FIELD OF THE INVENTION

The present invention is concerned with the production of compounds derived from oxaloacetate, such as ectoine or its biosynthetic precursor Ny-acetyl-L-2,4-diaminobutyric acid (NADA) or hydroxyectoine, in genetically modified microorganisms. The genetic modifications increase the obtainable yield by different mechanisms, such as by inhibiting the intracellular degradation of the compound, by increasing the extracellular accumulation of the compound, and by disrupting metabolic cycles, which are an obstacle for obtaining an increased yield. The invention is also concerned with the genetically modified microorganisms, and their use in the production of the desired compounds.

BACKGROUND

Chemical compounds derived from oxaloacetate, such as ectoine or its precursor Ng- acetyl-L-2,4-diaminobutyric acid (NADA), or hydroxyectoine, play an increasingly important role in several industries, for example as a cosmetics product and/or a medicinal product. Ectoine and hydroxyectoine have a lubricating and cell-protecting effect in creams and lotions, but can also protect the skin from harmful environmental influences such as UV radiation, allergies and fine dust. Furthermore, the precursor NADA is also a building block for polymers.

Alongside products in the cosmetics industry, ectoine is also a firm component in preparations which are used in the treatment of eye disorders. For example, ectoine in eye drops relieves irritation and inflammation by stabilizing the watery layer and the lipid layer of the tear film and therefore ensuring optimal lubrication.

In medicine, ectoine is used for irritation or inflammation of the skin or the mucous membranes, for example with a dry nose, allergic rhinitis (hay fever) or for inflammatory skin disorders such as neurodermatitis or psoriasis.

For use in the cosmetics industry, ectoine must have a purity of at least 80%. The degree of purity of ectoine for medicinal use is significantly higher. Currently, ectoine is an expensive and much sought-after product. Therefore there is a need for a method to provide ectoine, or its precursor NADA, in an efficient way and with high yields. Ectoine (1 ,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is a natural compound found in several species of bacteria. Ectoine is found in high concentrations in halophilic microorganisms and confers resistance towards salt and temperature stress. Ectoine was first identified in the microorganism Ectothiorhodospira halochloris, but has since been found in a wide range of Gram-negative and Gram-positive bacteria. Other species of bacteria in which ectoine was found include Brevibacterium linens, Halomonas elongata, Marinococcus halophilus, Pseudomonas stutzeri, Halomonas titanicae, Halorhodospira halophila, and Halomonas ventosae.

Ectoine is synthesized in three successive enzymatic reactions starting from aspartic b- semialdehyde. The enzyme ectoine synthase, encoded by the gene ectC, catalyzes the circularization of NADA to ectoine.

Ectoine is currently produced on an industrial scale by high-density fermentation of salt- tolerant bacteria such as Halomonas elongata, which in its wild-type status carries the genetic information to produce ectoine. Attempts have been made to increase the natural ectoine production by genetically modifying the microorganism.

It has been found that Halomonas elongata cells do not rely only on de novo synthesis of ectoine for adaptation to high saline environments, but can also take up compatible solutes or precursors thereof from the medium. To enable solute uptake, H. elongata is equipped with a set of compatible solute transporters of which only one accepts ectoine as a substrate, namely, the ectoine-specific transporter TeaABC. H. elongata with an inoperable TeaABC transporter constantly releases ectoine to the surrounding medium. Despite this, cells of this mutant are able to keep the internal ectoine concentration at the same level as the wild-type strain. Apparently, the mutation of teaABC not only causes excretion of ectoine to the medium but also results in overproduction of ectoine. An ectoine production strain of H. elongata with higher productivity in ectoine synthesis than the wild- type strain was designed (EP 1 409 707 B1 ). This mutant is also called“leaky mutant”, as the uptake mechanism is compromised.

A further attempted approach to increase ectoine production was to prevent ectoine degradation in the cells producing ectoine. It was found that the deletion of gene doeA, encoding the ectoine hydrolase protein, which catalyzes the first step in the ectoine degradation pathway, can lead to increased volumetric productivity of ectoine (Schwibbert et al. 2011 ).

