WO2006134374A2 - Procede de production d'acide l-ascorbique - Google Patents

Procede de production d'acide l-ascorbique Download PDF

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Publication number
WO2006134374A2
WO2006134374A2 PCT/GB2006/002203 GB2006002203W WO2006134374A2 WO 2006134374 A2 WO2006134374 A2 WO 2006134374A2 GB 2006002203 W GB2006002203 W GB 2006002203W WO 2006134374 A2 WO2006134374 A2 WO 2006134374A2
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Prior art keywords
micro
organism
ascorbic acid
tagatose
enzyme
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PCT/GB2006/002203
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English (en)
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WO2006134374A3 (fr
Inventor
Robert Douglas Hancock
Gary Davidson Hunter
Jane Shaw
Roberto Viola
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Scottish Crop Research Institute
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Publication of WO2006134374A2 publication Critical patent/WO2006134374A2/fr
Publication of WO2006134374A3 publication Critical patent/WO2006134374A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C

Definitions

  • the present invention provides an improved method for the production of L-ascorbic acid.
  • AsA L-Ascorbic acid
  • the Reichstein process is a chemical process in which L-sorbose, produced by bacterial fermentation is converted to AsA through a series of four chemical steps. Alternatively, L-sorbose is fermented to 2-keto-L- gulonic acid which is subsequently converted to AsA by chemical means. Significant investment in process improvement has resulted in relatively efficient syntheses with approximate AsA yields of 60% using either method. However both processes have significant limitations. The need for organic and inorganic solvents and heavy metal catalysts in the Reichstein process imposes a requirement for strict environmental controls resulting in significant waste disposal costs. Additionally, the high energy inputs required for many steps add to costs.
  • yeast such as Saccharomyces cerevisiae (Hancock et al., 2000) and Pichia pastoris that synthesise D- erythroascorbic acid from D-arabinose using the enzymes D-arabinose dehydrogenase and D-arabinono- 1,4-lactone oxidase and multicellular fungi such as Aspergillus nidulans, Basidiomycetes and Actinomycetes that additionally synthesise ⁇ -deoxy L-ascorbic acid (Okar ⁇ ura, 1998).
  • yeast such as Saccharomyces cerevisiae (Hancock et al., 2000) and Pichia pastoris that synthesise D- erythroascorbic acid from D-arabinose using the enzymes D-arabinose dehydrogenase and D-arabinono- 1,4-lactone oxidase and multicellular fungi such as Aspergillus nidulans, Basidiomycetes and Act
  • L-galactose is synthesised using the biological activity of bacterial enzymes.
  • the first enzyme required is any enzyme capable of epimerisation of L-ketohexoses at the C3 position (e.g. tagatose epimerase) and specifically epimerisation of L-sorbose to L- tagatose.
  • the second enzyme required is any enzyme capable of converting L-ketohexose to L-aldohexose and specifically L-tagatose to L-galacose (e.g. L- fucose isomerase; E. C. 5.3.1.25).
  • Enzymes endogenously expressed in a suitable organism that contain appropriate enzyme cofactors then convert L- galactose to AsA.
  • a putative mechanism is outlined in Fig. 1. An advantage of the methodology used is that L-AsA is produced using the inexpensive starting material L-sorbose.
  • Potential applications include but are not limited to: a) fermentation processes for the production of AsA; and/or b) a method for the production of AsA enhanced plants, algae, yeast and fungi.
  • the present invention provides a method of producing L-ascorbic acid, said method comprising co-incubating a source of the enzymes tagatose epimerase and fucose isomerase with a micro-organism able to convert L-galactose to L- ascorbic acid.
  • the micro-organism able to convert L-galactose to L-ascorbic acid is yeast.
  • yeast is Saccharomyces cerevlsiae.
  • Alternatives include fungus, for example Aspergillus nidulans.
  • the enzyme tagatose epimerase is obtained from Agrobacterium tumefaciens C58 (NCBI Accession No. AE008210) .
  • the enzyme fucose isomerase is obtained from E coli.
  • the enzyme originates from the micro-organism specified.
