LU501894B1 - Novel polypeptides having lactate dehydrogenase activity - Google Patents
Novel polypeptides having lactate dehydrogenase activity Download PDFInfo
- Publication number
- LU501894B1 LU501894B1 LU501894A LU501894A LU501894B1 LU 501894 B1 LU501894 B1 LU 501894B1 LU 501894 A LU501894 A LU 501894A LU 501894 A LU501894 A LU 501894A LU 501894 B1 LU501894 B1 LU 501894B1
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- Luxembourg
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- amino acid
- acid sequence
- seq
- dehydrogenase activity
- lactate dehydrogenase
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/56—Lactic acid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01028—D-Lactate dehydrogenase (1.1.1.28)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Abstract
The present invention relates to novel polypeptides having lactate dehydrogenase activity, and polynucleotides encoding said polypeptides. The present invention further relates to nucleic acid constructs and vectors encoding the polypeptides of the present invention as well as to recombinant host cells comprising (e.g., expressing) the polypeptides of the present invention. The present invention further provides methods for producing lactate (or lactic acid) employing such recombinant host cells.
Description
Novel polypeptides having lactate dehydrogenase activity
The present invention relates to novel polypeptides having lactate dehydrogenase activity, and polynucleotides encoding said polypeptides. The present invention further relates to nucleic acid constructs and vectors encoding the polypeptides of the present invention as well as to recombinant host cells comprising (e.g., expressing) the polypeptides of the present invention. The present invention further provides methods for producing lactate (or lactic acid) employing such recombinant host cells.
Lactic acid is a widely used building block for biodegradable polymer production. It is produced commercially by fermentation of carbohydrates by various bacteria, such as Bacillus coagulans. The carbohydrates, in particular C6 sugars, such as glucose are converted to lactate through an anaerobic or severely-oxygen limited process. The glucose is first imported into the host cell and metabolised to pyruvate, which is subsequently converted to lactate by the enzyme lactate dehydrogenase.
During fermentation, high lactate concentrations are produced, which is toxic to the production organism. To circumvent this problem, calcium hydroxide or calcium carbonate is used to regulate pH during the fermentation, which results in the formation of Ca-lactate salts. These are insoluble and precipitate during the fermentation, reducing the toxicity of the lactate. At the end of the process, the Ca-lactate is acidified with H,SO. to convert it to lactic acid, which is then further purified to the final product, producing gypsum (CaSQ.) as a by-product.
Two enantiomers of lactate can be produced by microorganisms, D-lactate, and L-lactate. The stereospecificity of the lactate is determined by the lactate dehydrogenase that converts pyruvate to lactate, and is generally organism specific. By replacing an L-lactate producing LDH with a D- lactate producing LDH through genetic methods, the final lactate form produced by the host can be controlled. L-lactic acid is currently the main commercially produced lactate form. Both lactic acid 1 enantiomers can be polymerized into polylactic acid (PLA) to form L-PLA and D-PLA, which can be mixed subsequently to form DL-PLA.
Bio-based products, such as lactic acid, can be produced from various substrates, such as carbohydrates and methanol. Carbohydrates have been the dominant substrate used for microbial chemical production due to their widespread availability. While carbohydrates may be a sustainable substrate in terms of atmospheric CO; release, they compete with food and feed chains. Because of the growing global population, carbohydrate-derived substrates will become less and less sustainable in the long term. On the other hand, bio-based methanol production from wood biomass or CO--sequestration does not interfere with food and feed production.
The present invention is based on the identification of novel polypeptides having lactate dehydrogenase activity derived from the methylotrophic bacteria, such as Methylobacillus flagellatus and Methylobacillus glycogenes.
The present invention thus provides in a first aspect an isolated polypeptide having lactate dehydrogenase activity and comprising an amino acid sequence which has at least 80 % sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8.
The present invention further provides an isolated polynucleotide which encoding a polypeptide of the present invention.
The present invention further provides a nucleic acid construct comprising a nucleotide sequence encoding a polypeptide of the present invention.
The present invention further provides an expression vector comprising a nucleotide sequence encoding a polypeptide of the present invention
The present invention further provides a recombinant host cell comprising (e.g. heterologously expressing) a polypeptide of the present invention.
The present invention further provides a method for the production of lactate (or lactic acid), preferably D-lactate (or D-lactic acid), comprising cultivating a recombinant host cell of the present invention under suitable culture conditions. 2
The present invention may be further summarized by the following items: 1. An isolated polypeptide having lactate dehydrogenase activity and comprising (or consisting of) an amino acid sequence which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: SEQ ID NO: 1, 2, 3, 4,5, 6, 7 or 8. 2. The isolated polypeptide according to item 1, comprising (or consisting of) the amino acid sequence of SEQ ID NO: SEQ ID NO: 1, 2, 3, 4,5, 6, 7 or 8. 3. The isolated polypeptide according to item 1, comprising (or consisting of) an amino acid sequence which has at least 80 % sequence identity with the amino acid sequence of SEQ ID
NO: 1. 4. The isolated polypeptide according to item 1, comprising (or consisting of) the amino acid sequence of SEQ ID NO: 1. 5. An isolated polynucleotide encoding a polypeptide having lactate dehydrogenase activity and comprising (or consisting of) an amino acid sequence which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: SEQ ID NO: 1, 2, 3, 4, 5,6, 7 or 8. 6. The isolated polynucleotide according to item 5, encoding a polypeptide having lactate dehydrogenase activity and comprising (or consisting of) an amino acid sequence which has at least 80 % sequence identity with the amino acid sequence of SEQ ID NO: 1. 7. The isolated polynucleotide according to item 6, encoding a polypeptide having lactate dehydrogenase activity and comprising (or consisting of) the amino acid sequence of SEQ ID
NO: 1. 8. A nucleic acid construct comprising a nucleotide sequence encoding a polypeptide a polypeptide having lactate dehydrogenase activity and comprising an amino acid sequence which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: SEQ
ID NO: 1, 2, 3, 4, 5,6,7 or 8. 9. The nucleic acid construct according to item 8, wherein the nucleotide sequence encodes a polypeptide having lactate dehydrogenase activity and comprising (or consisting of) an amino acid sequence which has at least 80 % sequence identity with the amino acid sequence of
SEQ ID NO: 1. 3