While these attempts managed to increase the yield obtainable from microorganisms producing ectoine, there is still a need for a method, which further increases this yield. Furthermore, there is a need for microorganisms, which are capable of producing ectoine at this high level to make the high yield obtainable. The present invention provides such an improved method for producing ectoine or other chemical compounds derived from oxaloacetate metabolism in microorganisms by providing microorganisms capable of producing the compound at this high level. The microorganisms comprise a novel combination of genetic modifications responsible for the high yield.

SUMMARY OF THE INVENTION

The present invention provides a method according to the appended claims.

The present invention provides a method for producing a chemical compound derived from oxaloacetate metabolism in microorganisms, wherein the microorganism comprises the genetic information for the production of the chemical compound, wherein the microorganism is genetically modified by

increasing the extracellular accumulation of the chemical compound by disruption of the uptake mechanism of the chemical compound or by introduction of efflux channels, or by reversible permeabilization of the cytoplasmic membrane, or by replacing passive channels with active transporters; and

disrupting the intracellular degradation mechanism of the chemical compound; and disrupting the PEP-pyruvate-oxaloacetate node to increase the production of the chemical compound.

In the method of the present invention the microorganism can be a gram negative bacterium, wherein optionally the bacterium can be a halophilic bacterium, wherein optionally the microorganism can be selected from the group comprising Halomonas elongata, Brevibacterium linens, Ectothiorhodospira halochloris, Marinococcus halophilus, Pseudomonas stutzeri, Halomonas titanicae, Halorhodospira halophila, and Halomonas ventosae.

The medium used to cultivate the mutated microorganisms of the present invention in the method of the present invention preferably comprises a sodium concentration of at least 0.5 M, preferably between to 0.5 M and 1 M.

The chemical compound derived from oxaloacetate metabolism can be ectoine or hydroxyectoine.

The chemical compound derived from oxaloacetate metabolism can be Ny-acetyl-L-2,4- diaminobutyric acid (NADA).

The microorganism can further comprise a deletion of the ectoine synthase gene ectC, when the desired end product is NADA.

In the method of the present invention, the uptake mechanism of the chemical compound can be disrupted by disruption of a TRAP transporter, wherein preferably the TRAP transporter can be TeaABC. TeaABC can be disrupted by deletion of at least one of the genes selected from the group comprising teaA, teaB, and teaC, wherein optionally all of the genes in the group can be deleted.

In the method of the present invention, the degradation mechanism of the chemical compound can be disrupted by deletion of the ectoine hydrolase gene doeA.

In the method of the present invention, the PEP-pyruvate-oxaloacetate node can be disrupted by deletion of the phosphoenolpyruvate carboxykinase gene Pck, and/or the deletion of PEP carboxylase gene Ppc, and/or the deletion of the malic enzyme gene maeB, and or maeA. Optionally, the genes Pck, Ppc, maeB, and maeA are deleted.

The present invention also provides a microorganism comprising the genetic information for the production of a chemical compound derived from oxaloacetate metabolism, comprising the following genetic modifications:

increase of the extracellular accumulation of the chemical compound by disruption of the uptake mechanism of the chemical compound or by introduction of efflux channels, or by reversible permeabilization of the cytoplasmic membrane, or by replacing passive channels with active transporters; and

disruption of the intracellular degradation mechanism of the chemical compound; and disruption of the PEP-pyruvate-oxaloacetate node to increase the production of the chemical compound.

In the microorganism of the present invention, the uptake mechanism of the chemical compound can be disrupted by disruption of a TRAP transporter, wherein preferably the TRAP transporter can be TeaABC. The degradation mechanism of the chemical compound can be disrupted by deletion of the ectoine hydrolase gene doeA. The PEP-pyruvate- oxaloacetate node can be disrupted by deletion of the phosphoenolpyruvate carboxykinase gene Pck, and/or the deletion of PEP carboxylase gene Ppc, and/or the deletion of the malic enzyme gene maeB, and or maeA.

The microorganism of the present invention can be a gram negative bacterium, wherein optionally the bacterium is a halophilic bacterium, or wherein the microorganism is selected from the group comprising Halomonas elongata, Brevibacterium linens, Ectothiorhodospira halochloris, Marinococcus halophilus, Pseudomonas stutzeri, Halomonas titanicae, Halorhodospira halophila, and Halomonas ventosae.

