CA2767259A1 - Engineered microorganisms with enhanced fermentation activity - Google Patents

Engineered microorganisms with enhanced fermentation activity Download PDF

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Publication number
CA2767259A1
CA2767259A1 CA2767259A CA2767259A CA2767259A1 CA 2767259 A1 CA2767259 A1 CA 2767259A1 CA 2767259 A CA2767259 A CA 2767259A CA 2767259 A CA2767259 A CA 2767259A CA 2767259 A1 CA2767259 A1 CA 2767259A1
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CA
Canada
Prior art keywords
composition
enzyme
microbe
yeast
spp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
CA2767259A
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French (fr)
Inventor
Stephen Picataggio
Kirsty Anne Lily Salmon
Jose Miguel Laplaza
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
EIDP Inc
Original Assignee
Verdezyne Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Verdezyne Inc filed Critical Verdezyne Inc
Publication of CA2767259A1 publication Critical patent/CA2767259A1/en
Abandoned legal-status Critical Current

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Classifications

    • 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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • 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
    • C12N9/88—Lyases (4.)
    • 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
    • C12N9/90—Isomerases (5.)
    • C12N9/92—Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Abstract

Provided herein are genetically modified microorganisms that have enhanced fermentation activity, and methods for making and using such microorganisms.

Claims (52)

