WO2014057008A1 - Ingénierie métabolique de l'acétyl-coenzyme a chez la levure - Google Patents

Ingénierie métabolique de l'acétyl-coenzyme a chez la levure Download PDF

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WO2014057008A1
WO2014057008A1 PCT/EP2013/071102 EP2013071102W WO2014057008A1 WO 2014057008 A1 WO2014057008 A1 WO 2014057008A1 EP 2013071102 W EP2013071102 W EP 2013071102W WO 2014057008 A1 WO2014057008 A1 WO 2014057008A1
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pfl
yeast
polypeptide
pyruvate
enzyme activity
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Verena Siewers
Anastasia KRIVORUCHKO
Yiming Zhang
Jens Nielsen
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Chalmers Intellectual Property Rights Ab
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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    • 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
    • C12P7/16—Butanols
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    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01002—Formate dehydrogenase (1.2.1.2)
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    • C12Y102/04—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with a disulfide as acceptor (1.2.4)
    • C12Y102/04001—Pyruvate dehydrogenase (acetyl-transferring) (1.2.4.1)
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    • C12Y102/07—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with an iron-sulfur protein as acceptor (1.2.7)
    • C12Y102/07001—Pyruvate synthase (1.2.7.1), i.e. pyruvate ferredoxin oxidoreductase
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    • C12Y118/00—Oxidoreductases acting on iron-sulfur proteins as donors (1.18)
    • C12Y118/01—Oxidoreductases acting on iron-sulfur proteins as donors (1.18) with NAD+ or NADP+ as acceptor (1.18.1)
    • C12Y118/01002—Ferredoxin-NADP+ reductase (1.18.1.2)
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    • C12Y197/00—Other oxidoreductases (1.97)
    • C12Y197/01—Other oxidoreductases (1.97) other oxidoreductases (1.97.1)
    • C12Y197/01004—[Formate-C-acetyltransferase]-activating enzyme (1.97.1.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01054—Formate C-acetyltransferase (2.3.1.54), i.e. pyruvate formate-lyase or PFL
    • 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

Definitions

  • the present invention relates to the development of metabolic engineering of yeast. More specifically this invention relates to a non-ethanol producing strain of the yeast Saccharomyces cerevisiae that can convert glucose to acetyl-Coenzyme A (CoA) in the cytosol.
  • yeast Saccharomyces cerevisiae that can convert glucose to acetyl-Coenzyme A (CoA) in the cytosol.
  • CoA acetyl-Coenzyme A
  • Biotechnology has been used for generations in the production of fermented beverages and food products, and in the last 60 years for the production of antibiotics, food ingredients and feed additives.
  • bioethanol for fuel use has increased rapidly.
  • novel cell factories for the production of chemicals and novel fuels there is extensive research on the development of novel cell factories for the production of chemicals and novel fuels, and it is expected that this will lead to implementation of several new biotech processes in the coming years.
  • metabolic engineering has evolved into a research field that encompasses detailed metabolic analysis with the objective to identify targets for metabolic engineering and the implementation of metabolic engineering strategies for improvement and/or design of novel cell factories.
  • synthetic biology has emerged as another research field that originally aimed at reconstruction of small, artificial biological systems, e.g. assembling a new biological regulon or oscillators that can be used to regulate gene expression in response to a specific input. But synthetic biology also includes the synthesis of DNA and complete chromosomes as illustrated in a recent work on reconstruction of a complete bacterial chromosome. Summary of the Invention
  • a primary object of the present invention is to generate a yeast platform cell factory with increased cytosolic acetyl-CoA (AcCoA) supply, by introduction of steps for the direct conversion of pyruvate to AcCoA.
  • the yeast platform cell factory of the present invention can efficiently convert pyruvate to acetyl-CoA and this feature is combined with elimination of pyruvate decarboxylase activity, resulting in an efficient cytosolic acetyl-CoA producer that cannot produce ethanol. This will result in high cytosolic levels of the important precursor acetyl-CoA.
  • Acetyl-CoA metabolism is highly compartmentalized in eukaryotic cells as this metabolite is used for metabolism in the cytosol, mitochondria, peroxisomes and the nucleus.
  • Acetyl-CoA serves as a key precursor metabolite for the production of important cellular constituents such as fatty acids, sterols, and amino acids as well as it is used for acetylation of proteins. Besides these important functions it is also precursor metabolite for many other biomolecules, such as polyketides, isoprenoids, 3-hydroxypropionic acid, 1 -butanol and polyhydroxyalkanoids, which encompass many industrially relevant chemicals.