10. The nucleic acid construct according to item 9, wherein the nucleotide sequence encodes a polypeptide having lactate dehydrogenase activity and comprising (or consisting of) the amino acid sequence of SEQ ID NO: 1. 11. The nucleic acid construct according to any one of items 8 to 10, comprising one or more (e.g., several) regulatory sequences operably linked to the nucleotide sequence encoding the polypeptide having lactate dehydrogenase activity. 12. The nucleic acid construct according to any one of items 8 to 11, comprises at least one transcriptional unit comprising, from 5’ to 3’, a promoter that is functional in a host cell to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide having lactate dehydrogenase activity, and optionally a transcriptional terminator sequence. 13. An expression vector comprises at least one transcriptional unit comprising, from 5’ to 3’, a promoter that is functional in a host cell to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding a polypeptide having lactate dehydrogenase activity and comprising (or consisting of) an amino acid sequence which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: SEQ ID NO: 1, 2, 3,4,5,6,7 or 8, and optionally a transcriptional terminator sequence. 14. The expression vector according to item 13, wherein the nucleotide sequence encodes a polypeptide having lactate dehydrogenase activity and comprising (or consisting of) an amino acid sequence which has at least 80 % sequence identity with the amino acid sequence of
SEQ ID NO: 1. 15. The expression vector according to item 13, wherein the nucleotide sequence encodes a polypeptide having lactate dehydrogenase activity and comprising (or consisting of) the amino acid sequence of SEQ ID NO: 1. 16. The expression vector according to any one of items 13 to 15, wherein the vector further comprises one or more (e.g., several) selectable markers that permit easy selection of transformed, transfected or transduced a host cell. 17. The expression vector according to any one of items 13 to 16, wherein the vector further comprises an element(s) that permits integration of the vector into a host cell's genome or autonomous replication of the vector in the cell independent of the genome. 4
18. A recombinant host cell comprising a nucleic acid construct according to any one of items 8 to 12 or an expression vector according to any one of items 13 to 17. 19. The recombinant host cell according to item 18, wherein the recombinant host cell expresses the polypeptide having lactate dehydrogenase activity 20. The recombinant host cell according to item 18 or 19, wherein the polypeptide having lactate dehydrogenase activity is heterologous to the recombinant host cell. 21. The recombinant host cell according to any one of items 18 to 20, wherein the recombinant host cell is selected from the group consisting of bacteria, yeasts, fungi, algae and plant cells. 22. The recombinant host cell according to any one of items 18 to 21, wherein the recombinant host cell is a bacterium. 23. The recombinant host cell according to item 22, wherein the bacterium is a bacterium of the genus Bacillus, Escherichia, Corynebacterium, Lactococcus, Lactobacillus, Clostridium,
Geobarcillus, Methylobacillus, Methylobacterium, Methylorubrum, Streptococcus,
Pseudomonas, Streptomyces, Shigella, Acinetobacter, Citrobacter, Salmonella, Klebsiella,
Enterobacter, Erwinia, Kluyvera, Serratia, Cedecea, Morganella, Hafnia, Edwardsiella,
Providencia, Proteus, or Yersinia. 24. The recombinant host cell according to item 22, wherein the bacterium is a bacterium of the genus Bacillus. 25. The recombinant host cell according to item 24, wherein the bacterium is Bacillus subtilis. 26. The recombinant host cell according to item 22, wherein the bacterium is a bacterium of the genus Escherichia. 27. The recombinant host cell according to item 26, wherein the bacterium is Escherichia coli. 28. The recombinant host cell according to item 22, wherein the bacterium is a bacterium of the genus Corynebacterium. 29. The recombinant host cell according to item 28, wherein the bacterium is Corynebacterium glutamicum. 30. The recombinant host cell according to any one of items 18 to 22, which is not a mesophilic, methylotrophic bacterium. 5
31. The recombinant host cell according to any one of items 18 to 21, wherein the recombinant host cell is a yeast. 32. The recombinant host cell according to item 31, wherein the yeast is of the genus
Saccharomyces, Pichia, Schizosacharomyces, Zygosaccharomyces, Hansenula, Pachyosolen,
Kluyveromyces, Debaryomyces, Yarrowia, Candida, Cryptococcus, Komagataella, Lipomyces,
Rhodospiridium, Rhodotorula, or Trichosporon. 33. The recombinant host cell according to any one of items 18 to 21, wherein the recombinant host cell is a fungus. 34. The recombinant host cell according to item 33, wherein the fungus is a fungus of the genus
Aspergillus. 35. The recombinant host cell according to any one of items 18 to 21, wherein the recombinant host cell is an algae cell. 36. The recombinant host cell according to item 35, wherein the algae cells is an algae cell of the genus Haematococcus, Phaedactylum, Volvox or Dunaliella. 37. The recombinant host cell according to any one of items 18 to 21, wherein the recombinant host cell is a plant cell. 38. The recombinant host cell according to item 37, wherein the plant cell is selected from the group consisting of soybean, rapeseed, sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum, lettuce, rice, broccoli, cauliflower, cabbage, parsnips, melons, carrots, celery, parsley, tomatoes, potatoes, strawberries, peanuts, grapes, grass seed crops, sugar beets, sugar cane, beans, peas, rye, flax, hardwood trees, softwood trees, and forage grasses. 39. Use of the recombinant host cell according to any one of items 18 to 38 in the production of lactate (or lactic acid). 40. Method for the production of lactate (or lactic acid), comprising cultivating a recombinant host cell according to any one of items 18 to 38 under suitable culture conditions, and optionally collecting the lactate from the culture medium. 41. Method for the production of a polypeptide as defined in any one of items 1 to 4, comprising (a) cultivating a recombinant host cell of any one of items 18 to 38 under conditions 6 conducive for production of the polypeptide; and (b) recovering the polypeptide from the cell.
Figure 1: Lactate production from methanol by the M. flagellatus strain OCB 354 that overexpressed the M. flagellatus LDH gene (SEQ ID NO: 11, encoding SEQ ID NO: 1). The experiment was performed in shake flasks as a duplicate.
Figure 2: Lactate production from glucose by the E. coli strain that overexpressed the M. flagellatus
LDH gene (SEQ ID NO: 11, encoding SEQ ID NO: 1). The experiment was performed in shake flasks as a duplicate.
The present invention is now described in more detail below.
Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled artisan in the fields of biochemistry, genetics, and microbiology.
All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, and recombinant DNA, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000,
Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press);
Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid 7
Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames &
S. J. Higgins eds. 1984); Culture Of Animal Cells (R. |. Freshney, Alan R. Liss, Inc, 1987); Immobilized
Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc, New
York), specifically, Vols.154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory).
Polypeptide having lactate dehydrogenase activity
As mentioned above, the present invention is based on the identification of novel polypeptides having lactate dehydrogenase activity, preferably D-lactate dehydrogenase activity, derived from the methylotrophs Methylobacillus flagellatus and Methylobacillus glycogenes.
A “polypeptide having lactate dehydrogenase activity” is a polypeptide that catalyzes the reaction: lactate + NAD(+) <=> pyruvate + NADH. Preferably, a polypeptide of the present invention has at least D-lactate dehydrogenase activity. A polypeptide having D-lactate dehydrogenase activity (EC 1.1.1.28) is a polypeptide that catalyzes the reaction: (R)-lactate + NAD(+) <=> pyruvate + NADH.