1. A composition comprising an engineered yeast that includes an alteration that adds or increases a phosphogluconate dehydratase activity, a 2-keto-3-deoxygluconate-6-phosphate aldolase activity and a xylose isomerase activity.
2. The composition of claim 1, wherein the yeast is a Saccharomyces spp.
yeast.
3. The composition of claim 2, wherein the yeast is a Saccharomyces cerevisiae yeast strain.
4. The composition of any one of claims 1 to 3 that includes heterologous polynucleotides that encode a phosphogluconate dehydratase enzyme, a 2-keto-3-deoxygluconate-6-phosphate aldolase enzyme and a xylose isomerase enzyme.
5. The composition of claim 4, wherein the polynucleotides encoding the phosphogluconate dehydratase enzyme and the 3-deoxygluconate-6-phosphate aldolase enzyme independently are from an Escherichia spp. microbe or Pseudomonas spp. microbe.
6. The composition of claim 5, wherein the Escherichia spp. microbe is an Escherichia coli strain.
7. The composition of claim 5, wherein the Pseudomonas spp. microbe is a Pseudomonas aeruginosa strain.
8. The composition of any one of claims 4 to 7, wherein the polynucleotide that encodes the phosphogluconate dehydratase enzyme is an EDD gene.
9. The composition of any one of claims 4 to 7, wherein the polynucleotide that encodes the 2-keto-3-deoxygluconate-6-phosphate aldolase enzyme is an EDA gene.
10. The composition of any one of claims 1 to 9, wherein the xylose isomerase enzyme is a chimeric enzyme.
11. The composition of claim 10, wherein a first portion of the polynucleotide that encodes the chimeric xylose isomerase enzyme is from a first microbe and a second portion of the polynucleotide that encodes the chimeric xylose isomerase enzyme is from a second microbe.
12. The composition of claim 10 or 11, wherein the first microbe, the second microbe, or the first microbe and the second microbe independently are selected from one or more of the group consisting of Clostridiales spp., Ruminococcus spp., Thermus spp., Bacillus spp., Clostridium spp., Orpinomyces spp., Escherichia spp. and Piromyces spp.
microbes.
13. The composition of claim 12, wherein the first microbe, the second microbe, or the first microbe and the second microbe independently are selected from one or more of the group consisting of Clostridiales_genomosp. BVAB3 str UP119-5, Ruminococcus flavefaciens, Ruminococcus_FD1, Ruminococcus_18P13, Thermus thermophilus, Bacillus stercoris, Clostridium cellulolyticum, Bacillus uniformis, Bacillus stearothermophilus, Bacteroides thetaiotaomicron, Clostridium thermohydrosulfuricum, Orpinomyces, Clostridium phytofermentans, Escherichia coli and Piromyces strain E2.
14. The composition of any one of claims 10 to 13, wherein 80% or more of the polynucleotide that encodes the xylose isomerase enzyme is from a Ruminococcus spp.
microbe xylose isomerase-encoding sequence.
15. The composition of claim 14, wherein all or a portion of the polynucleotide that encodes the xylose isomerase enzyme is from a Ruminococcus spp. microbe xylose isomerase-encoding sequence.
16. The composition of claim 15, wherein the Ruminococcus spp. microbe is a Ruminococcus flavefaciens strain.
17. The composition of any one of claims 10 to 16, wherein the polynucleotide that encodes the xylose isomerase enzyme is chimeric and includes a sequence that encodes a xylose isomerase from another microbe.
18. The composition of any one of claims 10 to 17, wherein the portion of the polynucleotide from the Ruminococcus spp. microbe xylose isomerase is 3' with respect to the portion of the polynucleotide from another microbe.
19. The composition of claim 17 or 18, wherein the other microbe is a fungus.
20. The composition of claim 19, wherein the fungus is an anaerobic fungus.
21. The composition of claim 20, wherein the fungus is a Piromyces spp.
fungus.
22. The composition of claim 21, wherein the Piromyces spp. fungus is a Piromyces strain E2.
23. The composition of any one of claims P1 to P22, wherein one or more of the following activities are added or increased: a glucose-6-phosphate dehydrogenase activity, a 6-phosphogluconolactonase activity, or a glucose-6-phosphate dehydrogenase activity and a 6-phosphogluconolactonase activity.
24. The composition of claim 23, wherein the yeast comprises one or more heterologous polynucleotides that encode one or more of the following enzymes, or wherein the yeast comprises multiple copies of endogenous polynucleotides that encode one or more of the following enzymes: glucose-6-phosphate dehydrogenase enzyme, 6-phosphogluconolactonase enzyme, or glucose-6-phosphate dehydrogenase enzyme and 6-phosphogluconolactonase enzyme.
25. The composition of claim 24, wherein the one or more polynucleotides that encode the glucose-6-phosphate dehydrogenase enzyme, the 6-phosphogluconolactonase enzyme, or the glucose-6-phosphate dehydrogenase enzyme and the 6-phosphogluconolactonase enzyme are from a yeast.
26. The composition of claim 25, wherein the yeast is a Saccharomyces spp.
yeast.
27. The composition of claim 26, wherein the yeast is a Saccharomyces cerevisiae strain.
28. The composition of any one of claims 24 to 27, wherein the glucose-6-phosphate dehydrogenase enzyme is expressed from a ZWF gene.
29. The composition of claim 28, wherein the ZWF gene is a ZWF1 gene.
30. The composition of any one of claims 24 to 29, wherein the 6-phosphogluconolactonase enzyme is expressed from a SOL gene.
31. The composition of claim 31, wherein the SOL gene is a SOL3 gene.
32. The composition of any one of claims 1 to 31, wherein the nucleic acid includes one or more polynucleotides that encode one or more glucose transporters.
33. The composition of claim 32, wherein the polynucleotide that encodes the one or more glucose transporters is from a yeast.
34. The composition of claim 32 or 33, wherein the one or more glucose transporters is encoded by a one or more of a GAL2, GSX1 and GXF1 gene.
35. The composition of any one of claims 1 to 34, wherein the yeast includes one or more added activities or increased activities selected from the group consisting of transketolase activity, transaldolase activity, or a transketolase activity and transaldolase activity.
36. The composition of claim 35, wherein the yeast includes one or more heterologous polynucleotides that encodes one or more of the following enzymes, or includes multiple copies of polynucleotides that encode one or more of the following enzymes:
transketolase enzyme, transaldolase enzyme, or a transketolase enzyme and transaldolase enzyme
37. The composition of claim 36, wherein the transketolase enzyme is encoded by a TKL1 coding sequence or a TKL2 coding sequence.
38. The composition of claim 36, wherein the transaldolase is encoded by a TAL
coding sequence.
39. The composition of any one of claims 36 to 38, wherein the transketolase enzyme or the transaldolase enzyme is from a yeast.
40. The composition of any one of claims 1 to 39, wherein the nucleic acid includes one or more promoters operable in a yeast, wherein the promoter is in operable connection with one or more of the polynucleotides.
41. The composition of claim 40, wherein the promoter is selected from promoters that regulate glucose phosphate dehydrogenase (GPD), translation elongation factor (TEF-1), phosphoglucokinase (PGK-1) and triose phosphate dehydrogenase (TDH-1).
42. The composition of any one of claims 1 to 41, wherein the yeast includes a reduction in one or more of the following activities: phosphofructokinase (PFK) activity, phosphoglucoisomerase (PGI) activity, 6-phosphogluconate dehydrogenase (decarboxylating) activity or combination thereof.
43. The composition of claim 42, wherein the yeast includes an alteration in one or more polynucleotides that inhibits production of one or more enzymes selected from the group consisting of phosphofructokinase (PFK) enzyme, phosphoglucoisomerase (PGI) enzyme, 6-phosphogluconate dehydrogenase (decarboxylating) enzyme or combination thereof.
44. The composition of claim 43, wherein the phosphofructokinase (PFK) enzyme is a PFK-2 enzyme or PFK-1 enzyme.
45. The composition of claim 43, wherein the 6-phosphogluconate dehydrogenase (decarboxylating) enzyme is encoded by a GND-1 gene or GND-2 gene.
46. The composition of claim 43, wherein the PGI is encoded by a PGI-1 gene.
47. The composition of any one of claims 1 to 46, wherein the polynucleotides, the promoters, or the polynucleotides and the promoters are not integrated in the yeast nucleic acid.
48. The composition of claim 47, wherein the polynucleotides, the promoters, or the polynucleotides and the promoters are in one or more plasmids.
49. The composition of any one of claims 1 to 48, wherein the polynucleotide subsequences, the promoters, or the polynucleotide subsequences and the promoters are integrated in genomic DNA of the yeast.
50. The composition of claim 49, wherein the polynucleotides, the promoters, or the polynucleotides and the promoters are integrated in a transposition integration event, in a homologous recombination integration event, or in a transposition integration event and a homologous recombination integration event.
51. The composition of claim 50, wherein the transposition integration event includes transposition of an operon comprising two or more of the polynucleotide subsequences, the promoters, or the polynucleotide subsequences and the promoters.
52. The composition of claim 50, wherein the homologous recombination integration event includes homologous recombination of an operon comprising two or more of the polynucleotide subsequences, the promoters, or the polynucleotide subsequences and the promoters.
CA2767259A 2009-07-09 2010-07-09 Engineered microorganisms with enhanced fermentation activity Abandoned CA2767259A1 (en)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US22443009P 2009-07-09 2009-07-09
US61/224,430 2009-07-09
US31678010P 2010-03-23 2010-03-23
US61/316,780 2010-03-23
US33409710P 2010-05-12 2010-05-12
US61/334,097 2010-05-12
PCT/US2010/041618 WO2011006136A2 (en) 2009-07-09 2010-07-09 Engineered microorganisms with enhanced fermentation activity

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US (6) US20120184007A1 (en)
EP (1) EP2451960A2 (en)
AP (1) AP3766A (en)
BR (1) BRPI1011934A8 (en)
CA (2) CA2767361A1 (en)
IN (1) IN2012DN00568A (en)
WO (2) WO2011006136A2 (en)
ZA (1) ZA201200506B (en)

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