The present invention relates to formation of highly enriched (R)-pantolactone from conversion of keto-pantolactone in a bio-catalytic process using NADPH- dependent enzymes in the presence of NADH as co-factor.

Pantothenate or vitamin B5 is a member of the B complex of vitamins which is essential for mammals including humans. It has to be supplied as food or feed additive. In cells, pantothenate is used primarily for the biosynthesis of coenzyme A and acyl carrier protein. These essential coenzymes function in the metabolism of acyl moieties, which form thioesters with the sulfhydryl group of the 4'-phosphopantethein portion of these molecules. Importantly, only (R)- pantothenate is biologically active as vitamin.

Pantothenate has been synthesized conventionally via chemical synthesis from bulk chemicals. However, the substrates required for chemical synthesis are expensive and the racemic intermediates have to be optically resolved. Bacterial or microbial systems, if available, that produce enzymes useful in pantothenate biosynthesis processes, are not yet economically viable for industrial production because of insufficient yield, poor selectivity of the used enzymes, undesired side-reactions in cheap crude enzyme preparations, or too high co-factor cost For example, Si et al. (Appl Microbiol Biotechnol (2012) 93:1619-1625) describes the use of Stenotrophomonas maltophilia and its application to stereospecific production of D-pantoic acid. The enzyme belongs to the enzyme family of pantoate 4-dehydrogenase (EC 1.1.1.106). While only few members of this family e.g. from Pseudomonas accept NADH as co-factor (Mybhanen, T.; Mantsala, P.; Biochim. Biophys. Acta 614, 266-273 (1980)) the enzyme from Stenotrophomoas was classified as NADPH dependent according to Si et al. (Appl Microbiol Biotechnol (2012) 93:1619-1625). The same co-factor dependency for NADPH is known for the pantoate 4-dehydrogenase from Candida parapsilosis described by Shimizu et a. (Appl Microbiol Biotechnol (2004) 64: 359-366). An obvious drawback of the described processed is the use of expensive NADPH as cofactor.

Thus, there is a need for more effective and simpler methods towards enantioselective production of (R)-pantolactone with a high enantiomeric excess (e.e.) to be used in an industrial scale, with no need for further resolution and/or purification steps and with the avoidance of expensive co-factors such as e.g. NADPH.

Surprisingly, we now identified a novel process for production of (R)- pantolactone via enantioselective catalytic reduction of keto-pantolactone (KPL), comprising the use of NADH as co-factors with known NADPH-dependent reductases leading to a highly efficient enantioselective conversion of KPL.

Particularly, the present invention is directed to enantioselective reduction of KPL into (R)- panto lactone, wherein the reduction of KPL is catalyzed by enantioselective NADPH dependent alcohol dehydrogenase (ADH) enzymes (EC 1.1.1.2) or pantoate 4-dehydrogenase (EC 1.1.1.106) which surprisingly accept NADH as co-factor, wherein an e.e. in the range of about 80-99% could be achieved.

For the purpose of the present invention, i.e. enantioselective conversion of KPL into (R)-pantolactone as described herein, any NADPH dependent ADH isolated from various sources can be used, particularly such enzymes isolated from archaea, bacteria, fungi including yeast, or higher eukaryotes. Preferably, the enzyme is selected from yeast, such as Candida or Saccharomyces, or from bacteria, such as Bacillus or Escherichia, e.g. Stenotrophomonas, preferably a NADPH-dependent reductase selected from Candida parapsilosis, Saccharomyces cerevisiae or Stenotrophomonas maltophilia, more preferably Sma_KPR from Stenotrophomonas maltophilia NBRC 14161, Cpa_PKR_2 from Candida parapsilosis IFO 0708, ADH 440 (evo-1.1.440.S, evoXX technology GmbH, Germany; for specification see Table 3) originated from E. coli, most preferably Sma_KPR from Stenotrophomonas maltophilia NBRC 14161 or Cpa_PKR_2 from Candida parapsilosis IFO 0708 or enzymes having equivalent activity of Sma_KPR from Stenotrophomonas maltophilia NBRC 14161 or Cpa_PKR_2 from Candida parapsilosis IFO 0708 or ADH 440 from E. coli.

As used herein, the term "keto-pantolactone" also includes the respective ring- opened form, i.e. ketopantoic acid. As used herein, terms like "alcohol dehydrogenase", "pantoate 4- dehydrogenase", "reductase" or "enantioselective alcohol dehydrogenase (ADH) enzymes" are used interchangeably herein and define suitable enzymes to be used for the performance of the present invention, i.e. conversion of KPL into pantolactone with a high percentage, i.e. at least about 80%, of (R)- pantolactone.