The present invention thus provides in a first aspect an isolated polypeptide having lactate dehydrogenase activity and comprising an amino acid sequence which has least 80% sequence identity, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, with the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence which has least 84% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, 4,5, 6, 707 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence which has least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, 4,5, 6, 707 8. 8
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence which has least 86% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5,6, 7 or 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence which has least 87% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5,6, 7 or 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence which has least 88% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5,6, 7 or 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence which has least 89% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5,6, 7 or 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence which has least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5,6, 7 or 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence which has least 91% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5,6, 7 or 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence which has least 92% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5,6, 7 or 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence which has least 93% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5,6, 7 or 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence which has least 94% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5,6, 7 or 8. 9
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence which has least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5,6, 7 or 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence which has least 96% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5,6, 7 or 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence which has least 97% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5,6, 7 or 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence which has least 98% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5,6, 7 or 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence which has least 99% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5,6, 7 or 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) the amino acid sequence of SEQ ID NO: 1, 2, 3, 4,5, 6, 7 or 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with the amino acid sequence of SEQ ID NO: 1.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 84% sequence identity with the amino acid sequence of SEQ ID NO: 1. 10
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 93 % sequence identity with the amino acid sequence of SEQ ID NO: 1.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 1.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) the amino acid sequence of SEQ ID NO: 1.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity consists of the amino acid sequence of SEQ ID NO: 1.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with the amino acid sequence of SEQ ID NO: 2.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 84% sequence identity with the amino acid sequence of SEQ ID NO: 2. 11
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 2.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 2.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 93% sequence identity with the amino acid sequence of SEQ ID NO: 2.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 2.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 2.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) the amino acid sequence of SEQ ID NO: 2.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity consists of the amino acid sequence of SEQ ID NO: 2.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with the amino acid sequence of SEQ ID NO: 3.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 84% sequence identity with the amino acid sequence of SEQ ID NO: 3. 12
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 3.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 3.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 93% sequence identity with the amino acid sequence of SEQ ID NO: 3.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 3.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 3.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) the amino acid sequence of SEQ ID NO: 3.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity consists of the amino acid sequence of SEQ ID NO: 3.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with the amino acid sequence of SEQ ID NO: 4.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 84% sequence identity with the amino acid sequence of SEQ ID NO: 4. 13
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 85 % sequence identity with the amino acid sequence of SEQ ID NO: 4.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 93% sequence identity with the amino acid sequence of SEQ ID NO: 4.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 4.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) the amino acid sequence of SEQ ID NO: 4.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity consists of the amino acid sequence of SEQ ID NO: 4.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with the amino acid sequence of SEQ ID NO: 5.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 84% sequence identity with the amino acid sequence of SEQ ID NO: 5. 14
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 85 % sequence identity with the amino acid sequence of SEQ ID NO: 5.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 5.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 93% sequence identity with the amino acid sequence of SEQ ID NO: 5.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 5.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) the amino acid sequence of SEQ ID NO: 5.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity consists of the amino acid sequence of SEQ ID NO: 5.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with the amino acid sequence of SEQ ID NO: 6.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 84% sequence identity with the amino acid sequence of SEQ ID NO: 6. 15
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 85 % sequence identity with the amino acid sequence of SEQ ID NO: 6.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 6.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 93% sequence identity with the amino acid sequence of SEQ ID NO: 6.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 6.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 6.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) the amino acid sequence of SEQ ID NO: 6.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity consists of the amino acid sequence of SEQ ID NO: 6.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with the amino acid sequence of SEQ ID NO: 7.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 84% sequence identity with the amino acid sequence of SEQ ID NO: 7. 16
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 85 % sequence identity with the amino acid sequence of SEQ ID NO: 7.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 7.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 93% sequence identity with the amino acid sequence of SEQ ID NO: 7.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 7.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 7.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) the amino acid sequence of SEQ ID NO: 7.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity consists of the amino acid sequence of SEQ ID NO: 7.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with the amino acid sequence of SEQ ID NO: 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 84% sequence identity with the amino acid sequence of SEQ ID NO: 8. 17
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 85 % sequence identity with the amino acid sequence of SEQ ID NO: 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 93% sequence identity with the amino acid sequence of SEQ ID NO: 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity comprises (or consists of) the amino acid sequence of SEQ ID NO: 8.
According to some embodiments, the isolated polypeptide having lactate dehydrogenase activity consists of the amino acid sequence of SEQ ID NO: 8.
Also contemplated within the scope of the present invention are truncated variants of the polypeptides of the invention, which are characterized in that 1 to 20, such as 1 to 15, 1 to 10,1 to 5, or 1 to 3, amino acids have been deleted from the N- and/or C-terminus of the original polypeptide.
Techniques for determining lactate dehydrogenase activity are well known to the skilled person.
The lactate dehydrogenase activity may for instance be determined in accordance with the following method: 18
The cells expressing lactate dehydrogenase are centrifuged and lysed to release the intracellular contents using B-PER (Thermo Scientific) according to manufacturer instructions. Briefly, 4 mL B-
PER is mixed with 1 g wet cell biomass and incubated for 15 minutes at room temperature. After the cells have been disrupted, the lysate is clarified using centrifugation. The clear cell lysate is used for determination of lactate dehydrogenase activity. The enzymatic reaction is performed at 37°C.
The reaction contains 2.8 mL of 0.13 mM (B NADH) prepared in 100 mM Sodium phosphate buffer (pH 7.5) and 0.1 mL of 34 mM sodium pyruvate prepared in 100 mM Sodium phosphate buffer (pH 7.5). The components are mixed, and A340 is measured until constant. After stabilization, 0.1 mL cell free extract is added to the reaction, and mixed. The decrease in A340 is continuously measured for 5 minutes. The linear slope of the reaction is used to calculate the lactate dehydrogenase activity according to the formula U/ml =(AA340/min)*3*dilution factor/(6.22*0.1).
As shown in Example 4, the polypeptide isolated from M. flagellatus produces D-lactic acid as main product. However, some amounts of L-lactic acid was also produced, indicating thatthe polypeptide also has some L-lactate dehydrogenase activity. Accordingly, the polypeptide of the present invention may further have L-lactate dehydrogenase activity. A polypeptide having L-lactate dehydrogenase activity (EC 1.1.1.27) is a polypeptide that catalyzes the reaction: (S)-lactate +
NAD(+) <=> pyruvate + NADH.
Polynucleotides, nucleic acid constructs and expression vectors
The present invention also provides an isolated polynucleotide encoding a polypeptide as described herein. More specifically, the present invention provides an isolated polynucleotide encoding a polypeptide having lactate dehydrogenase activity as detailed above.
By way of example, the isolated polynucleotide may encode a polypeptide having lactate dehydrogenase activity (preferably D-lactate hydrogenase activity) and comprising an amino acid which has at least 80%%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with the amino acid sequence of SEQ ID NO: 1.
The present invention also provides a nucleic acid construct, such as DNA construct, comprising a nucleotide sequence encoding a polypeptide as described above. 19
By way of example, the DNA construct may comprise a nucleotide sequence encoding a polypeptide having lactate dehydrogenase activity (preferably D-lactate hydrogenase activity) and comprising an amino acid which has at least 80%%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with the amino acid sequence of SEQ ID NO: 1.