  • the enzyme may be present in a purified or partially purified form, but this may not always be essential and a crude cell extract containing the enzyme may also be suitable in some circumstances.
  • Use of the native genes encoding the enzyme from that organism to express the enzyme by genetic engineering means is also included.
  • the genes encoding these enzymes may be cloned and expressed in any suitable host micro- organism. Mention may be made of E coli or other bacteria such as Gluconobacter oxydans, Corynebacter ⁇ um sp. , Acetobacter liquifaciens; yeasts or fungi such as Saccharomyces cerevisiae, Pichia pastoris, Candida blankii, Aspergillus nidulans, as exemplary hosts micro-organisms.
  • the coding sequences for fucose isomerase and/or tagatose epimerase is optimised for expression in the expression in the selected host micro-organism.
  • the enzymes expressed are transported to the outer surface of the host cell or are exported therefrom.
  • the micro-organism able to convert L-galactose to L-ascorbic acid is itself transformed to additionally express one or both of tagatose epimerase and/or fucose isomerase, for example from recombinant genetic construct (s) .
  • the present invention also provides a recombinant polynucleotide comprising a sequence as set out in SEQ ID Nos 1 or 3, or homologs thereof.
  • homologs with reference to a polynucleotide, we refer to a polynucleotide modified by deletion, substitution or addition of nucleic acids to have at least 80% homology, preferably 85% homology, to the nucleotide sequence (s) as set out in the sequence listing. In one embodiment the homolog will have 90% or more homology, for example 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, to the nucleotide sequence (s) as set out in the sequence listing and when assessed by direct sequence alignment and comparison sequence.
  • Sequence homology can be determined by direct best- fit sequence alignment and comparison, or by using any suitable homology algorithm, such as BLAST.
  • BLAST is described by Altschul et al., in J MoI Biol 25:403 (1990).
  • S is calculated as the sum of substitution and gap scores.
  • Substantial homology when assessed by BLAST refers to low Expectation (E) values. Expectation value is the number of different alignments with scores equivalent or better than S that are expected to occur in a database search by chance. The lower the E value, the more significant the score.
  • homologs also includes a polynucleotide capable of hybridising to a polynucleotide comprising 15 contiguous bases from any one of SEQ ID Nos 1 or 3, preferably under stringent conditions.
  • the polynucleotide hybridises to a polynucleotide comprising 20 or more contiguous bases (for example 25 to 50 contiguous bases) from any one of SEQ ID Nos 1 or 3, preferably under stringent conditions.
  • Stable hybridisation of polynucleic acids is a function of hydrogen base pairing. Hydrogen base pairing is affected by the degree to which the two polynucleotide strands in the duplex are complementary to each other and also the conditions under which hybridisation occurs. In particular salt concentration and temperature affect hybridisation.
  • E Tm effective melting temperature
  • stringent conditions refers to IM Na + at 65 to 68°C.
  • the polynucleotide can be DNA or RNA and can be single stranded or double stranded. Double stranded DNA (eg. cDNA) is usually convenient for most applications.
  • the polynucleotide can be in the form of a vector, for example an expression vector.
  • polynucleotides of the present invention can be isolated polynucleotides or can be incorporated into expression or cloning vectors. Such vectors can be used to transfect or transform host cells and the host cells cultured in conventional culture media according to methods described in the art.
  • Suitable host cells include bacterial, yeast, mammalian and plant cells. Generally the host cell will be selected to be compatible with the vector used.
  • the present invention provides a polynucleotide comprising the nucleotide sequence as set out in SEQ ID No 1 or homologs thereof, and the protein expressed therefrom.
  • the present invention provides a polynucleotide comprising the nucleotide sequence as set out in SEQ ID No 3 or homologs thereof, and the protein expressed therefrom.