  • cerevisiae is a very important cell factory as it is already widely used for production of biofuels, chemicals and pharmaceuticals, and there is therefore much interest in developing platform strains of this yeast that can be used for production of a whole range of different products. It is however a problem that such a platform cell factory for efficient production of cytosolic acetyl-CoA is not as efficient as needed for good industrial application.
  • Our invention is a multiple gene modification approach of the yeast generating higher yield of acetyl-CoA, by combining pathways for direct conversion from pyruvate to acetyl-CoA together with elimination of ethanol production.
  • Figure 1 provides a simplified overview of acetyl-CoA metabolism in S.
  • Acetyl-CoA is key metabolite in three different compartments: the cytosol
  • acetyl-CoA is formed from pyruvate by the pyruvate dehydrogenase complex.
  • acetyl-CoA is formed from acetate by acetyl-CoA synthase.
  • acetyl-CoA can be formed from both acetate (also by acetyl-CoA synthase, not shown) and from fatty acids by beta-oxidation.
  • TCA tricarboxylic acid
  • Acetyl-CoA in the peroxisomes can via the glyoxylate cycle (GYC) be converted to C4 organic acids (malic and succinic acid) that can be transferred to the mitochondria for oxidation via malic enzyme and the TCA cycle.
  • GYC glyoxylate cycle
  • C4 organic acids malic and succinic acid
  • the primary fate of acetyl-CoA in the cytosol is to serve as precursor for cellular lipids (fatty acids and ergosterol).
  • biotechnological products are derived from acetyl-CoA and the biosynthesis of most of these occurs in the cytosol.
  • a platform yeast cell factory for all these products should therefore redirect carbon towards the acetyl-CoA in the cytosol.
  • Figure 2 provides an overview of the strategy that can be used in the invention.
  • the normal route for conversion of pyruvate to acetyl-CoA in the cytosol is blocked through deletion of the three structural genes encoding pyruvate
  • yeast decarboxylase activity in yeast (PDC1, PDC5 and PDC6).
  • This strain is auxotrophic for C 2 carbon sources such as acetate or ethanol, and it has been shown that this requirement is solely to fulfil the need for acetyl-CoA in the cytosol (required for production of cellular lipids).
  • the yeast cells By removing pyruvate decarboxylase activity the yeast cells also cannot produce ethanol from glucose.
  • a cytosoiic route for production of acetyl-CoA pyruvate formate lyase (PFL) can be expressed in the cytosol.
  • the strains with the novel pathway for production of cytosoiic acetyl-CoA can be used for production of fatty acids, 3-hydroxypropionic acid, isoprenoids, polyhydroxyalkanoates and 1 -butanol.
  • Figure 3 shows the plasmid pYZ01 described in Example 2.
  • Figure 4 shows the plasmid pKB01 described in Example 2.
  • Figure 5 shows the plasmid pKB02 described in Example 2
  • Figure 6 shows growth curves on minimal media for strains evolved for increased growth on glucose described in Example 3. Data from 3-4 independently evolved replicates is shown. Detailed Description of the Invention
  • the invention herein relies, unless otherwise indicated, on the use of conventional techniques of biochemistry, molecular biology, microbiology, cell biology, genomics and recombinant technology.
  • nucleic acid DNA or RNA
  • RNA DNA or RNA
  • cloning, restriction, and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems.
  • acetyl-CoA in a host cell and indicates that the host cell is producing more acetyl-CoA by virtue of the introduction of recombinant/heterologous nucleic acid sequences encoding polypeptides that alter the host cell's normal metabolic pathways or as a result of other modifications (e.g., altering the expression of one or more endogenous polynucleotides) as compared with, for example, the host cell that is not modified/transformed with the recombinant polynucleotides as described herein.
  • protein and “polypeptide” refer to compounds comprising amino acids joined via peptide bonds and are used interchangeably.
  • This increase can be observed by comparing said increase in a yeast transformed with, for example, recombinant polynucleotides encoding a polypeptide having the enzyme activity of pyruvate formate lyase (PFL) and a polypeptide having the enzyme activity of pyruvate formate lyase-activating enzyme and a recombinant polynucleotide encoding a polypeptide having the enzyme activity of formate dehydrogenase and one or more recombinant polynucleotides encoding a ferredoxin or a flavodoxin polypeptide and a ferredoxin reductase polypeptide compared to the yeast not transformed with the recombinant polynucleotides.
