S17-1 to R. eutropha strain H16. Transconjugants were selected on MSM agar plates containing 1 % (wt/vol) sodium gluconate and tetracycline (12.5 pg/ml), and were subsequently transferred to MSM plates containing 1 % (wt/vol) D-xylose as sole carbon source. Recombinant strains harboring plasmid pBBR1 MCS-3::xy//ABFGH exhibited weak growth after 5 days of incubation. Evolutionary optimization of the plasmid pBBR1 MCS-3::xyMSFGH-harboring strain of R. eutropha H16. Although the recombinant plasmid pBBRI MCS- 3::xy//ABFGH-harbohng strain of R. eutropha H16 was engineered for D-xylose utilization, this strain exhibited only weak growth after 5 days of incubation. A single colony of this recombinant strain was cultivated in fluid MSM containing 1 % (wt/vol) D-xylose as a sole carbon source for 2 days at 30 °C. Dilutions were transferred on solid MSM containing 1 % (wt/vol) D-xylose as a sole carbon source and incubated at 30 °C. A single colony which showed fastest growth was again cultivated in fluid MSM containing 1 % (wt vol) D-xylose as a sole carbon source, and a dilution was again transferred on solid MSM containing 1 % (wt vol) D-xylose as a sole carbon source. After repetition of this procedure for several times, a recombinant plasmid pBBR1 MCS-3::xy//4BFGH-harboring strain of R. eutropha H16 was obtained exhibiting comparably good growth on solid MSM containing 1 % (wt/vol) D-xylose as a sole carbon source after 2 days of incubation.

Investigations on utilization of several carbon sources by the recombinant plasmid pBBR1MCS-3::xy/A6FGH-harboring strain of R. eutropha H16 and of the respective optimized strain. Cells of the recombinant strain of H16 harboring pBBR1 MCS-3::xy//4BFGH and of the respective optimized recombinant strain of H16, of R. eutropha strain G+1 , a glucose-utilizing mutant of R. eutropha strain H16 (Schlegel and Gottschalk 1965), and of R. eutropha strain H16 were cultivated on solid MSM agar plates containing different carbon sources (1 % [wt/vol] L- arabinose, D-fructose, D-galactose, D-gluconate, D-glucose, D-maltose, D- mannose, N-acetylglucosamine, D-ribose, D-trehalose, D-xylose) to compare the growth of the strains on several carbon sources. The wild type of R. eutropha H16 served as negative control. All the strains were able to use D-fructose, D- gluconate, and N-acetylglucosamine as sole carbon sources. The plasmid pBBR1 MCS-3::xy//4BFGH containing strain of R. eutropha strain H16 was also able to use glucose as sole carbon source showing a similar growth as R. eutropha H16 strain G+1 . The plasmid pBBR1 MCS-3::xy//ABFGH containing strain was also able to use D-xylose, L-arabinose and D-galactose as sole carbon sources.

Growth of the recombinant plasmid pBBR1 MCS-3::xyMSFGH-harboring strain of R. eutropha H16 with glucose, gluconate or xylose as sole carbon source. Cells of the recombinant plasmid pBBR1 MCS-3::xy//4BFGH-harboring strain of R. eutropha H16 were cultivated in fluid MSM containing different concentrations (0.5%, 1 %, 2%, and 4% [wt/vol]) of D-xylose, 1 % (wt/vol) D-glucose and 1 % (wt/vol) Na-gluconate as sole carbon source. Also R. eutropha strain H16 wild type was cultivated with 1 % (wt/vol) Na-gluconate as sole carbon source to compare the growth rate. The recombinant strain showed a similar growth on 1 % (wt/vol) Na-gluconate (μ = 0.47 h ^"1 ) and also on 1 % (wt/vol) glucose (μ = 0.55 h ^"1 ) as sole carbon source in comparison to the wild type strain on 1 % (wt/vol) Na- gluconate (μ = 0.47 h ^"1 ) (Fig. 9). After almost 22 h, the stationary growth phase was reached. Growth of the recombinant strain on D-xylose as sole carbon source was slower. After almost 40 h, the stationary growth phase was reached for the cultures containing 0.5% (wt/vol) (μ = 0.20 h ^"1 ) to 2% (wt/vol) (μ = 0.18 h ^"1 ) of D- xylose as sole carbon source. The culture containing 4% (wt/vol) of D-xylose as sole carbon source reached the stationary growth phase after almost 50 h (μ = 0.17 h ^"1 ) (Fig. 9).