The terms "conversion", "enantioselective reduction", "biocatalytic process", "enantioselective catalytic reduction" or "enzymatic conversion" are used interchangeably herein and describe the reduction of KPL into (R)-pantolactone catalyzed by the ADHs as defined herein.

As used herein, the term "used in the presence of NADH" or "together with NADH as cofactor" is used interchangeably herein and refers to a reaction wherein NAD (NAD ⁺ ) is added but the reduced form, i.e. NADH, is actually used by the enzyme as co-factor.

The enzymatic conversion of KPL as defined herein into (R)-pantolactone, as described herein can be performed with isolated enzymes, such as e.g. dehydrogenase enzymes as defined herein, particularly ADHs, such as e.g. ADHs as defined herein originating from Stenotrophomonas maltophilia, or Candida parapsilosis, particularly Sma_KPR or Cpa_PKR_2, that might be expressed in a suitable host cell, such as e.g. yeast or bacteria, preferably Pichia or Escherichia, such as e.g. as heterologous enzymes. Particularly, the enantioselective enzymes as described herein are heterologous expressed in Escherichia coli. Isolation of suitable enzymes can be done by known methods, the respective enzymes might be further used in isolated form or in the form of a cell free extract (cfe), powder or liquid formulations, i.e. in immobilized or non-immobilized form. Preferably, the enzymes as defined herein, such as e.g. the reductases from Stenotrophomonas, Candida, as defined herein, are used as cfe. If the enzymes are used in isolated form, such as e.g. in liquid or immobilized form, they might be either covalently bound or adsorbed, e.g. on epoxy carrier, depending on the supplier. When using immobilized forms, the enzymes might be used several times with more or less the same performance (so-called recycling). Suitable enzymes to be used for the purpose of the present invention might be sourced from commercially available sources, such as e.g. ADH 440 (evo-1.1.440.S; evoXX technology GmbH, Germany).

The enzymatic conversion of KPL as defined herein into (R)-pantolactone, as described herein can be performed in a process wherein a suitable host cell expressing said enzymes, such as e.g. dehydrogenase enzymes as defined herein, particularly recombinant host cell expressing heterologous enzymes, is cultivated under suitable conditions in the presence of a substrate allowing the conversion of KPL into (R)- panto lactone as described herein via catalytic action of said expressed enzymes. Such expression system wherein recombinant host cell expressing the respective enzymes as defined herein is acting on the substrate, i.e. KPL, is also called a "recombinant whole cell biocatalyst" to be used for conversion of KPL into (R)-pantolactone.

The enzymes to be used for the present invention are known as NADPH- dependent alcohol dehydrogenases, i.e., using NADPH as co-factor for their catalytic activity and have not been disclosed to use other co-factors such as e.g. NADH, NAD or NAD+ as co-factor. As used herein, the term "co-factor" and "co-enzyme" are used interchangeably herein. As it is known in biocatalysis, the performance of an enzyme is normally very much related /restricted to the corresponding co-factor.

The process according to the present invention comprises incubation of KPL in the presence of a suitable enzyme as defined herein said process being furthermore carried out in the presence of NADH as co-factor. Such process might be conducted at a pH in the range of about 7.5 to about 4.5, such as e.g. a pH of about 7.4, 7.3, 7.2, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.0, 4.5.

The process according to the present invention wherein KPL is enantioselectively converted via action of a reductase together with NADH as defined herein and performed at a pH between about 7.5 and 4.5 might be combined with an in-situ co-factor regeneration system, such as e.g. coexpression of co-factor regeneration enzymes, including but not limited to glucose dehydrogenase (GDH) converting D-glucose to D-gluconic acid and glucose as substrate, such as e.g. co-expression of E. coli GDH. Alternatively, GDH (CAS Number 9028-53-9) is available from various suppliers as known in the art.

In one embodiment, the process according to the present invention is performed with Sma_KPR from Stenotrophomonas maltophilia NBRC 14161, preferably a polypeptide with at least about 80%, such as e.g. 85, 90, 95, 98 or 100% identity to SEQ ID NO:1, including polynucleotides encoding said polypeptide such as e.g. the polynucleotide of SEQ ID NO:2, in the presence of NADH, resulting in (R)-pantolactone with an e.e. of at least about 85%, particularly with the process being carried out at pH of about 5.5. The e.e. might be further increased to about 100% with a process carried out at pH of about 7.5. Using such process, the yield of converted (R)-pantolactone might be in the range of at least about 87%.