In order to facilitate expression of a polypeptide having lactate dehydrogenase activity in a host cell, the nucleic acid construct may comprise suitable regulatory elements such as a promoter that is functional in the host cell to cause the production of an mRNA molecule and that is operably linked to the nucleotide sequence encoding said polypeptide.
Thus, according to some embodiments, the nucleic acid construct comprises one or more (e.g., several) regulatory elements operably linked to the nucleotide sequence encoding the polypeptide having lactate dehydrogenase activity.
According to some embodiments, the nucleic acid construct comprises at least one transcriptional unit comprising, from 5’ to 3’, a promoter that is functional in a host cell to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide having lactate dehydrogenase activity, and optionally a transcriptional terminator sequence.
According to some embodiments, the nucleic acid construct is an expression cassette or a vector.
According to some embodiments, the nucleic acid construct is an expression cassette.
According to some embodiments, the nucleic acid construct is a vector, such as an expression vector.
Thus, the present invention provides an expression vector comprises at least one transcriptional unit comprising, from 5’ to 3’, a promoter that is functional in a host cell to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding a polypeptide having lactate dehydrogenase activity as described herein, and optionally a transcriptional terminator sequence.
By way of example, the expression vector may comprise a nucleotide sequence encoding a polypeptide having lactate dehydrogenase activity (preferably D-lactate hydrogenase activity) and 20 comprising an amino acid which has at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with the amino acid sequence of SEQ ID NO: 1.
The expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide of the present invention. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.
The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.
The vector may contain one or more (e.g., several) selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
Examples of bacterial selectable markers are Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB (phosphoribosyl- aminoimidazole synthase), amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate 21 reductase), pyrG (orotidine-5" -phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are
Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and a Streptomyces hygroscopicus bar gene. Preferred for use in a Trichoderma cell are adeA, adeB, amdS, hph, and pyrG genes.
The selectable marker may be a dual selectable marker system such as a hph-tk dual selectable marker system.
The vector may further contain an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo. Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMB1 permitting replication in Bacillus. Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and
CEN3, and the combination of ARS4 and CEN6. Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANS1 (WO 00/24883).
Promoters useful in accordance with the invention are any known promoters that are functional in a given host cell to cause the production of an mRNA molecule. Many such promoters are known to the skilled person. Such promoters include promoters normally associated with other genes, and/or promoters isolated from any bacteria, yeast, fungi, alga or plant cell. The use of promoters for protein expression is generally known to those of skilled in the art of molecular biology, for example, see Sambrook et al., Molecular cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. , 1989. The promoter employed may be inducible. The term “inducible” used in the context of a promoter means that the promoter only directs transcription of an operably linked nucleotide sequence if a stimulus is present, such as a change in temperature or the presence of a chemical substance (“chemical inducer”). As used herein, “chemical induction” according to the present invention refers to the physical application of a exogenous or endogenous 22 substance (incl. macromolecules, e.g., proteins or nucleic acids) to a host cell. This has the effect of causing the target promoter present in the host cell to increase the rate of transcription.
Alternatively, the promoter employed may be constitutive. The term “constitutive” used in the context of a promoter means that the promoter is capable of directing transcription of an operably linked nucleotide sequence in the absence of stimulus (such as heat shock, chemicals etc.).
Non-limiting examples of promoters functional in bacteria, such as Bacillus subtilis, Lactococcus lactis or Escherichia coli, include both constitutive and inducible promoters such as T7 promoter, the beta-lactamase and lactose promoter systems; alkaline phosphatase (phoA) promoter, a tryptophan (trp) promoter system, tetracycline promoter, lambda-phage promoter, ribosomal protein promoters; and hybrid promoters such as the tac promoter. Other bacterial and synthetic promoters are also suitable. Specific examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyl), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB),
Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis crylllA gene, E. coli lac operon, E. coli trc promoter, Streptomyces coelicolor agarase gene (dagA), and prokaryotic beta-lactamase gene, as well as the tac promoter. Further promoters are described in “Useful proteins from recombinant bacteria” in Gilbert et al., 1980, Scientific American 242: 74-94; and in Sambrook et al., 1989, supra.
Examples of tandem promoters are disclosed in WO 99/43835.
Non-limiting examples of promoters functional in yeast, such as Saccharomyces cerevisiae, include promoters derived from the gene encoding Saccharomyces cerevisiae enolase (ENO-1),
Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and
Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are the GAL10 promoters, TEF1 promoter, and pgkl promoter.
Non-limiting examples of promoters functional in fungi, such as Aspergillus Oryzae or Aspergillus niger, include promotors derived from the gene encoding Aspergillus oryzae TAKA amylase,
Aspergillus niger neutral a-amylase, Aspergillus niger acid stable a-amylase, Aspergillus niger or 23
Aspergillus awamsii glucoamylase (gluA), Aspergillus niger acetamidase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphatase isomerase, Rhizopus meihei aspartic proteinase, and Rhizopus meihei lipase.
Non-limiting examples of promoters functional in alga, such as Haematococcus pluvialis, include the CaMV35S promoter, the SV40 promoter, and promoter of the Chlamydomonas reinhardtii
RBCS2 gene and the promoter of the Volvox carteri ARS gene.
Non-limiting examples of promoters functional in plant cells include the Lactuca sative psbA promoter, the tabacco psbA promoter, the tobacco rrn16 PEP+NEP promoter, the CaMV 35S promoter, the 19S promoter, the tomate E8 promoter, the nos promoter, the Mac promoter, and the pet E promoter or the ACT1 promoter.
Besides a promoter, the nucleic acid construct or expression vector may further comprise at least one transcriptional terminator sequence, which refer to a nucleic acid sequence which causes transcription to cease (i.e. it defines the end of a transcriptional unit).
Transcriptional terminators useful in accordance with the invention are any known terminators that are functional in a given host cell to cause transcription to cease. Many such transcriptional terminator sequences are known to the skilled person. Such transcriptional terminators include transcriptional terminator sequences normally associated with genes found in any bacteria, yeast, fungi, alga or plant cell, and can be derived from such cells.
Transcriptional terminator sequences may include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3’ end of the mRNA precursor.
The nucleic acid construct or expression vector may further comprise at least one regulatory element selected from a 5’ untranslated region (5’UTR) and 3’ untranslated region (3’ UTR). Many such 5’ UTRs and 3’ UTRs derived from prokaryotes and eukaryotes are well known to the skilled person. Such regulatory elements include 5’ UTRs and 3’ UTRs normally associated with other genes, and/or 5’ UTRs and 3’ UTRs isolated from any bacteria, yeast, fungi, alga or plant cell. 24
If the host cell is a prokaryotic organism, the 5’ UTR usually contains a ribosome binding site (RBS), also known as the Shine Dalgarno sequence which is usually 3-10 base pairs upstream from the initiation codon. Meanwhile, if the host cell is a eukaryotic organism the 5’ UTR usually contains the
Kozak consensus sequence. A eukaryotic 5’ UTR may also contain cis-acting regulatory elements.
Host cells
The present invention also provides recombinant host cells, comprising a nucleic acid construct according to present invention.