  • the present invention provides a protein comprising the amino acid sequence of SEQ ID Nos 2 or 4 or homologs thereof.
  • homologs with reference to a protein, we refer to a protein modified by deletion, substitution or addition of amino acids to have at least 80% homology, preferably 85% homology, to the amino acid sequence as set out in the sequence listing.
  • the homolog will have 90% or more homology, for example 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, to an amino acid sequence as set out in the sequence listing.
  • the present invention provides a protein expressed from a polynucleotide comprising the nucleotide sequence of SEQ ID Nos 1 or 3 or homologs of such polynucleotides.
  • the present invention provides a protein comprising the amino acid sequence as set out in SEQ ID No 2 or homologs thereof.
  • the present invention provides a protein comprising the amino acid sequence as set out in SEQ ID No 4 or homologs thereof.
  • protein is used herein to refer to a peptide and polypeptide and does not denote any particular size of the polymer.
  • a vector including such a recombinant polynucleotide is also encompassed by this invention.
  • the present invention provides a vector encoding tagatose epimerase from Agrobacterium tumefaciens.
  • the present invention provides a vector encoding fucose isomerase from E coli.
  • the same vector may encode both enzymes.
  • the present invention also provides a host cell containing a vector as indicated above.
  • the host cell expresses the enzyme (s) in the presence of a micro-organism able to convert L- galactose ' to L-ascorbic acid, such that the provision of L-sorbose results in the production of L-ascorbic acid.
  • the L-ascorbic acid produced by the method of the invention may be purified and used as required.
  • the L-ascorbic acid enriched biomass produced prior to purification of the L-ascorbic acid may itself find utility in specific applications.
  • the present invention provides a micro-organism (such as a microalgae, bacteria, plant cell culture or yeast) having an enhanced L- ascorbic acid content.
  • the micro- organism is able to express tagatose epimerase and/or fucose isomerase (optionally from a recombinant genetic construct) and to convert L- galactose to L-ascorbic acid.
  • tagatose epimerase and/or fucose isomerase optionally from a recombinant genetic construct
  • L- galactose to L-ascorbic acid.
  • Such micro-organisms are expected to be oxidatively stabilised and hence be of interest for the bioprocess industry. Additionally or alternatively the increased L- ascorbic acid content may give enhanced nutritional value when present in foodstuffs. Consequently the micro-organisms may find utility in the food or beverage industry.
  • Figure 1 shows a putative mechanism for L-asorbic acid production
  • Figure 2 gives the gene sequence of tagatose epimerase from Agrobacterium tumefaciens C58; underlined sections represent bases to which primers were generated in order to isolate and clone the gene.
  • Figure 3 is an SDS-PAGE gel showing the purification of recombinant tagatose epimerase; approximate MW (kDa) is shown on the left.
  • Lane 1 Crude protein extract
  • Lane 2 Desalted protein extract
  • Lane 3 Unbound protein
  • Lane 4 - 125 mM imidazole eluate
  • Lane 5 500 mM imidazole eluate.
  • Figure 4 gives the gene sequence of fucose isomerase from E coli Kl2; underlined sections represent bases to which primers were generated in order to isolate and clone the gene.
  • Figure 5 is an SDS-PAGE gel showing the purification of recombinant fucose isomerase; approximate MW (kDa) is shown on the left.
  • Lane 1 Crude protein extract
  • Lane 2 Desalted protein extract
  • Lane 3 Unbound protein
  • Lane 4 - 125 mM imidazole eluate
  • Lane 5 - 250 mM imidazole eluate
  • Lane 6 - 375 mM imidazole eluate
  • Figure 6 shows HPLC analyis of L-sorbose incubated with tagatose epimerase and fucose isomerase; 20% L- sorbose was incubated for 3 days at 30 0 C with equal amounts of recombinant TE and FI. At the end of incubation, an aliquot was taken and the reaction stopped by boiling. After removal of precipitated proteins by centrifugation, reaction products were analysed by HPLC; and
  • Figure 7 shows HPLC analysis of L-sorbose incubated with tagatose epimerase, fucose isomerase and yeast.