Control of expression of xylA and xylB in the recombinant plasmid pBBR1 MCS-3::xy/4SFGH-containing strain of R. eutropha H16. The presence of functional active XylA and XylB in the recombinant strain was confirmed by enzymatic analyses. Coupled enzyme assays employing the soluble protein fractions obtained from cells of the recombinant strain of H16 harboring pBBRI MCS- 3::xylABFGH cultivated in MSM containing 1 % (wt/vol) xylose as sole carbon source demonstrated the presence of active XylA (0.035 U mg ^"1 ) and XylB (0.364 U mg ^"1 ), respectively, whereas these activities were absent in the soluble protein fractions of the negative control of strain H16 harboring plasmid pBBR1 MCS-3, thus indicating the absence of XylA and XylB in the negative control strain.

Analysis of PHB content of recombinant R. eutropha cells by GC. Lyophilized cell material was subjected to methanolysis in presence of methanol and sulfuric acid (85%, vol/vol, MeOH and 15%, vol/vol, H ₂ SO ₄ ) for 4 h at 100 °C, and the resulting methylesters of the PHA constituents were characterized by gas chromatography using an Agilent 6850 GC (Agilent Technologies, Waldbronn, Germany) equipped with a BP21 capillary column (50 m by 0.22 mm; film thickness, 250 nm; SGE, Darmstadt, Germany) and a flame ionization detector (Agilent Technologies). A 2 μΙ portion of the organic phase was analyzed after split injection (split ratio, 1 :5), and a constant hydrogen flow of 0.6 ml min ^"1 was used as carrier gas. The temperatures of the injector were 250 °C and 220 °C, respectively. The following temperature program was applied: 120 °C for 5 min, increase of 3 °C min ^"1 to 180 °C and increase of 10 °C min ^"1 to 220 °C and 220 °C for 31 min. Substances were identified by comparison of their retention times to those of standard fatty acid methyl ester. Construction of different vectors suitable for expression of transketolase (Tkt) and transaldolase (TalB) in strains of the genera Ralstonia and Cu- priavidus. The coding regions of genes encoding a transketolase (Tkt) and transaldolase (TalB) homologous protein were amplified by PCR from genomic gDNA of the respective host strain. The PCR products can comprise beside the coding regions also suitable restriction sites for cloning, suitable regulatory elements as e.g. a promoter sequence, and ribosome binding sites for strains of the genera Ralstonia and Cupriavidus upstream of the respective start codon to enable expression in this host. These features can either be provided by synthetic oligonu- cleotides or by fusion PCR. These fragments can be cloned via cloning vectors into vectors suitable for expression in strains of the genera Ralstonia and Cupriavidus under the control of a suitable promoter, which allows expression of the cloned genes in the respective host strain. The coding regions of genes encoding a transketolase (Tkt) and transaldolase (TalB) homologous protein can either be cloned separately or in combination with different genes, e.g. xylA, xylB, xylB, and xylFGH. Accurate construction of these plasmids can be confirmed by sequencing. The constructed plasmids can be either transferred via conjugation from E. coli S17-1 or by electroporation to strains of the genera Ralstonia and Cupriavidus. Transconjugants or transformants, respectively, can be selected on MSM agar plates containing 1 % (wt/vol) sodium gluconate and a suitable selection for plas- mid stability. Recombinant strains of the genera Ralstonia and Cupriavidus can be characterized concerning their growth on different carbon sources.