According to some embodiments, the present invention provides a recombinant host cell comprising an expression vector according to the present invention.
According to some embodiments, the recombinant host cell expresses the polypeptide having lactate dehydrogenase activity.
According to some embodiments, the polypeptide having lactate dehydrogenase activity is heterologous to the recombinant host cell.
Generally, a recombinant host cell according to the invention has been genetically modified to comprise a nucleic acid construct or vector according to present invention, which means that said nucleic acid construct has been introduced in the host cell. Techniques for introducing exogenous nucleic acid molecules into the various host cells are well-known to those of skill in the art, and include transformation (e.g., heat shock or natural transformation), transfection, conjugation, electroporation, microinjection and microparticle bombardment.
A construct or vector comprising is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
Recombinant host cells in accordance with the invention can be produced from any suitable host organism, including single-celled or multicellular microorganisms such as bacteria, yeast, fungi, 25 algae and plant, and higher eukaryotic organisms including nematodes, insects, reptiles, birds, amphibians and mammals.
According to some embodiments, a recombinant host cells in accordance with the invention is selected from the group consisting of bacteria, yeast, fungi, algae and plant.
According to some embodiments, a recombinant host cells in accordance with the invention is selected from the group consisting of bacteria, yeast, fungi, and algae.
According to some embodiments, a recombinant host cells in accordance with the invention is selected from the group consisting of bacteria, yeast and fungi.
According to some embodiments, a recombinant host cells in accordance with the invention is selected from the group consisting of bacteria and yeast.
According to some embodiments, a recombinant host cells in accordance with the invention is not a plant cell.
Bacterial host cells are selected from Gram-positive and Gram-negative bacteria. Non-limiting examples for Gram-negative bacterial host cells include species from the genera Escherichia,
Methylobacillus, Methylobacterium, Erwinia, Klebsiella and Citrobacter. Non-limiting examples of
Gram-positive bacterial host cells include species from the genera Bacillus, Lactococcus,
Lactobacillus, Clostridium, Corynebacterium, Streptomyces, Streptococcus, and Cellulomonas.
According to certain embodiment, the recombinant host cell is a bacterium of the family selected from the group consisting of Enterobacteriaceae, Bacillaceae, Lactobacillaceae, Methylophilaceae,
Methylobacteriaceae and Corynebacteriaceae. According to some embodiments, the recombinant host cell is a bacterium of the family Enterobacteriaceae. According to some embodiments, the recombinant host cell is a bacterium of the family Bacillaceae. According to some embodiments, the recombinant host cell is a bacterium of the family Corynebacteriaceae.
According to certain embodiments, the recombinant host cell is a bacterium, which may be a bacterium of the genus Escherichia, Bacillus, Lactococcus, Lactobacillus, Clostridium,
Corynebacterium, Methylobacillus, Methylobacterium, Methylorubrum, Geobacillus,
Thermoanaerobacterium, Streptococcus, Pseudomonas, Streptomyces, Shigella, Acinetobacter, 26
Citrobacter, Salmonella, Klebsiella, Enterobacter, Erwinia, Kluyvera, Serratia, Cedecea, Morganella,
Hafnia, Edwardsiella, Providencia, Proteus, or Yersinia.
According to some embodiments, the recombinant host cell is a bacterium of the genus Bacillus.
Non-limiting examples of a bacteria of the genus Bacillus are Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus mojavensis. According to some embodiments, the recombinant host cell is Bacillus subtilis.
According to some embodiments, the recombinant host cell is a bacterium of the genus
Lactococcus. A non-limiting example of a bacterium of the genus Lactococcus is Lactococcus lactis.
According to some embodiments, the recombinant host cell is Lactococcus lactis.
According to some embodiments, the recombinant host cell is a bacterium of the genus
Corynebacterium. A non-limiting example of a bacterium of the genus Corynebacterium is
Corynebacterium glutamicum. According to some embodiments, the recombinant host cell is
Corynebacterium glutamicum.
According to some embodiments, the recombinant host cell is a bacterium of the genus Escherichia.
A non-limiting example of a bacterium of the genus Escherichia is Escherichia coli. According to some embodiments, the recombinant host cell is Escherichia coli.
According to some embodiments, the recombinant host cell is a bacterium of genus
Methylobacillus. Non-limiting examples of a bacterium of the genus Methylobacillus are
Methylobacillus flagellatus, Methylobacillus glycogenes, Methylobacillus pratensis, Methylobacillus rhizosphaerae, Methylobacillus gramineus, Methylobacillus arboreus, Methylobacillus caricics,
Methylobacillus methilovorans, and Methylobacillus sp. According to some embodiments, the recombinant host cell is Methylobacillus flagellutes. According to some embodiments, the recombinant host cell is Methylobacillus glycogenes. According to some embodiments, the recombinant host cell is Methylobacillus rhizosphaerae.
According to some embodiments, the recombinant host cell is a bacterium of the genus
Methylobacterium. Non-limiting examples of a bacterium of the genus Methylobacterium are
Methylobacterium extorquens and Methylobacterium organophilum. According to some embodiments, the recombinant host cell is Methylobacterium extorquens. According to some embodiments, the recombinant host cell is Methylobacterium organophilum. 27
According to some embodiments, the recombinant host cell is a bacterium of the genus
Methylorubrum. A non-limiting example of a bacterium of the genus Methylorubrum is
Methylorubrum extorquens. According to some embodiments, the recombinant host cell is
Methylorubrum extorquens.
According to some embodiments, the recombinant host cell is not a methylotrophic bacterium.
According to some embodiments, the recombinant host cell is not a methylotrophic bacterium which belongs to the family Methylophilaceae or Methylobacteriaceae. According to some embodiments, the recombinant host cell is not a methylotrophic bacterium which belongs to the genus Methylobacillus, Methylobacterium or Methylorubrum. According to some embodiments, the recombinant host cell is not a methylotrophic bacterium selected from Methylobacillus flagellatus, Methylobacillus glycogenes, Methylobacillus pratensis, Methylobacillus rhizosphaerae,
Methylobacillus gramineus, Methylobacillus arboreus, Methylobacillus caricics, Methylobacillus methilovorans, Methylobacillus sp, Methylobacterium extorquens, Methylobacterium organophilum and Methylorubrum extorquens.
Yeast host cells may be derived from e.g., Saccharomyces, Pichia, Schizosacharomyces,
Zygosaccharomyces, Hansenula, Pachyosolen, Kluyveromyces, Debaryomyces, Yarrowia, Candida,
Cryptococcus, Komagataella, Lipomyces, Rhodospiridium, Rhodotorula, or Trichosporon.
According to some embodiments, the recombinant host cell is a yeast, which may be a yeast is of the genus Saccharomyces, Pichia, Schizosacharomyces, Zygosaccharomyces, Hansenula,
Pachyosolen, Kluyveromyces, Debaryomyces, Yarrowia, Candida, Cryptococcus, Komagataella,
Lipomyces, Rhodospiridium, Rhodotorula, or Trichosporon.