The present invention also provides the use of the microorganism of the invention for producing a chemical compound derived from oxaloacetate metabolism, such as ectoine, hydroxyectoine, or NADA. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the PEP-Pyr-OAA node and the glyoxylate shunt in H. elongata as it has been determined by genomic analysis.

Figure 2 shows ectoine concentrations in medium in overnight cultures of three strains of Halomonas elongata in mineral medium (MM63); each value is the average of three cultures. Error bars represent two standard deviations. The strains are WT, which is the “leaky mutant” (ATeaABCADoeA), KHI (ATeaABCADoeAAPck) and KH2(ATeaABCADoeAAPckAMaeB). A measurement was removed as an outlier according to the interquartile range criterion (x> Q3+1.5 ^* IQR).

Figure 3 shows a diagram of the culture media used in preculture steps.

Figure 4 shows the growth curves of three strains, the “leaky mutant” (ATeaABCADoeA), ApckA (ATeaABCADoeAAPck) and ApckA AmaeB

(ATeaABCADoeAAPckAMaeB). The strain ApckA corresponds to strain KH1 in Figure 2, and the strain ApckA AmaeB corresponds to strain KH2 in Figure 2.

Figure 5 shows the growth rate m per hour of the three strains, the“leaky mutant” (ATeaABCADoeA), ApckA (ATeaABCADoeAAPck) and ApckA AmaeB

(DT eaABCADoeAAPckAMaeB).

Figure 6 shows the final ectoine concentration measured one week after inoculation in the three strains, the“leaky mutant” (ATeaABCADoeA), ApckA (ATeaABCADoeAAPck) and ApckA AmaeB (ATeaABCADoeAAPckAMaeB).

Figure 7 shows the ectoine concentration over time in the three strains, the“leaky mutant” (ATeaABCADoeA), ApckA (ATeaABCADoeAAPck) and ApckA AmaeB (DT eaABCADoeAAPckAMaeB).

DESCRIPTION OF THE INVENTION

It has been surprisingly found that the obtainable yield of a chemical compound derived from oxaloacetate metabolism such as ectoine in microorganisms can be significantly increased by introducing at least one genetic modification which disrupts the PEP-pyruvate- oxaloacetate node into microorganisms already comprising genetic modifications to increase the extracellular accumulation of the chemical compound, for example by disrupting the uptake mechanism of the chemical compound, and disrupting the intracellular degradation mechanism of the chemical compound.

It has been found that the synthesis of ectoine from its precursor oxaloacetate (OAA) withdraws a high amount of carbon out of the tricarboxylic acid (TCA) cycle, which must then be replenished by anaplerotic reactions. The PEP-pyruvate-oxaloacetate node has been found to play a major role in these anaplerotic reactions. Figure 1 shows the PEP-Pyr-OAA node and the glyoxylate shunt in H. elongata as it has been determined by genomic analysis. It has been shown that two of the four enzymes shown in Figure 1 contribute to the anaplerotic flux: PEP carboxylase (Ppc) and oxaloacetate decarboxylase (Oad). The other two enzymes: Phosphoenolpyruvate carboxykinase (Pck) and malic enzyme (Mae) carry flux in the opposite direction when needed, be it for gluconeogenesis or, in the case of Mae, as part of a bypass to malic dehydrogenase (also shown in Figure 1 ).

It has now been surprisingly found that disrupting the PEP-Pyr-OAA node by deletion of Pck, Ppc and/or Mae, mutant strains can be obtained, which are capable of a further increased yield of chemical compounds derived from OAA. Mae can be present in two isoforms, MaeA and MaeB. Both are targets for deletion according to the present invention.

It has been found that these mutations lead to an improved ectoine production in two different ways.

The first effect of the mutations introduced, separately or simultaneously, is to increase the supply of OAA and to suppress futile cycles. A futile cycle, also known as a substrate cycle, occurs when two metabolic pathways run simultaneously in opposite directions and have no overall effect other than to dissipate energy in the form of heat. For example, if glycolysis and gluconeogenesis were to be active at the same time, glucose would be converted to pyruvate by glycolysis and then converted back to glucose by gluconeogenesis, with an overall consumption of ATP.