Example 3

Bacterial strains, plasmids, oligonuclotides and cultivation conditions. All bacteria, plasmids and primers used in this application are listed in Table 5. Cells of E. coli were cultivated in Lysogeny Broth (LB) (Sambrook et al., 1989) or in mineral salts medium (MSM) supplemented with 1 % (w/v) glucose according to Schlegel et al. (1961 ). Cells of R. eutropha were cultivated in Nutrient Broth (NB) (Sambrook et al., 1989) or in MSM supplemented with 1 % (w/v) sodium gluconate and sodium oleate (0.4%, w/v) for FAEE synthesis. Liquid cultures in baffled Er- lenmeyer flasks were incubated on a horizontal rotary shaker at an agitation of 1 10 rpm at 37°C (E. coli) or 30°C (R. eutropha strains). Antibiotics were applied according to Sambrook et al. (1989) and as indicated in the text.

Table 5. Bacterial strains, plasmids and oligonucleotides

Strain, plasmid or Source or

Relevant characteristics

primer reference

Strains

E. coli TOP10 F- mcrk, (mrr-hsdRMS-mcrBC)†80lacZ ΔΜ15,

NacX74, deoR, recA1, araD139, A(ara-/eu)7697, Invitrogen galil, galK, rpsL, endA 1, nupG recA1 , thft , /?sc/R17(rk-, mk+), proA, fra-genes of Simon et

E. coli S17-1 RP4 plasmid chromosomally integrated (mobilization strain) al- 1983

Peplinski et

R. eutropha phaCI PHB-negative mutant of R. eutropha

al., 2010

Plasmids

Kovach et pBBR1 MCS-3 TcR lacZa, mob, rep

al., 1995 pMicrodiesel AmpR atfA, adhB, pdc Kalscheuer et al., 2006 pBBR1 MCS-3::

with atfA/pdc/adhB as Xba\/Apa\ fragment in this example atfA/pdc/adhB

Oligonucleotides

M13 fwd 5' GTAAAACGACGGCCAGT 3' (SEQ ID NO:94)

5' AACAGCTATGACCATG 3' (SEQ ID NO: 95)

M13 rev Isolation, analysis and modification of DNA. Plasmid DNA from R. eutropha strains was prepared from crude lysates by the alkaline extraction method (Birn- boim and Doly, 1979). Plasmid DNA from E. coli was prepared with a commercial plasmid preparation kit (Roti Prep Plasmid Kit, Roth, Karlsruhe) applying the con- ditions recommended by the manufacturer. DNA was restricted with restriction en- donucleases (Fermentas GmbH, St. Leon-Rot) under conditions recommended by the manufacturer. All other genetic procedures and manipulations were conducted as described by Sambrook et al. (1989).

Transfer of DNA by conjugation. Transfer of plasmids pBBR1 MCS-3 and pBBW MCS-3..atfA/pdc/adhB was performed by conjugation applying the protocol described by Friedrich et al. (1981 ). E. coli S17-1 was used as donor strain and R. eutropha strains AphaCI as the recipient.

Preparation of soluble protein fractions from R. eutropha strains and E. coli.

Cells of recombinant strains of R. eutropha or E. coli were cultivated as described above in presence of 1 % (w/v) sodium gluconate or 1 % (w/v) glucose, respectively. Cells were harvested by 15 min of centrifugation at 4°C and 3500 χ g, washed in a suitable buffer as described below and resuspended in two volumes of the same buffer. The cells were disrupted by sonication employing a MS72 probe and a Sonoplus GM200 sonicator (Bandelin, Berlin) with an amplitude of 10 μιτι (2 min/ml; 50% output control). During ultrasonication the samples were cooled with ice. Protein concentrations in cell crude exracts were determined as described by Bradford (1976).