In one embodiment, the process according to the present invention is performed with Cpa_PKR_2 from Candida parapsilosis IFO 0708, preferably a polypeptide with at least about 80%, such as e.g. 85, 90, 95, 98 or 100% identity to SEQ ID NO:3 including polynucleotides encoding said polypeptide such as e.g. the polynucleotide of SEQ ID NO:4, in the presence of NADH, resulting in (R)- pantolactone with an e.e. of at least about 84%, particularly with the process being carried out at pH of about 5.5. The e.e. might be further increased to at least about 99% with a process carried out at pH of about 7.5. Using such process, the yield of converted (R)- panto lactone might be in the range of at least about 39%.

The "enantiomeric excess" (e.e.) as used herein refers to the degree to which a sample contains one enantiomer (such as e.g. (R)-pantolactone) in higher amount than the other enantiomer (such as e.g. (S)-pantolactone), see Helpfile on selectivity, Uni Graz, 2012: httD://biocatalvsis.uni- graz.at/enantio/DataFiles/Selectivitv-HelD.Ddf.

The terms "sequence identity", "% identity" are used interchangeable herein. For the purpose of this invention, it is defined here that in order to determine the percentage of sequence identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids/bases or amino acids. The sequence identity is the percentage of identical matches between the two sequences over the reported aligned region. The percent sequence identity between two amino acid sequences or between two nucleotide sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose of this invention the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, Longden and Bleasby, Trends in Genetics 16, (6) pp276— 277, http://emboss.bioinformatics.nl/). For protein sequences EBLOSUM62 is used for the substitution matrix. For nucleotide sequence, EDNAFULL is used. The optional parameters used are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.

After alignment by the program NEEDLE as described above the percentage of sequence identity between a query sequence and a sequence of the invention is calculated as follows: number of corresponding positions in the alignment showing an identical amino acid or identical nucleotide in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. The identity as defined herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled in the output of the program as "longest identity". If both amino acid sequences which are compared do not differ in any of their amino acids, they are identical or have 100% identity.

As used herein, the term "specific activity" or "activity" with regards to enzymes means its catalytic activity, i.e. its ability to catalyze formation of a product from a given substrate. The specific activity defines the amount of substrate consumed and/or product produced in a given time period and per defined amount of protein at a defined temperature. Typically, specific activity is expressed in pmol substrate consumed or product formed per min per mg of protein. Typically, activity is given per mg of the specific enzyme; if specified otherwise, activity can also be given on mg enzyme preparation (e.g. total protein, total soluble protein etc.). Typically, pmol/min is abbreviated by U (= unit). Therefore, the unit definitions for specific activity of pmol/min/(mg of protein) or U/(mg of protein) are used interchangeably throughout this document. An enzyme is active, if it performs its catalytic activity in vivo, i.e. within the host cell as defined herein or within an in vitro system in the presence of a suitable substrate. The skilled person knows how to measure enzyme activity, in particular activity of ADHs as defined herein, such as in particular KPL-reductase activity. Analytical methods to evaluate the capability of a suitable enzyme as defined herein for formation of (R)-pantolactone from conversion of KPL as defined herein are known in the art. The present invention is directed to a process as defined herein for enantioselective reduction of KPL, wherein the KPL is enzymatically converted into (R)-pantolactone in the presence of NADH as co-factor, said process being carried out at a pH between about 7.5 and 4.5, with an e.e. in the range of at least about 84-100%, and a yield of (R)-pantolactone of at least about 39%, depending on the used enzyme and the pH conditions.

In one aspect of the present invention, a host cell comprising and expressing an enantioselective enzyme as defined herein, is used in a process for production of (R)-pantolactone from conversion of KPL in the presence of NADH as co-factor as defined herein. The host cell comprising the enzyme may be cultured in an aqueous medium supplemented with appropriate nutrients under aerobic or anaerobic conditions and as known by the skilled person for the respective host cells. Optionally, such cultivation is carried out at a pH of about 7.5 to about 4.5. The cultivation/growth of the host cell may be conducted in batch, fed-batch, semi-continuous or continuous mode. (R)-pantolactone might be isolated and/or optionally further purified.

In one embodiment of the invention the conversion as described herein is carried out using an immobilized enzyme for enantioselective conversion of KPL into (R)-pantolactone. Such process might be performed under conditions known in the art, e.g. such as in an aqueous medium or under conditions mentioned herein. Particularly, the process might be carried out at pH between about 7.5 and 4.5, using NADH and GDH/glucose as in situ co-factor regeneration system.