According to some embodiments, the recombinant host cell is a yeast of the genus Saccharomyces.
A non-limiting example of a yeast of the genus Saccharomyces is Saccharomyces cerevisiae.
According to some embodiments, the recombinant host cell is Saccharomyces cerevisiae.
According to some embodiments, the recombinant host cell is a yeast of the genus Pichia. Non- limiting example of a yeast of the genus Pichia are Pichia pastoris and Pichia kudriavzevii. According some embodiments, the recombinant host cell is Pichia pastoris. According to some embodiments, the recombinant host cell is Pichia kudriavzevii. 28
According to some embodiments, the recombinant host cell is a yeast of the genus Yarrowia. A non- limiting example of a yeast of the genus Yarrowia is Yarrowia lipolytica. According to some embodiments, the recombinant host cell is Yarrowia lipolytica.
Fungi host cells may be derived from, e.g., Aspergillus.
According to some embodiments, the recombinant host cell is a fungus, such as a fungi of the genus
Aspergillus. Non-limiting examples of a fungus of the genus Aspergillus are Aspergillus Oryzae,
Aspergillus niger or Aspergillus awamsii. According to some embodiments, the recombinant host cell is Aspergillus Oryzae. According to some embodiments, the recombinant host cell is Aspergillus niger. According to some embodiments, the recombinant host cell is Aspergillus awamsii.
Algae host cells may be derived from, e.g., Chlamydomonas, Haematococcus, Phaedactylum, Volvox or Dunadliella.
According to some embodiments, the recombinant host cell is an alga, which may be an algae of the genus Chlamydomonas, Haematococcus, Phaedactylum, Volvox or Dunaliella.
According to some embodiments, the recombinant host cell is an alga cell of the genus
Chlamydomonas. A non-limiting example of an alga of the genus Chlamydomonas is
Chlamydomonas reinhardtii.
According to some embodiments, the recombinant host cell is an alga cell of the genus
Haematococcus. A non-limiting example of an alga of the genus Haematococcus is Haematococcus pluvialis.
According to some embodiments, the recombinant host cell is an alga cell of the genus
Phaedactylum. A non-limiting example of an alga of the genus Phaedactylum is Phaedactylum tricornatum.
A plant host cell may be derived from, e.g., soybean, rapeseed, sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum, lettuce, rice, broccoli, cauliflower, cabbage, parsnips, melons, carrots, celery, parsley, tomatoes, potatoes, strawberries, peanuts, grapes, grass seed crops, sugar beets, sugar cane, beans, peas, rye, flax, hardwood trees, softwood trees, and forage grasses. 29
According to some embodiments, the recombinant host cell is a plant cell, such as a plant cell selected from the group consisting of soybean, rapeseed, sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum, lettuce, rice, broccoli, cauliflower, cabbage, parsnips, melons, carrots, celery, parsley, tomatoes, potatoes, strawberries, peanuts, grapes, grass seed crops, sugar beets, sugar cane, beans, peas, rye, flax, hardwood trees, softwood trees, and forage grasses.
Methods and uses
The present invention also provides a method of producing a polypeptide of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide from the host cell.
The present invention also provides a method for producing lactate, such as D-lactate, comprising cultivating a recombinant host cell according to the invention under suitable culture conditions.
The method may further comprise collecting lactate from the culture medium.
The culture medium employed may be any conventional medium suitable for culturing a host cell in question, and may be composed according to the principles of the prior art. The medium will usually contain all nutrients necessary for the growth and survival of the respective host cell, such as carbon and nitrogen sources and other inorganic salts. Suitable media, e.g. minimal or complex media, are available from commercial suppliers, or may be prepared according to published receipts, e.g. the American Type Culture Collection (ATCC) Catalogue of strains. Non-limiting standard medium well known to the skilled person include Luria Bertani (LB) broth, Sabouraud
Dextrose (SD) broth, MS broth, Yeast Peptone Dextrose, BMMY, GMMY, or Yeast Malt Extract (YM) broth, which are all commercially available. A non-limiting example of suitable media for culturing bacterial cells, such as E. coli cells, including minimal media and rich media such as Luria Broth (LB),
M9 media, M17 media, SA media, MOPS media, Terrific Broth, YT and others. Suitable media for culturing eukaryotic cells, such as yeast cells, are RPMI 1640, MEM, DMEM, all of which may be supplemented with serum and/or growth factors as required by the particular host cell being cultured. The medium for culturing eukaryotic cells may also be any kind of minimal media such as
Yeast minimal media.
The carbon source may be any suitable carbon substrate known in the art, and in particularly any carbon substrate commonly used in the cultivation of microorganisms and/ or fermentation. Non- limiting examples of suitable fermentable carbon substates include carbohydrates (e.g., C5 sugars such as arabinose or xylose, or C6 sugars such as glucose), glycerol, glycerine, acetate, dihydroxyacetone, one-carbon source, methanol, methane, oils, animal fats, animal oils, plant oils, fatty acids, lipids, phospholipids, glycerolipids, monoglycerides, diglycerides, triglycerides, renewable carbon sources, polypeptides (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, peptone, casaminoacids or any combination of two or more of the foregoing.
As the nitrogen source, various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean- hydrolysate, and digested fermentative microorganism can be used. As minerals, potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like can be used.
Suitably, the host cell is cultivated under suitable conditions for the production of the desired product. Suitable conditions for culturing the respective host cell are well known to the skilled person. Typically, the recombinant host cell is cultured at a temperature ranging from about 20 to about 60°C, such as from about 20 to about 45°C, such as from about 30 to about 38°C, such as at about 37°C. The cultivation can be preferably performed under aerobic conditions, such as by a shaking culture, by a stirring culture or in a bioreactor with aeration, at a temperature of about 20 to about 45 °C, such as about 30 to 38 °C, preferably at about 37°C. The pH of the culture is usually above 5, such as in a range from about 6 to about 8, preferably from about 6.5 to about 7.5, more preferably from about 6.8 to about 7.2. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. The cultivation may be carried out for a period in the range from 10 to 70 h, preferably in a range from 24 to 60 h, more preferably in a range from 36 to 50 h.
After cultivation, solids such as cells can be removed from the culture medium by centrifugation or membrane filtration. The lactate can be collected by conventional method for isolation and purification chemical compounds from a medium. Well-known purification procedures include, but are not limited to, centrifugation or filtration, precipitation, ion exchange, chromatographic 31 methods such as e.g. ion exchange chromatography or gel filtration chromatography, and crystallization methods. The method may further comprise collecting lactate from the culture medium. Similarly, the polypeptide can be recovered from the host cell by conventional method for isolation and purification of polypeptides. Well-known purification procedures include, but are not limited to, centrifugation or filtration, precipitation, ion exchange, chromatographic methods such as e.g. ion exchange chromatography or gel filtration chromatography, and crystallization methods.
The present invention also provides the of a recombinant host cell according to present invention in the production of lactate (or lactic acid), preferably D-lactate (or D-lactic acid).
The present invention also provides lactate (or lactic acid) obtainable by a method as detailed above.