DNA sequence analysis. DNA was sequenced in a 48-capillary 3730 DNA Analyzer electrophoresis system (Applied Biosystems, Foster City, CA). For sequenc- ing of inserts in plasmid pBBR1 MCS-3 (Kovach et al. 1995) the universal "M13 rev"- and "M13 fwd" primers were used.

Construction of the used plasmids. As the expression vector in R. eutropha H16 the vector pBBR1 MCS-3 was chosen allowing a stable expression of the containing genes in R. eutropha. The necessary genes for FAEE synthesis were cut from the plasmid pMicrodiesel (Kalscheuer et al., 2006) by restriction with Xba\ and Apa\ resulting in a 4.8 kbp fragment containing genes atfA, pdc and adhB. This fragment was ligated to the Xba\ and Apa\ restricted plasmid pBBR1 MCS-3 using T4 DNA ligase (Fermentas GmbH, St. Leon-Rot) resulting in the 9.9 kbp plasmid pBBR1 MCS-3: :atfA/pdc/adhB. The constructed plasmid was transferred by transformation (Hanahan 1983) to E. coli Top10 for verification and finally to E. coli strain S17-1 serving as the donor for conjugation experiments.

WS/DGAT, PDC and ADHB activity assays. Soluble protein fractions of recombinant R. eutropha strains were applied for WS/DGAT, PDC and ADHB activity measurements at 30°C using a Nicolet Evolution 100 UV/VIS spectrophotometer (Thermo Electron Corporation, Cambridge, UK). Activity of PDC was assayed with an NADH-coupled system using ADH as an auxiliary enzyme measuring NAD ⁺ production by the decrease of absorption at 340 nm. The buffered reaction mixture (50 mM potassium phosphate (KP) buffer, pH 6.5) contained 5 mM MgSO , 0.1 mM thiamine pyrophosphate (TPP), 0.175 mM NADH, 17.5 mM pyruvate, 0.1 U/ml ADH and 100 g/ml protein solution of the soluble extract. ADH activity was measured directly by the decrease of NADH due to the reduction of acetic aldehyde. The same reaction mixture as for the PDC activity assay was used, though pyruvate was replaced by 0.1 mM acetic aldehyde and ADH was replaced by KP buffer.

WS/DGAT activity was measured radiometrically by the synthesis of labelled wax esters from hexadecanol or ethanol and 1 - ¹⁴ C-palmitoyl coenzyme A. The buffered reaction mixture (125 mM sodium phosphate buffer, pH 7.4) contained 3.75 mM hexadecanol or ethanol, 12.5 g/ml bovine serum albumine (BSA), 4.72 μΜ 1 - ¹⁴ C- palmitoyl CoA and 200 g/ml protein crude extract. Prior to use a double concentrated emulsion of hexadecanol, BSA and sodium-phosphate buffer was prepared by ultrasonication (1 min/ml). The reaction mixture was incubated for 30 min at 35°C and the reaction was stopped by the addition of one volume of chloroform- methanol (1 :1 ; v/v). After vigorous vortexing for 1 min phases were separated by centrifugation for 2 min at 16000 x g. The organic phase was removed and applied to a thin layer chromatography (TLC-) plate (Silica gel 60, 20x20 cm aluminium sheets, Merck KGaA, Darmstadt) which was then developed with hexane:diethyl ether:acetic acid (90:7.5:1 ; (v/v/v) as the solvent. Radioactively marked wax ester spots were visualized by a PharosFX Phospho-lmager (Bio-Rad Laboratories GmbH, Munich) after exposing the developed TLC plate to a phospor screen. As standard substances for TLC oleyloleate or ethyloleate were used which were visualized by iodine vapour staining.

Quantitative and qualitative determination of synthesized ethanol and FAEE. Ethanol amounts in cell free culture media were determined by high performance liquid chromatography (HPLC). HPLC analysis of 20 μΙ samples was carried out with a LaChrom Elite HPLC (VWR-Hitachi International GmbH, Darmstadt) equipped with a Metacarb 67H advanced C-column (length: 300 mm; inner diameter 6.5 mm; Varian, Palo Alto, USA) and a Type 2490 refractive index (Rl) detector (VWR-Hitachi International GmbH, Darmstadt).