The increase in supply of OAA has been shown to disrupt the normal regulation of the PEP-Pyr-OAA node in other gram negative bacteria and would not be expected to lead to an increase of ectoine production by itself. Rather, the increase in supply could lead to a general decrease in metabolic activity due partly to the inhibition of Ppc, as observed in £. coli. It has however been unexpectedly found that this decrease in metabolic activity can be mitigated by combining two strategies.

The first strategy is to ensure that an alternative anaplerotic enzyme that is regulated by different effectors is present in the cell. In the particular case of H. elongata the wild type already possesses such an enzyme, Oad, which has been shown to be regulated by sodium concentration in the medium. In the presence of a significant salt concentration such as 0.5 M to 1 M a significant anaplerotic flux is thus guaranteed in H. elongata. The presence of the alternative anaplerotic enzyme can also be ensured by introduction of the genetic information for the enzyme into the cell by artificial means.

The second strategy is to increase the demand for the desired end product to at least partially compensate the effects of increased supply on the metabolic state of the cell. This can be done by several mechanisms, which result in a reduction of the concentration of the desired end product in the cell.

It can either be ensured that the desired end product is removed from the cell, and/or it can be ensured that desired end product already removed from the cell cannot be taken up again by the cell.

Removing the desired end product from the cell can for example be achieved by introduction of efflux channels into the cell, or by permeabilization of the membrane by chemical agents such as organic solvents, e.g. methanol, acetone, or detergents such as saponin, triton or tween, or by replacement of passive channels with active transporters. Furthermore, genes encoding proteins acting as active transporters could be overexpressed in a mutant strain to achieve increased removal of the desired end product from the cell.

To ensure that any desired end product already removed from the cell into the medium is not taken up again by the cell, the uptake mechanism can be disrupted, e.g. by deleting genes encoding the proteins which facilitate the uptake. In the case of H. elongata, the disruption of the uptake mechanism can be carried out by deletion of the genes encoding the TeaABC transporter. This has already been done in the art and the resulting mutant H. elongata strain has been termed“leaky mutant” (ATeaABCADoeA).

The second effect of the mutations introduced, separately or simultaneously, is to optimize the process by decoupling growth and ectoine production. Independently of the effect of increased OAA supply, the deletion of the genes Pck, Ppc, maeB and/or maeA eliminates the two reactions that are important for gluconeogenesis. This will hinder growth on several carbon sources such as malate and acetate, since any decarboxylation of TCA intermediates would have to proceed through reactions that are thermodynamically unfavorable. Thus, mutant strains according to the invention can use glucose for both growth and ectoine production, but gluconeogenic carbon sources will only be usable for ectoine production. It has been shown by the applicants of the present invention that a microorganism which is genetically modified as defined above allows an increase of the ectoine yield of about at least 50 % (see also Figure 2).

The present invention is therefore concerned with a method for producing a chemical compound derived from oxaloacetate metabolism in microorganisms, wherein the microorganism comprises the genetic information for the production of the chemical compound, wherein the microorganism is genetically modified by

• increasing the extracellular accumulation of the chemical compound by disruption of the uptake mechanism of the chemical compound or by introduction of efflux channels, or by reversible permeabilization of the cytoplasmic membrane, or by replacing passive channels with active transporters; and

• disruption of the intracellular degradation mechanism of the chemical compound; and

• disruption of the PEP-pyruvate-oxaloacetate node to increase the production of the chemical compound.

Disruption of the PEP-pyruvate-oxaloacetate node to increase the production of the chemical compound can be achieved by silencing the function of enzymes involved in the PEP-pyruvate-oxaloacetate node. Preferred enzymes are Ppc, Pck, MaeA, and MaeB. Their function can for example be silenced by deletion of the sequence comprising the genetic information for the enzyme.

The PEP-pyruvate-oxaloacetate node can be disrupted by deletion of the phosphoenolpyruvate carboxykinase gene Pck, and/or by deletion of the PEP carboxylase gene Ppc and/or the deletion of the malic enzyme gene maeB, and/or maeA.

In a preferred embodiment the Pck gene and the maeB gene are both deleted.