Certain other definitions “Polypeptide" and "protein" are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.).
Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids. "Nucleic acid" or "polynucleotide" are used interchangeably herein to denote a polymer of at least two nucleic acid monomer units or bases (e.g., adenine, cytosine, guanine, thymine) covalently linked by a phosphodiester bond, regardless of length or base modification. "Recombinant" or "non-naturally occurring" when used with reference to, e.g., a host cell, nucleic acid, or polypeptide, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature, or is identical thereto but produced or derived from synthetic materials and/or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant bacterial cells expressing genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise expressed at a different level. "Heterologous" or "exogenous" as used herein in the context of a gene or nucleic acid molecule refer to a gene or nucleic acid molecule (i.e. DNA or RNA molecule) that does not occur naturally as part of the genome of the host cell in which it is present or which is found in a location or locations 32 in the genome that differ from that in which it occurs in nature. Thus, a “heterologous” or “exogenous” gene or nucleic acid molecule is not endogenous to the host cell and has been exogenously introduced into the host cell. A “heterologous” gene or nucleic acid molecule DNA molecule may be from a different organism, a different species, a different genus or a different kingdom, as the host DNA. “Heterologous” as used herein in the context of a polypeptide means that a polypeptide is normally not found in or made (i.e. expressed) by the host cell but derived from a different organism, a different species, a different genus or a different kingdom.
As used herein, the term "ortholog" or “orthologs” refers to genes, nucleic acid molecules encoded thereby, i.e., mRNA, or proteins encoded thereby that are derived from a common ancestor gene but are present in different species. "Expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
As used herein, "regulatory element" refers to a nucleic acid sequence that affects the expression of a coding sequence. Regulatory elements are known in the art and include, but are not limited to, promoters, enhancers, transcription terminators, polyadenylation sites, matrix attachment regions and/or other elements that regulate expression of a coding sequence. Each regulatory element may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native or foreign to each other. “Substitution” or “substituted” refers to modification of the polypeptide by replacing one amino acid residue with another, for instance the replacement of an Serine residue with a Glycine or
Alanine residue in a polypeptide sequence is an amino acid substitution. When used with reference to a polynucleotide, “substitution” or “substituted” refers to modification of the polynucleotide by replacing one nucleotide with another, for instance the replacement of a cytosine with a thymine in a polynucleotide sequence is a nucleotide substitution. "Conservative substitution”, when used with reference to a polypeptide, refers to a substitution of an amino acid residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar class 33 of amino acids. By way of example and not limitation, an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid, e.g., alanine, valine, leucine, and isoleucine; an amino acid with hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain, e.g., serine and threonine; an amino acid having an aromatic side chain is substituted with another amino acid having an aromatic side chain, e.g., phenylalanine, tyrosine, tryptophan, and histidine; an amino acid with a basic side chain is substituted with another amino acid with a basic side chain, e.g., lysine and arginine; an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain, e.g., aspartic acid or glutamic acid; and a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively. "Non-conservative substitution”, when used with reference to a polypeptide, refers to a substitution of an amino acid in a polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., serine for glycine), (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.
As used herein, "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded nucleic acid loop into which additional nucleic acid segments can be ligated. Certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". Certain other vectors are capable of facilitating the insertion of an exogenous nucleic acid molecule into a genome of a bacterium. Such vectors are referred to herein as "transformation vectors". In general, vectors of utility in recombinant nucleic acid techniques are often in the form of plasmids.
In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of a vector. Large numbers of suitable vectors are known to those of skill in the art and commercially available. 34
As used herein, "promoter" refers to a sequence of DNA, usually upstream (5') of the coding region of a structural gene, which controls the expression of the coding region by providing recognition and binding sites for RNA polymerase and other factors which may be required for initiation of transcription. The selection of the promoter will depend upon the nucleic acid sequence of interest.
A suitable “promoter” is generally one which is capable of supporting the initiation of transcription in a host cell of the invention, causing the production of an mRNA molecule.
As used herein, a “transcriptional terminator sequence” is a nucleic acid sequence that stops transcription.
As used herein, "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory element "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory element. A promoter sequence is "operably-linked" to a coding sequence when it is in sufficient proximity to the transcription start site of a gene to regulate transcription of the coding sequence. A transcriptional terminator sequence is considered “operably linked” to a nucleotide sequence if it can stop transcription of the sequence to which it is linked.
The term “isolated” means that a substance, such as polypeptide or polynucleotide as described herein, is in a form or environment that does not occur in nature. For example, it may mean that a polypeptide or polynucleotide as described may have been at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature.
Alternatively, it may mean that a polypeptide or polynucleotide as described herein has been prepared by chemical synthesis. "Percentage of sequence identity," "% sequence identity" and "percent identity" refers to sequence identity between a nucleotide sequence and a reference nucleotide sequence or between an amino acid sequence and a reference amino acid sequence. Sequence identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. A degree of identity between nucleotide or amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at positions shared by the nucleotide or amino acid sequences, respectively. Various alignment algorithms and/or programs may be used to calculate the identity between two sequences, including FASTA or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin,
Madison, Wis.), and can be used with default settings. "Reference sequence" or “reference amino acid sequence” refers to a defined sequence to which another sequence is compared. In the context of the present invention a reference amino acid sequence may, for example, be an amino acid sequence set forth in SEQ ID NO: 1.
As used herein, the term "about" means plus or minus 10% of the numerical value of the number with which it is being used.
Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and sub ranges within a numerical limit or range are specifically included as if explicitly written out.
As used herein, the indefinite articles "a" and "an" mean "at least one" or "one or more" unless the context clearly dictates otherwise.
As used herein, the terms "comprising", "including", "having" and grammatical variants thereof are to be taken as specifying the stated features, steps or components but do not preclude the addition of one or more additional features, steps, components or groups thereof.
Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.
Example 1: Bioinformatic analysis
While investigating the potential of Methylobacillus flagellatus for use in industrial biotechnology, we noticed it produced significantly higher titers of lactic acid under certain bioreactor fermentation conditions compared to other common overflow products such as acetate. This was unexpected because the published Methylobacillus flagellatus genome (Chistoserdova, Ludmila, et al. "Genome of Methylobacillus flagellatus, molecular basis for obligate methylotrophy, and polyphyletic origin of methylotrophy." Journal of bacteriology 189.11 (2007): 4020-4027.) 36 contained no annotation for a lactate dehydrogenase enzyme. To identify which gene was responsible for lactic acid production the amino acid sequences of two well-characterized lactate dehydrogenase genes from Escherichia coli, ldhA (SEQ ID NO: 9) and IldD (SEQ. ID NO: 10), were used to perform a BLAST search against the Methylobacillus flagellatus genome. The search resulted in only three hits, summarized in Table 1. The hit described as “2-hydroxyacid dehydrogenase” with the locus tag Mfla_0399 (SEQ ID NO: 11, encoding SEQ ID NO: 1) was deemed as the only plausible candidate.