  • PFL pyruvate formate lyase
  • a recombinant polynucleotide encoding a polypeptide having the enzyme activity of pyruvate formate lyase-
  • the terms “increase,” “increasing,” “increased,” “enhance,” “enhanced,” “enhancing,” and “enhancement” indicate an elevation of at least about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 0%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500% or more, or any range therein, as compared to a control.
  • “diminish,” “suppress,” and “decrease” describe, for example, a decrease in the pyruvate decarboxylase activity in a yeast (e.g., a yeast having deletions in the polynucleotides PDC1, PDC5 and PDC6) as compared to a control (e.g., a yeast not having said deletions in the polynucleotides PDC1, PDC5 and PDC6).
  • a yeast e.g., a yeast having deletions in the polynucleotides PDC1, PDC5 and PDC6
  • a control e.g., a yeast not having said deletions in the polynucleotides PDC1, PDC5 and PDC6.
  • the terms “reduce,” “reduces,” “reduced,” “reduction,” “diminish,” “suppress,” and “decrease” and similar terms mean a decrease of at least about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500% or more, or any range therein, as compared to a control.
  • overexpress refers to higher levels of activity of a gene (e.g. transcription of the gene); higher levels of translation of mRNA into protein; and/or higher levels of production of a gene product (e.g., polypeptide) than would be in the cell in its native (or control (e.g., not transformed with the particular heterologous or recombinant polypeptides being overexpressed)) state.
  • gene product e.g., polypeptide
  • Overexpression can result in levels that are 25%, 50%, 100%, 200%, 500%, 1000%, 2000% or higher in the cell, as compared to control levels.
  • Saccharomyces cerevisiae can be a host for carrying out the invention, as it is a popular host in basic and applied research apart from being a good ethanol producer, a precursor of esters and specifically of fatty acid ethyl esters.
  • other yeast cells useful with the present invention include, but are not limited to, other Saccharomyces species, Hansenula polymorpha, Kluyveromyces species, Pichia species, Candida species, Trichoderma species, Yarrowia lipolytica, etc.
  • platform cell factories In industry, there is much interest in applying a limited number of platform cell factories for production of a wide range of fuels and chemicals as this allows for flexible use of production facilities, which are very capital intensive.
  • One of these platform cell factories is the yeast Saccharomyces cerevisiae, which is widely used for the production of beer, bread, wine, bioethanol, nutraceuticals, chemicals and pharmaceuticals.
  • These platform cell factories can efficiently convert raw materials, today typically glucose/fructose derived from starch or sucrose, but in the future also pentoses derived from lignocellulose, into so-called precursor metabolites can then be further converted into a product of interest.
  • acetyl-CoA that is used as precursor for the production of a wide range of industrially very interesting products (see Fig. 1 ).
  • Several of these products are produced by pathways that drain acetyl-CoA from the cytosol and in connection with
  • acetyl-CoA is, however, produced and used in several different cellular compartments, i.e. the cytosol, mitochondria and the peroxisomes, and in S. cerevisiae it cannot be transported directly between the different compartments (S. cerevisiae holds all the components of the carnitine transport system, but it cannot synthesize carnitine and in industrial fermentations it would be too expensive to add this component to the medium).
  • acetyl-CoA in the cytosol is produced from acetate that is derived from acetaldehyde, that is formed by de-carboxyiation of pyruvate.
  • Acetaldehyde can also be converted to ethanol by alcohol dehydrogenase, and during growth on glucose the majority of the glycolytic flux is directed towards ethanol due to the so-called Crabtree effect in yeast.
  • Alcohol dehydrogenase Adhl p
  • the normal route for conversion of pyruvate to acetyl- CoA in the cytosol is blocked through deletion of the three structural genes encoding pyruvate decarboxylase activity in yeast ⁇ PDC1, PDC5 and PDC6),
  • This strain is auxotrophic for C 2 carbon sources such as acetate or ethanol, and it has been shown that this requirement is solely to fulfil the need for acetyl-CoA in the cytosol (required for production of cellular lipids).
  • the yeast cells can also not produce ethanol from glucose. This has been shown by MT Flikweert et. al ((1999) Growth requirements of pyruvate decarboxylase-negative Saccharomyces cerevisiae. FEMS Micorobio. Lett. 174, 73-79).