In a further preferred embodiment the Pck gene, the Ppc gene and the maeB gene are deleted. In a yet further preferred embodiment the Pck gene, the Ppc gene, the maeB gene, and the maeA gene are deleted.

The chemical compound derived from oxaloacetate metabolism can be ectoine or hydroxyectoine. The chemical compound derived from oxaloacetate metabolism can also be Ny-acetyl-L-2,4-diaminobutyric acid (NADA). In this embodiment, the microorganism can further comprise a deletion of the ectoine synthase gene ectC to allow accumulation of NADA. The chemical compound derived from oxaloacetate metabolism can also be one of a whole family of amino acids, also known as the oxaloacetate family of amino acids. These amino acids are derived from oxaloacetate and comprise aspartate, asparagine, methionine, lysine, threonine, and isoleucine. The person of skill in the art will know how to disrupt the degradation mechanism of these compounds, and the person of skill in the art will also know how to increase extracellular accumulation of these compounds, and will therefore be able to adapt the teachings of this application to a method directed at increasing the yield of chemical compounds derived from oxaloacetate metabolism other than NADA, ectoine, or hydroxyectoine.

The microorganism can be a gram negative bacterium, wherein optionally the bacterium is a halophilic bacterium. The microorganism can be selected from the group comprising Halomonas elongata, Brevi bacterium linens, Ectothiorhodospira halochloris, Marinococcus halophilus, Pseudomonas stutzeri, Halomonas titanicae, Halorhodospira halophila, and Halomonas ventosae. The uptake mechanism of the chemical compound can be disrupted by disruption of a TRAP transporter, wherein preferably the TRAP transporter is TeaABC. TeaABC can be disrupted by deletion of at least one of the genes selected from the group comprising teaA, teaB, and teaC, wherein optionally all of the genes in the group are deleted. Based on the information provided in this application the person of skill in the art will be able to identify similar targets in other microorganisms, which have the same function as the TRAP transporter TeaABC in H. elongata.

Disruption of the intracellular degradation of the compound can be achieved by several mechanisms known to the person of skill in the art. For example, genes in the degradation pathway can be knocked out or knocked down. The translation of these proteins can also be inhibited or silenced by antisense or interference RNA targeting the mRNA of the genes encoding the proteins involved in the degradation pathway. The gene expression can also be downregulated for example by promoter replacement or by using the CRISPR/CAS technique. In the case of H. elongata disruption of the intracellular degradation of ectoine has already been achieved by deletion of the ectoine hydrolase gene doeA, which catalyzes the first step in the ectoine degradation pathway. Based on the information provided in this application the person of skill in the art will be able to identify similar targets in other microorganisms, which have the same function as the doeA in H. elongata.

The medium used to cultivate the mutated microorganisms of the present invention preferably comprises a significant sodium concentration, such as 0.5 M to 1 M. Sea water has a sodium concentration of about 0.5 M and can therefore be used as a base medium to grow the mutants of the present invention. E. coli exhibits reduced growth at a sodium concentration of about 0.5 M and cannot grow when the sodium concentration exceeds 0.9 M. The significant sodium concentration ensures that the anaplerotic flux is maintained even when OAA supply is increased and therefore further improves the method of the present invention.

The present invention is also concerned with a microorganism obtained by the genetic modifications as defined above.

The present invention is also concerned with the use of the genetically modified microorganism for producing a chemical compound derived from oxaloacetate metabolism.

EXAMPLES

Example 1

Strains, plasmids, and growth conditions

Halomonas elongata DSM2581T was routinely grown in minimal medium MM63 or LB medium containing 1 M NaCI at 30 °C under shaking. All Escherichia coli strains were grown in LB medium at 37 °C amended with the antibiotic necessary for maintenance of the plasmid. All plasmids and genetic manipulations were performed on E. coli DH5a or E. coli DH5a l-pir.