With regards to the present invention, it is understood that organisms, such as e.g. animals, microorganisms, fungi, algae or plants also include synonyms or basonyms of such species having the same physiological properties, as defined by the International Code of Zoological Nomenclature, International Code of Nomenclature of Prokaryotes or the International Code of Nomenclature for algae, fungi, and plants (Melbourne Code).

The process as described herein might be carried out at temperatures of about 21°C to about 45°C, such as e.g. between about 21°C to about 40°C, particularly between about 21°C to about 35°C, preferably between about 25°C to about 31°C, more preferably about 28°C.

In particular, the present invention is directed to the following embodiments (1) to (9): (1) Process for enantioselective conversion of keto-pantolactone into R- pantolactone, comprising incubation of keto-pantolactone in the presence of a NADPH dependent reducing enzyme and NADH as co-factor.

(2) Process according to embodiment (1), wherein the enzyme is selected from alcohol dehydrogenase EC enzymes (EC 1.1.1.2) or pantoate 4-dehydrogenase (EC 1.1.1.106).

(3) Process according to embodiment (1) or (2), wherein the pH is in the range of about 7.5 to about 4.5.

(4) Process according to embodiment (1), (2) or (3), wherein the reducing enzyme is an alcohol dehydrogenase originating from Candida or Stenotrophomonas, particularly selected from Candida parapsilosis or Stenotrophomonas maltophilia, preferably Sma_KPR from Stenotrophomonas maltophilia NBRC 14161, Cpa_PKR_2 from Candida parapsilosis IFO 0708, most preferably Sma_KPR from Stenotrophomonas maltophilia NBRC 14161 with at least about 80% identity to SEQ ID NO:1 or Cpa_PKR_2 from Candida parapsilosis IFO 0708 with at least about 80% identity to SEQ ID NO:3.

(5) Process according to embodiment (4), wherein the reducing enzyme is a commercially available NADPH-dependent alcohol dehydrogenase, particularly ADH 440 (catalogue it evo 440, evoXX GmbH, Germany).

(6) Process according to embodiment (1), (2), (3), (4) or (5), wherein the enzyme is used as a recombinant whole cell biocatalyst or as cell free extract.

(7) Process according to embodiment (1), (2), (3), (4), (5) or (6), further comprising an in-situ co-factor regeneration system, particularly comprising glucose dehydrogenase and glucose.

(8) Process for production of (R)- panto lactone with e.e. of at least about 99%, comprising enantioselective reduction of keto-pantolactone in the presence of an enantioselective alcohol dehydrogenase (ADH), particularly an ADH enzyme selected from Candida parapsilosis or Stenotrophomonas maltophilia, preferably Sma_KPR from Stenotrophomonas maltophilia NBRC 14161 or Cpa_PKR_2 from Candida parapsilosis IFO 0708, most preferably an enzyme with at least about 80% identity to SEQ ID NOs: 1 or 3, at a pH of about 7.5.

(9) Process for production of (R)- panto lactone with e.e. of at least about 94%, comprising enantioselective reduction of keto-pantolactone in the presence of the enantioselective alcohol dehydrogenase ADH 440 (catalogue it evo 1.1.440, evoXX GmbH, Germany) at a pH of about 5.5, preferably wherein the yield of (R)- pantolactone is at least about 59%.

The following examples are illustrative only and are not intended to limit the scope of the invention in any way.

Examples

Example 1: General methods and media

All basic molecular biology and DNA manipulation procedures described herein were generally performed according to Sambrook et al. (1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York) or Ausubel et al. (1998. Current Protocols in Molecular Biology. Wiley: New York). Media used for protein production are listed in Table 1.

Table 1: Media composition.

Example 2: Expression of enzymes in Escherichia coli

Gene encoding sequences 1, 3, 5, 7, were ordered codon-optimized for expression in E. coli as synthetic DNA at ATUM (Newark, California, US). All constructs were used for transformation of E. coli RV308ArhaBAD.