  • S. cerevisiae were cultured for 24 hours in a medium containing 2% L-sorbose that had been preincubated for 3 days with TE and FI.
  • the lower trace shows authentic AsA and the upper trace shows clarified yeast extract.
  • Insets shows the absorption spectra of the peaks indicated.
  • TE tagatose epimerase
  • FI fucose isomerase
  • the TE primers were 5' CAC CAT GAA ACA CGG CAT CTA TTA TTC TTA CTG GG 3' (forward) and 5' GCC ACC AAG AAC GAA GCG GGA G 3' (reverse) and primers used to facilitate the cloning of FI were 5' CAC CAT GAA AAA AAT CAG CTT ACC G 3' (forward) and 5' TTA ACG CTT GTA CAA CGG ACC G 3' (reverse) .
  • PCR products were generated and inserted into the entry vector following the manufacturer' s instructions.
  • E. coli TOPlO chemically competent cells were transformed with the vector and transformants selected on LB containing kanamycin (50 ⁇ g ml "1 ) .
  • E. coli DH5 ⁇ chemically competent cells were transformed with the reaction as described within the manual and transformants selected on LB containing ampicillin (100 ⁇ gml "1 ) . Clones were analysed by restriction digestion of plasmid DNA.
  • the TE and FI genes were functionally expressed in E. coli BL21 by transforming the chemically competent cells with plasmid DNA of the positive pDESTl7 clones.
  • the transformed cells were transferred to 1 1 of Overnight ExpressTM Instant TB Medium to maximixe the expression of the proteins.
  • the cultures were incubated at 3O 0 C on a shaker at 200 rpm until the cells reached stationary phase (-24-36 hrs) and then harvested by centrifugation (600Og, 10 minutes at 4°C) .
  • Cells were resuspended 2:1 (v/w) in either 50 mM Tris pH 7.5, 2 mM tris (2-carboxyethyl) phosphine HCl, 1 mM EDTA, 1 mM EGTA, 1 mM benzamidine hydrochloride, 0.5 mM PMSF (TE expressing cells) or 50 mM KPO 4 pH 7.6, 10 mM magnesium acetate, 1 mM EDTA, 10 mM ⁇ -mercaptoethanol (FI expressing cells) .
  • Cells were homogenised in a hand homogeniser and passed through a one shot cell disrupter at 20 kPsi (Constant Cell Disruption Systems, Northampshire, UK) .
  • Cell extracts were centrifuged (20000 g, 30 min, 1°C) and cleared lysates desalted through sephadex PD-IO columns (Amersham Biosciences, Buckinghamshire, UK) equilibrated with 2OmM NaPO 4 , 0.5 M NaCl, 20 mM Imidazole pH7.4. Desalted protein extracts were loaded onto 1 ml His-TrapTM columns (Amersham Biosciences) at 1 ml min "1 using a Kontron 420 pump.
  • Protein fractions collected from the purification of his ⁇ -TE and his 6 -FI were analysed using the NuPAGE ® electrophoresis Bis-Tris buffer system under denaturing conditions (Invitrogen, Paisley, UK) . Approximately equal concentrations of total protein per sample were prepared as detailed in the NuPAGE ® Technical guide (Invitrogen, Paisley, UK) . Samples and SeeBlue Plus2 Prestained Standard (Invitrogen, Paisley, UK) were loaded and resolved on a Pre-Cast 4-12% NuPAGE ® Novex Bis-Tris gel.
  • the gels were run in MES running buffer containing NuPAGE ® antioxidant in a Novex XCELL IITM Mini Cell under the recommended conditions (200 V, 35 min) using a Novex Powerease 500 power pack.
  • the gels were removed and stained according to the protocol for Bis-Tris gels provided with the Colloidal Blue Staining Kit (Invitrogen, Paisley, UK) .
  • Tagatose epimerase activity was estimated in a reaction mixture containing 50 mM Tris pH 7.5, 100 mM of the appropriate sugar and an appropriate volume of enzyme extract in a final reaction volume of 100 ⁇ l. The reaction was allowed to proceed at 37°C and was stopped by boiling for 1 min.
  • Precipitated protein was removed by centrifugation (1600Og, I 0 C, 5 min) and the supernatant was diluted 100 times with distilled H2O prior to analysis of reaction products by HPLC as described below.
  • Fucose isomerase activity was estimated in a reaction mixture containing 50 mM potassium phosphate buffer pH 7.6, 10 mM CH 3 COOMg, 25 ⁇ M MnCl 2 , 100 mM of the appropriate sugar and an appropriate volume of enzyme extract in a final reaction volume of 100 ⁇ l.
  • the reaction mixture was incubated at 37°C and the reaction stopped by boiling for 1 min. After removal of precipitated protein, the mixture was diluted 100 times with distilled H 2 O and the reaction products analysed by HPLC.