Example 2

Construction of plasmids pSEVA212S-pckA::Sm, pSEVA212S-ApckA,

pSEVA212S-maeB::Sm, pSEVA212S-AmaeB, and pSEVA212S-AmaeA

Replacement of the genes pckA, maeB, and maeA with aadA, encoding a Streptomycin resistance cassette was performed by adapting the method previously reported for Pseudomonas putida (Martinez-Garcia et al., 2011 ). The plasmids were constructed by Gibson Assembly (NEB) according to the suppliers manual. Oligonucleotides used for the gene amplification are shown in Table 1. A DNA segment containing about 500 bp of the genomic sequence upstream of the target gene ( pckA , maeB, or maeA) followed by the sequence of the aadA gene, encoding the streptomycin resistance cassette, and about 500 bp of the downstream chromosomal region, was inserted into pSEVA212S, carrying the R6K suicide origin of replication (Martinez-Garcia et al., 2015) previously linearized with EcoRI. The plasmid was transformed into E. coli DH5a l-pir and clones carrying it were selected on LB agar plates supplemented with 200 pg/ml streptomycin (Sm 200). The correct assembly of pSEVA212S-pckA::Sm, pSEVA212S-ApckA, pSEVA212S-maeB::Sm, pSEVA212S-AmaeB, and pSEVA212S-AmaeA was verified by isolation of the plasmids and sequencing.

Example 3

Conjugal transfer of plasmids into H. elongata by triparental mating

Two plasmids had to be transferred to H. elongata using E. coli HBH101 (pRK600) as helper in the mating procedure. First, plasmid pSW-2 encoding the 1-Scel endonuclease was transferred to H. elongata. Therefore, the donor strain E. coli DH5a l-pir (pSW-2), the recipient strain H. elongata, and the helper strain E. coli HBH101 (pRK600) were grown in LB medium, amended with the antibiotic necessary to assure plasmid maintenance (pSW-2: 10 pg/ml gentamycin and pRK600: 35 pg/ml chloramphenicol) to the stationary phase (overnight). The next day, optical densities at 600 nm were measured and 1 ml of the donor culture was mixed with the amount of recipient and helper culture to obtain OD ratios of 1 :1 :1 and 1 :2:2. These suspensions were centrifuged; the supernatant was discarded by decanting and the cell pellet was resuspended in the remaining medium rest. This condensed cell suspension was pipetted on one spot of a LB agar plate containing 0.5 M NaCI, which was incubated for 5 h at 30 °C. Afterwards, the cells were resuspended in 600 pi of a 0.5 M NaCI solution, an aliquot of 100 mI was plated on LB agar plates containing 0.5 M NaCI, 500 pg/ml ampicillin and 50 pg/ml gentamycin and the plates were incubated for 48 h at 30 °C. Colonies were picked and the presence of pSW-2 was verified by PCR with pSW2-F and pSW2-R (see Table 1 ).

Next, pS E VA212S-pckA: :S m , pSEVA212S-ApckA, pSEVA212S-maeB::Sm, pSEVA212S-AmaeB, and pSEVA212S-AmaeA had to be transferred to H. elongata (pSW- 2) again by triparental mating. This was performed as described above but using H. elongata (pSW-2) as recipient, E. coli DH5a l-pir (pSEVA212S-pckA::Sm, pSEVA212S- ApckA, pSEVA212S-maeB::Sm, pSEVA212S-AmaeB, and pSEVA212S-AmaeA) as donor and £. coli HBH101 (pRK600) as helper. The cointegrates were selected by plating different dilutions of the mating mixture on LB agar plates containing 0.5 M NaCI, 500 pg/ml ampicillin, 60 pg/ml kanamycin, and 50 pg/ml gentamycin.

Example 4

Resolution of cointegrates to obtain H. elongata ApckA::Sm, AMaeB::Sm and AMaeA::Sm

To resolve the cointegrates a single colony was picked from the selective agar plate and grown in 3 ml LB medium with 1 M NaCI, 500 pg/ml ampicillin, 200 pg/ml streptomycin and 50pg/ml gentamycin overnight at 30 °C. The next day, cells were pelleted by

centrifugation, washed once in LB medium containing 1 M NaCI, and subsequently resuspended to an OD600 of 1 with LB medium with 1 M NaCI, 500 pg/ml ampicillin, and 50pg/ml gentamycin. Four ml of that culture were induced with 80 mI of 3-methylbenzoate to a final concentration of 10 mM and incubated at 30 °C under shaking.