FAEE in lyophilised cells were analysed by TLC, gas chromatography (GC) and coupled gas chromatography mass spectrometry (GC/MS). For qualitative TLC analysis 100 mg cell dry matter were mixed with 1 ml chloroform :methanol (2:1 ; v/v) and broken up with a type MM301 bead mill (Retsch, Haan). After centrifuga- tion for 10 min at 16000 x g the organic supernatant was separated by TLC as described for the WS/DGAT activity assays. Ethyloleate served as the standard substance. Spots were visualized by iodine vapour staining and spots of interest were scraped of the TLC plate, eluted with chloroform:methanol (2:1 , v/v) and analyzed by GC or GC/MS. GC analyses of 10-100 mg cell extracts were performed with an Agilent 6850 capillary gas chromatograph (Agilent Technologies GmbH, Waldbronn) equipped with a BP21 capillary column (length: 50 m; inner diameter: 0.22 mm; film thickness 250 nm; SGE GmbH, Darmstadt) and a flame ionisation detector (FID). At a split ratio of 1 :15 2 μΙ of sample were injected by an Agilent 6850 Series Auto Sampler (Agilent Technologies GmbH, Waldbronn). As the car- er gas H ₂ (99.999 %) was used at a flow rate of 0.6 ml/min. Inlet and detector temperatures were 250°C and 275°C, respectively, the colum temperature was kept at 120°C for 5 min, followed by an increase of 3°C/min to 180°C, a second increase of 10°C/min to 220°C and was finally kept at 220°C for 31 min.

GC/MS analyses were carried out on a Series 6890 GC System (Hewlet Packard, Waldbronn) equipped with a BPX35 capillary column (length: 50 m; inner diameter: 0.25 mm; film thickness 250 nm; SGE GmbH, Darmstadt). GC was coupled with a Series 5973 electron ionisation mass selective detector (EI-MSD) (Hewlett Packard, Waldbronn). Helium (99.999%) served as the carrier gas with a constant flow rate of 0.6 ml/min. 3 μΙ of sample were injected by a Series 7683 autoinjector (Hewlett Packard, Waldbronn) with a split ratio of 1 :20. The inlet temperature was 250°C, that of the outlet was 240°C. The following temperature programme was applied: 5 min at 120°C, increase of 3°C/min to 180°C, followed by an increase of 5°C/min to 210°C and finally the temperature was kept for 29 min at 210°C. Data were analysed with the NIST mass spectral search programme (Stein et al., 1998)

Transfer of plasmids to R. eutropha AphaCI and determination of enzyme activities. Both plasmids, pBBR1 MCS-3 as the control vector and pBBRI MCS- 3::atfA/pdc/adhB, were transferred via conjugation from E. coli S17-1 to R. eutropha AphaCI resulting in the strains R. eutropha AphaCI pBBR1 MCS-3 and R. eutropha AphaCI pBBR1 MCS-3: :atfA/pdc/adhB. Transconjugants could be selected on MSM agar plates containing 1 % (wt/vol) sodium gluconate and tetracy- clin (12.5 pg/ml) after 12 h of incubation at 30°C. Confirmation of the presence of functional active WS/DGAT, PDC and ADHB.

The presence of functional active WS/DGAT, PDC and ADHB in the recombinant strains was confirmed by enzymatic analyses.