Transformations were done as follows:

Competent cells were thawed on ice. Subsequently, 1 pl plasmid DNA was added to the cells and the mix was incubated for 5 minutes on ice. After a heat shock treatment (60 sec, 42°C) cells were immediately placed on ice again for 5 minutes. 300 pl SOC medium (Catalogue it 15544034, Thermo Fisher Scientific, USA) were added and the cells were incubated for 1-2 hours at 37°C and gentle shaking (300 rpm). Subsequently, 50 pl of the reaction mix was plated on LB medium containing 50 pg/ml neomycin. Alternatively, the full reaction mix was centrifuged (2 min, 6000 rpm) and the cell pellet was resuspended in 100 pl SOC medium and completely plated on the selective agar plates. Selection plates were incubated overnight at 37°C.

Recombinant strains were cultivated for ADH expression as follows: 20 ml of LB medium containing 100 mg/l neomycin were inoculated with the glycerol stock of the respective clone. The culture was incubated over-night at 28°C and with shaking at 110 rpm. The next day, 100 ml 2xTY medium containing 100 mg/l neomycin in a 500 ml baffled Erlenmeyer flask were inoculated with 1 ml of the preculture (initial OD620 -0.05). The culture was incubated on an orbital shaker (180 rpm) at 28°C. When the OD620 reached -0.5, the protein expression was induced by addition of 100 pl 20% (v/v) L-rhamnose (final concentration 0.02%). The cultivation of the strains was continued over-night (180 rpm, 28°C). On the next day, cells were harvested by centrifugation at 4°C for 15 min and 5000 rpm. Cell pellets were stored at -20° C.

For further studies cell free extract was prepared. Wet cells were resuspended in 200 vol% of 100 mM potassium phosphate buffer pH 7.0 with respect to their wet weight. The cell suspension was sonicated on ice until the OD620 of the suspension was below 10% of the starting value. Cell debris was subsequently removed by centrifugation (4°C, 5000 rpm for 10 min). The clear supernatant (cell free extract, CFE) was stored until further use at -20° C.

Example 3: Enantioselective reduction of KPL into (R)-pantolactone in the presence of NADH

Formation of (R)-pantolactone was tested with different enzymes using NAD ⁺ as co-factor at constant pH 5.5 or 7.5. Reactions on a 10 ml scale were performed with 100 mM KPL, MgCL ₂ (imM final cone.) ZnSOz, (imM final concentration), 5% (vol/vol) enzyme (CFE; see Ex. 2), 150 mM (final cone.) D-glucose & 20 U/ml (final cone.) of GDH (Codexis, GDH 105). NAD ⁺ (0.7 mM final cone.) was added.

Incubations were done at 28°C, shaken (stirrer speed 10) for 1 -3 hours. 250 pl sample was acidified with 25 pl of HCl (37%), heated at 80°C for 15 min and extracted with 900 pl ethyl acetate with internal standard (anisole, ca. 1 mg/ml). After 10 min of shaking, the phases separated within 10 min and a sample of the ethyl acetate layer was taken for analysis.

Measurements were carried out on an Agilent HP 6890 gas chromatograph equipped with hot-split injector, autosampler and FID detector. Column used was Chirasil DEX CB, dimensions 25 m x 250 pm x 0.25 pm film thickness. The isothermal temperature was 125°C during 12 minutes. As a carrier gas Helium with a flow of 1.5 ml/min was used. Injection volume was 1 pl, with a split ratio of 1 : 30 at a hot split injection at 220°C. A flame ionisation detector was used at 250°C, with a hydrogen flow of 40 mL/min, an air flow of 450 mL/min and a make-up flow of 30 mL/min. The results for keto panto lactone reductase from S. maltophila or polyketone reductase from C. parapsilosis (Cpa_PKR_2) compared to ADH 440 (evo-1.1.440.S, evoXX technology GmbH, Germany; see Table 3) are shown in Table 2 below.

Table 2A; (R)-selectivity of selected enzymes at pH 7.5. "ee R-PL" means ee of (R)-pantolactone measured at the end, "yield R-PL" means the percentage of (R)-pantolactone based on mol. All enzymes are used as CFE. The bio blank contains CFE of E. coli RV308ArhaBAD (no plasmid). The chemical blank contains all components of the reaction but no CFE. For more details, see text.

Table 2B; (R)-se lectivitv of selected enzymes at pH 5.5. "ee R-PL" means ee of (R)-pantolactone measured at the end, "yield R-PL" means the percentage of (R)-pantolactone based on mol. All enzymes are used as CFE, except for ADH 440 which is in powder form. For more details, see text or legend to Table 2A.

Table 3; enzyme specification of ADH 440 (evo-1.1.440.S) according to the supplier (evoXX technology GmbH, Germany). "U" is defined as 1 unit reducing 1 pmol cyclohexanone per minute at pH 7.0 and 30°C.