  • Metabolite Analysis was estimated in a reaction mixture containing 50 mM potassium phosphate buffer pH 7.6, 10 mM CH 3 COOMg, 25 ⁇ M MnCl 2 , 100 mM of the appropriate sugar and an appropriate volume of enzyme extract in a final
  • Sorbose, tagatose and galactose were identified and quantified using a carbopak PA-I analytical column (4 X 250 mm) fitted with a PA-I guard column (4 X 50 mm) (Dionex UK Ltd. , Surrey, UK) .
  • Mobile phase was 16 mM NaOH pumped at a flow rate of 1 ml min '1 using a GP40 quarternary gradient pump (Dionex) with the post-column addition of IM NaOH using a Dionex pneumatic controller set to 80 psi.
  • Sugars were detected and quantified by pulsed amperometry using an ED40 electrochemical detector with a quadruple waveform as recommended for the analysis of carbohydrates by Dionex.
  • the column was regenerated after each 30 min analysis by washing for 10 min with 200 mM NaOH followed by 10 min equilibration to the start conditions. Under these conditions, galactose, tagatose and sorbose were baseline separated with retention times of 14.5, 16.2 and 21.8 min respectively.
  • Organic acids were extracted from yeast by resuspending cells in a known volume of 5% (w/v) metaphosphoric acid containing 5 mM tris(2- carboxyethyl) phosphine hydrochloride. Cells were briefly vortexed then freeze-thawed three times and cell debris removed by centrifugation (1600Og, 5 min, 1°C) .
  • the gene was then cloned into pDEST17 (Invitrogen, Paisley, UK) under the control of the T7 promoter and the plasmid introduced into E. coli strain BL21.
  • Cells were grown to stationary phase in a medium suitable for gene induction (medium 2) and total protein extracted. Proteins were applied to a Ni + -containing metal chelate column and unbound protein collected. The column was washed with 125 mM imidazole in 20 ⁇ iM NaPO 4 , 0.5 M NaCl pH 7.4 and eluting proteins collected. Finally, strongly bound proteins were eluted with 500 mM imidazole in 20 mM NaPO 4 , 0.5 M NaCl pH 7.4.
  • Fig. 3 shows a strongly expressed protein of the predicted molecular weight ( ⁇ 33 kDa; Ishida et al . , 1997) that bound tightly to the Ni + column suggesting that it was modified with a his ⁇ -tag.
  • the purified enzyme (Fig. 3, lane 5) had a specific activity of 0.834 ⁇ mol mg protein ⁇ 1 h ⁇ 1 with D-tagatose as substrate and also catalysed the conversion of D-psicose to D-fructose and L-sorbose to L-tagatose. It is concluded that the gene cloned from A. tumefaciens is an active tagatose epimerase with the capacity to convert L- sorbose to L-tagatose.
  • the gene encoding fucose isomerase was cloned from E. coli strain K12, and the gene sequenced. Sequence data corresponding to the first 500 bases from the 5' and 3' ends suggested that the gene cloned corresponded to fucose isomerase as shown in Fig. 4. This sequence was cloned into pDESTl7 and the plasmid was then introduced into E. coli BL21. Soluble proteins were extracted and applied to a Ni + column and sequentially eluted with 125 mM, 250 mM, 375 mM and 500 mM imidazole in 20 mM NaPO 4 , 0.5 M NaCl pH 7.4.
  • Protein fractions were run on SDS-PAGE gels and stained with coomassie brilliant blue (Fig. 5) .
  • a strongly expressed protein band was present that eluted in all fractions and had a molecular weight corresponding to that of E. coli fucose isomerase ( ⁇ 65 kDa; Seeman and Schulz, 1997).
  • the fractions eluting in 250-500 mM imidazole (lanes 5-7) were combined and observed to catalyse the conversion of D-ribulose to D-arabinose with a specific activity of 265.5 ⁇ mol mg protein "1 h "1 .
  • the enzyme also catalysed the conversion of L- galactose to L-tagatose.
  • L-galactose was 5.9:1.0:2.0.
  • the culture was incubated at 3O 0 C and
  • FIG. 7 demonstrates that yeast cells cultured in
  • both enzymes were combined either in a buffer consisting of 50 mM tris pH 7.5 or in a dialysis sack in a medium suitable for the culture of yeast cells, they catalysed the conversion of L-sorbose to L- galactose.
  • yeast cells were added to such a reaction mixture they become competent for the synthesis of AsA.
  • the method would be suitable for the production of AsA using any other biological system that contain enzymes and their appropriate co-factors for the synthesis of AsA from L-galactose, such as plant cell cultures, filamentous fungi and microalgae.