Direct or coupled photometrical enzyme assays employing the soluble protein fractions obtained from cells of the recombinant strains of R. eutropha AphaCI pBBR1 MCS-3 and R. eutropha AphaCI pBBR1 MCS-3..atfA/pdc/adhB cultivated for 12 h at 30°C in MSM containing 1 % (wt/vol) sodium gluconate as sole carbon source, demonstrated the presence of active PDC (18.73 U/mg) and ADH B (1 .51 U/mg) in R. eutropha AphaCI pBBR1 MCS-3: :atfA/pdc/adhB, in contrast to the control strain R. eutropha AphaCI pBBR1 MCS-3 (PDC: 3.52 U/mg and ADH B: 0.25 U/mg). The radiometrical enzyme assays for WS/DGAT revealed the synthesis of radiolabeled wax esters and ethyl esters from hexadecanol or ethanol, respectively, and 1 - ¹⁴ C-palmitoyl CoA in the soluble protein fractions of R. eutropha AphaCI pBBR1 MCS-3..atfA/pdc/adhB but not of R. eutropha AphaCI pBBR1 MCS-3, thus indicating the absence of WS/DGAT in the negative control strain. For WS/DGAT activity no specific activities were determined. Analysis of ethanol and FAEE biosynthesis by HPLC, GC, GC/MS and TLC.

Ethanol and FAEE biosynthesis was analysed in the strains R. eutropha AphaCI pBBR1 MCS-3 and R. eutropha AphaCI pBBR1 MCS-3..atfA/pdc/adhB. The cells were grown in MSM-precultures containing 2% gluconate (w/v) and tetracycline or NB-medium with tetracycline for 12 h at 30°C and were used to inoculate main cultures of the same media in baffeled 11 Erlenmeyer flasks with 2.5% of the culture volume of 200 ml. One additional MSM culture (400 ml) additionally contained 0.4% (w/v) Na-oleate. Every 4-8 h samples were withdrawn and centrifuged. The supernatant was used for ethanol determination by HPLC. The cell pellets were washed twice with sterile saline, lyophilized and used for ethylester determination by GC, GC/MS and TLC.

Without Na-oleate as the cosubstrate maximum ethanol amounts of 1 .51 g/l were found after 22 h in R. eutropha AphaCI pBBR1 MCS-3..atfA/pdc/adhB grown in NB, while the control strain reached 1 .16 g/l after 14 h. With MSM medium the maximum ethanol amounts reached 0.85 g/l for R. eutropha AphaCI pBBRI MCS- 3::atfA/pdc/adhB after 32 h and 0.44 g/l for the control strain after 8 h. If Na-oleate was added to the MSM medium, the maximum ethanol concentration reached 4.38 g/l with R. eutropha AphaCI pBBR1 MCS-3..atfA/pdc/adhB after 36 h and 1 .34 g/l with the control strain after 30 h. Thus, it could be shown that R. eutropha AphaCI pBBRM^\CS-3::atfA/pdc/adhB is able to synthesize three times higher ethanol concentrations as the control strain if grown in MSM with gluconate and Na-oleate.

Possibly FAEE occurrences were analyzed by TLC and GC from lyophilized cell mass. Without the addition of Na-oleate neither cells grown in NB nor those grown in MSM showed ethyloleate synthesis as revealed by GC and TLC analysis. If Na- oleate was added to the medium cell dry mass of R. eutropha AphaCI pBBR1 MCS-3..atfA/pdc/adhB showed ethyloleate concentrations of 0.45% (w/w) after 36 h and of 0.23% (w/w) after 48 h. The cell dry mass of the control strains gave no peak corresponding to ethyloleate in GC analyses. Comparable to the GC analyses, the TLC analyses of crude extracts of the respective cell dry masses revealed the occurrence of substances corresponding to ethyloleate only in the pBBR1 MCS-3::atfA/pdc/adhB containing strain R. eutropha AphaCI. While GC/MS analyses of the putative ethyloleate spot scraped from TLC plates gave no results, GC/MS analyses of the ethyloleate containing 36 h sample and the 48 h sample revealed the occurrence of ethylpalmitoleate in the cell dry mass. Other FAEE could not be detected. Thus, if supplemented with Na-oleate strain R. eutropha AphaCI pBBR1 MCS-3: :atfA/pdc/adhB is able to synthesize FAEE.

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