Table 1: Summary of BLAST search of E. coli lactate dehydrogenases against the Methylobacillus flagellatus genome.
Query Hit description Query Coverage Identity
IdhA 2-hydroxyacid dehydrogenase 100% 51%
IdhA phosphoglycerate dehydrogenase 70% 30%
IldD FMN-binding glutamate synthase family protein 28% 34%
The amino acid sequence of this gene was used to perform a BLAST search against all known genomes in the Methylobacillus genus, resulting in a similar “2-hydroxyacid dehydrogenase” hit in each genome (SEQ ID NO: 2-8) with a high degree of similarity. A protein alignment using the Clustal
Omega service was performed on the identified sequences, revealing a high degree of conservation across all Methylobacilli strains, with several 100% conserved regions and an overall similarity of over 84% (data not shown).
Example 2: Expression of novel identified enzyme and production of lactate in methylotrophs
To test whether the identified gene (Mfla_0399, SEQ ID NO: 11, encoding SEQ ID NO: 1) is responsible for lactate production by Methylobacillus flagellatus, we prepared constructs that enable controlled gene expression in M. flagellatus and other Methylobacilli. The gene was amplified from the genome of M. flagellatus by PCR and cloned into an expression vector under the control of the IPTG-inducible lacO/trc promotor. The prepared plasmids were checked by sequencing and transformed into M. flagellatus via electroporation to create strain OCB 354.
To test lactate production, the transformants were cultivated in 250 mL shake flasks in a mineral medium mineral medium containing methanol, KH:PO4, Na;HPO4, MgSO4, NH4SO4, and trace elements. The shake flasks were inoculated with 5 mL liquid overnight culture. After 4 h of growth, 37
0.5 g/L IPTG was added to induce gene expression. The cultures were sampled after 24 h of growth.
Lactate was measured by HPLC.
The strain OCB 354, expressing the Mfla_0399 gene (SEQ ID NO: 11) produced 2 g/L lactate from methanol after 24 h of cultivation (Figure 1). Under the same conditions, the control strain that did not express Mfla_0399 did not produce detectable amounts of lactic acid. This confirmed that
Mfla_0399 is responsible for converting methanol into lactate in M. flagellatus.
Example 3: Expression of Mfla_0399 in Escherichia coli
We tested whether Mfla_0399 could also induce lactate production in Escherichia coli. The
Mfla_0399 gene was amplified from the genome of M. flagellatus by PCR and cloned into an expression vector under the control of the IPTG-inducible lacO/trc promotor. The prepared plasmids were checked by sequencing and transformed into E. coli BW25113 where lactate production was disrupted. This ensured that any lactate production was only due to the activity of
Mfla_0399.
To test lactate production, the transformants were cultivated in 250 mL shake flasks in a medium containing glucose, KH2PO4, Na:HPO4, MgSO4, NH4S04, yeast extract, and trace elements. The shake flasks were inoculated with 5 mL liquid overnight culture. After 4 h of growth, 0.5 g/L IPTG was added to induce gene expression. The cultures were sampled after 24 h of growth. Lactate was measured by HPLC. The transformed E. coli strain produced approx. 1.25 g/L lactate (Figure 2).
Example 4: Determination of the lactate chirality
After confirming that Mfla_0399 is responsible for lactate formation in M. flagellatus, we sought to determine the chirality of the lactate produced. We tested the clarified cell broth at the end of the fermentation by using the enzymatic L-lactic acid and D-lactic acid specific kits from Megazyme (K-
LATE and K-DATE, respectively). The assays were performed according to the manufacturer instructions. We confirmed that D-lactic acid was the main product of the Mfla_0340 gene (Table 2) and constituted more than 98% of the total produced lactic acid. This indicates that Mfla_0399 (SEQ ID NO: 1) is a D-lactic acid producing lactate dehydrogenase. 38
Table 2: Chirality determination of the lactic acid produced by Mfla_0399.
D-lactate | L-lactate 98.3% 39
Claims (18)
1. An isolated polypeptide having lactate dehydrogenase activity and comprising (or consisting of) an amino acid sequence which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8.
2. The isolated polypeptide according to claim 1, comprising (or consisting of) the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8.
3. The isolated polypeptide according to claim 1, comprising (or consisting of) an amino acid sequence which has at least 80 % sequence identity with the amino acid sequence of SEQ ID NO: 1.
4. The isolated polypeptide according to claim 1, comprising (or consisting of) the amino acid sequence of SEQ ID NO: 1.
5. An isolated polynucleotide encoding a polypeptide having lactate dehydrogenase activity and comprising (or consisting of) an amino acid which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3, 4,5, 6, 7 or 8.
6. The isolated polynucleotide according to claim 5, encoding a polypeptide having lactate dehydrogenase activity and comprising an amino acid sequence which has at least 80 % sequence identity with the amino acid sequence of SEQ ID NO: 1.
7. The isolated polynucleotide according to claim 6, encoding a polypeptide having lactate dehydrogenase activity and comprising the amino acid sequence of SEQ ID NO: 1.
8. A nucleic acid construct comprising a nucleotide sequence encoding a polypeptide a polypeptide having lactate dehydrogenase activity and comprising an amino acid sequence which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3,4,5,6,7or8.
9. The nucleic acid construct according to claim 8, wherein the nucleotide sequence encodes a polypeptide having lactate dehydrogenase activity and comprising an amino acid sequence which has at least 80 % sequence identity with the amino acid sequence of SEQ ID NO: 1.
10. The nucleic acid construct according to claim 9, wherein the nucleotide sequence encodes a polypeptide having lactate dehydrogenase activity and comprising the amino acid sequence of SEQ ID NO: 1.
11. An expression vector comprises at least one transcriptional unit comprising, from 5’ to 3’, a promoter that is functional in a host cell to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding a polypeptide having lactate dehydrogenase activity and comprising an amino acid sequence which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: SEQ ID NO: 1, 2,3,4,5,6,7 or 8, and optionally a transcriptional terminator sequence.
12. The expression vector according to claim 11, wherein the nucleotide sequence encodes a polypeptide having lactate dehydrogenase activity and comprising an amino acid sequence which has at least 80 % sequence identity with the amino acid sequence of SEQ ID NO: 1.
13. The expression vector according to claim 13, wherein the nucleotide sequence encodes a polypeptide having lactate dehydrogenase activity and comprising the amino acid sequence of SEQ ID NO: 1.
14. A recombinant host cell comprising a nucleic acid construct according to any one of claims 8 to 10 or an expression vector according to any one of items 11 to 13.
15. The recombinant host cell according to claim 14, wherein the recombinant host cell is selected from the group consisting of bacteria, yeasts, fungi, algae and plant cells.
16. The recombinant host cell according to claim 14, wherein the recombinant host cell is a bacterium.
17. Use of the recombinant host cell according to any one of claims 14 to 16 in the production of lactate (or lactic acid).
18. Method for the production of lactate (or lactic acid), comprising cultivating a recombinant host cell according to any one of claims 14 to 16 under suitable culture conditions, and optionally collecting the lactate from the culture medium. 41
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