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Abstract

Cette invention concerne un procédé servant à produire de l'acide L-ascorbique (vitamine C) dans lequel les enzymes tagatose épimérase et fucose isomérase sont incubées conjointement avec un micro-organisme capable de convertir le L-galactose en acide L-ascorbique. Ce micro-organisme peut être une levure ou un champignon. Un polypeptide recombiné comprenant la séquence codant la tagatose épimérase ou la fucose isomérase est également présenté, et cette invention concerne également un vecteur contenant un tel polynucléotide et des cellules hôtes transformées par ce polynucléotide.
PCT/GB2006/002203 2005-06-15 2006-06-15 Procede de production d'acide l-ascorbique WO2006134374A2 (fr)

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GB0512150A GB0512150D0 (en) 2005-06-15 2005-06-15 Production of L-ascorbic acid
GB0512150.4 2005-06-15

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
WO2014049373A1 (fr) * 2012-09-27 2014-04-03 Tate & Lyle Ingredients Americas Llc 3-épimérase
WO2014168697A1 (fr) * 2013-04-11 2014-10-16 California Institute Of Technology Conversion du glucose en sorbose
EP2749645A4 (fr) * 2011-08-24 2015-06-10 Cj Cheiljedang Corp Mutant de d-psicose 3-épimérase ayant une stabilité thermique améliorée, et production continue de d-psicose l'utilisant
WO2018116266A1 (fr) * 2016-12-23 2018-06-28 Petiva Private Ltd. Mutant de d-psicose 3-épimérase et ses utilisations
EP3684800A4 (fr) * 2017-09-22 2021-07-21 Modern Meadow, Inc. Souches de levure recombinées

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WO2004029267A1 (fr) * 2002-09-27 2004-04-08 Dsm Ip Assets B.V. Procede pour produire de l'acide l-ascorbique

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2749645A4 (fr) * 2011-08-24 2015-06-10 Cj Cheiljedang Corp Mutant de d-psicose 3-épimérase ayant une stabilité thermique améliorée, et production continue de d-psicose l'utilisant
EP3192867A1 (fr) * 2011-08-24 2017-07-19 CJ CheilJedang Corporation Mutant de d-psicose 3-épimérase ayant une stabilité thermique améliorée, et production continue de d-psicose l'utilisant
WO2014049373A1 (fr) * 2012-09-27 2014-04-03 Tate & Lyle Ingredients Americas Llc 3-épimérase
US9725707B2 (en) 2012-09-27 2017-08-08 Tate & Lyle Ingredients Americas Llc 3-epimerase
EP3336194A1 (fr) * 2012-09-27 2018-06-20 Tate & Lyle Ingredients Americas LLC 3-epimerase
US10294469B2 (en) 2012-09-27 2019-05-21 Tate & Lyle Ingredients Americas Llc 3-epimerase
US11859224B2 (en) 2012-09-27 2024-01-02 Tate & Lyle Solutions Usa Llc Methods for manufacturing a product using a 3-epimerase
WO2014168697A1 (fr) * 2013-04-11 2014-10-16 California Institute Of Technology Conversion du glucose en sorbose
US9255120B2 (en) 2013-04-11 2016-02-09 California Institute Of Technology Conversion of glucose to sorbose
WO2018116266A1 (fr) * 2016-12-23 2018-06-28 Petiva Private Ltd. Mutant de d-psicose 3-épimérase et ses utilisations
EP3684800A4 (fr) * 2017-09-22 2021-07-21 Modern Meadow, Inc. Souches de levure recombinées
US11384135B2 (en) 2017-09-22 2022-07-12 Modern Meadow, Inc. Recombinant yeast strains

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