After 3.5 h of induction of 1 -Seel expression an aliquot was plated on LB agar with 1 M NaCI, 500 pg/ml ampicillin and 200 pg/ml streptomycin and incubated at 30 °C for 48 hours. Single colonies were picked and gridded on LB plates with 1 M NaCI, 500 pg/ml ampicillin, and either 60 pg/ml kanamycin or 200 pg/ml of streptomycin to identify those clones, that lost the kanamycin resistance cassette but maintained the streptomycin cassette. Clones showing the desired phenotype should represent the desired mutants. This was verified by PCR using the primers as shown in Table 1 that hybridized to sequences outside the 500 bp flanks used in pSEVA ApckA::Sm, AMaeB::Sm or AMaeA::Sm and by sequencing of the whole fragment.

Table 1: Oligonucleotides used in this work.

Example 5

Ectoine production by H. elongata mutants of the present invention compared to ectoine production by an H. elongata mutant of the prior art

The result of this experiment is shown in Figure 2.

Figure 2 shows ectoine concentrations in medium in overnight cultures of three strains of Halomonas elongata in mineral medium (MM63) each value is the average of three cultures. Error bars represent two standard deviations. The strains are WT, the“leaky mutant” (ATeaABCADoeA) of the prior art, and KHI (ATeaABCADoeAAPck) and KH2(ATeaABCADoeAAPckAMaeB) of the present invention. A measurement was removed as an outlier according to the interquartile range criterion (x> Q3+1.5 ^* IQR).

It is shown that the mutants of the present invention exhibit a significantly increased yield in ectoine production compared to the mutant of the prior art.

Example 6

Strains H. elongata ApckA and ApckA AmaeB - Ectoine Analytics

Preculture Steps:

The culture media used in the preculture steps are shown in Figure 3.

Composition of the main culture medium:

A modified medium MM63 with lower amount of sulfate than normal was used. The concentration of sulfate was calculated to allow the formation of biomass up to OD ₆ oo 2 (theoretical value).

Growth curves:

The growth curves of three strains are shown in Figure 4. The growth curve of the reference strain (“leaky mutant”) shows a sharp bend after reaching an OD ₆ oo of 2 which was to be expected due to the limitation of sulfate in the medium. In contrast to this, both mutant strains reach higher OD ₆ oo values than expected. It is possible that the introduced deletions cause a change in the relation of biomass to OD ₆ oo- This way, the amount of biomass produced by the mutant strains might be slightly overestimated, which means that the actual yields are higher than what is reported here.

Figure 5 and the table below show the growth rates of the three strains. The lower values for the inventive strains reflect the shift of carbon flow from biomass to ectoine production.

Ectoine Analytics:

1 ) End-point measurement comparable with already submitted data (see Figure 2):

All strains are incapable of metabolizing ectoine due to the deletion of the ectoine hydrolase (DoeA). The extracellular ectoine concentration was measured as shown in Figure 6 and in the table below. However, since the end-point samples were taken one week after inoculation it is possible that a high amount of the intracellular ectoine was already released due to cell lysis during death phase. An increase in the ectoine concentration of about 43.5% was observed in the inventive strains compared to the reference strain of the prior art, the“leaky mutant”.

Ectoine concentration over time

For the reference strain (“leaky mutant”) and the double mutant strain (ApckA AmaeB) five to six measurements of ectoine were performed (see Figure 7). Due to its longer lag phase, for the ApckA mutant only three samples contained enough ectoine above the measuring limit for HPLC analysis. The first samples of this strain were taken before OD ₆ oo 1 , so this was to be expected. The results already normalized per ash-free dry weight (DW (af)) look very promising. Accumulation of ectoine for each strain in g ect / g DW / h was as follows: leaky mutant 0.043 +/- 0.001 , pckA 0.058 +/- 0.009, pckA maeB 0.056 +/- 0.002. The rates are means from three biological replicates, each obtained by linear regression (R ² > 0.85) except for pcKA strains where the number of points was too low for regression.

For all strains the same correlation of dry weight to OD ₆ oo was used. Since the differences in maximal OD ₆ oo values suggest that the biomass for the mutant strains are overestimated, higher values for ectoine per DW (af) might be achievable and the shown data represents the rate of production in the worst case scenario.

REFERENCES

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