  • yeast platform cell factory that can efficiently convert pyruvate to acetyl-CoA in one step and combine this feature with elimination of pyruvate de-carboxylase activity, thereby establishing an efficient cytosolic acetyl-CoA producer that overproduces acetyl-CoA and at the same time cannot produce ethanol.
  • the present invention also relates to strain cultivation and evolution.
  • the strains containing introduced pyruvate to AcCoA conversion system can be cultivated, for example on yeast extract peptone dextrose liquid media.
  • the strains can then be evolved to increase growth, for example on glucose as the sole carbon source.
  • This step involves two phases. In the first phase strains are cultivated in a medium, e.g. YP medium.
  • the strains can be transferred with ethanol concentration being gradually decreased until the only carbon source in the media is glucose.
  • glucose tolerant strains When the fast-growing, glucose tolerant strains are obtained they can be further evolved for growth on minimal media.
  • PFL Expressing pyruvate formate lyase
  • PFL (encoded by pfIB) is a homodimer and catalyzes the non-oxidative conversion of pyruvate to acetyl-CoA and formate under anaerobic conditions.
  • the catalytic mechanism involves a glycyl radical, which is part of the enzyme and which is sensitive to oxygen. Thus, the enzyme is not active under aerobic conditions.
  • the radical is formed by pyruvate formate lyase-activating enzyme (PFL-AE) (encoded by pfIA), a monomeric iron- sulfur cluster protein, through cleavage of S-adenosylmethionine.
  • rheinhardtii or Neocallmastix frontalis examples include E. coli, Lactobacillus plantarum, Bifidobacterium longum, Bacteroides thetaiotaomicron, Clostridium pasteurianum, Staphylococcus aureus, Zymomonas mobilis.
  • archaeal PFL sources are Archaeoglobus fulgidus, Methanobacterium
  • thermoautotrophicus
  • WO09143495 provides a method of increasing production of formate in a modified yeast comprising inserting genes encoding the E. coli pyruvate formate lyase enzyme complex (PFL) into a 58Oa triple auxotrophic strain of Saccharomyces cerevisiae yeast. Unlike the present invention, WO09143495 provides a S. cerevisiae in which FDH1 and FDH2 are deleted, in contrast in the present invention it is beneficial to over-express FDH1 and FDH2. Further, neither pfIA and pfIB
  • WO09143495 coexpressed with fdx, fldA and fpr in the yeast strains of WO09143495.
  • the modified S. cerevisiae of WO09143495 is intended to increase ethanol production, unlike the present invention where ethanol production is eliminated.
  • a further embodiment of the invention provides a method of producing a yeast having increased production of cytosolic acetyl-CoA, comprising introducing into a yeast: (a) a deletion of the endogenous polynucleotides encoding pyruvate decarboxylase (PDC) (e.g., a deletion of PDC1, PDC5 and PDC6); (b) one or more recombinant polynucleotides encodes a polypeptide having the enzyme activity of pyruvate formate lyase (PFL) which is selected from the group of polypeptides consisting of: E. coli PfIB Chlamydomonas rheinhardtii Pfl;
  • PDC pyruvate decarboxylase
  • PFL pyruvate formate lyase
  • Neocallimastix frontalis PFL Lactobacillus plantarum PFL, Bifidobacterium longum PFL; Bacteroides thetaiotaomicron PFL; Clostridium pasteurianum PFL;
  • Staphylococcus aureus PFL Zymomonas mobilis PFL; Archaeoglobus fulgidus PFL; or Methanobacterium thermoautotrophicus PFL or a polypeptide having at least 50 % identity to any of the polypeptides mentioned above and (ii) a polypeptide having the enzyme activity of pyruvate formate lyase-activating enzyme (PFL-AE), which is selected from the group of polypeptides consisting of: E.
  • coli PfIA Chlamydomonas rheinhardtii PfIA; Neocallimastix frontalis PFL-AE; Lactobacillus plantarum PFL-AE; Bifidobacterium longum PFL-AE; Bacteroides thetaiotaomicron PFL-AE; Clostridium pasteurianum PFL-AE; Staphylococcus aureus PFL-AE; Zymomonas mobilis PFL- AE; Archaeoglobus fulgidus PFL-AE; or Methanobacterium thermoautotrophicus PFL-AE or a polypeptide having at least 50 % identity to any of the polypeptides mentioned above; (c) a polynucleotide encoding a polypeptide having the enzyme activity of formate dehydrogenase (e.g., S.
  • formate dehydrogenase e.g., S.
  • the polynucleotide encoding a polypeptide having the enzyme activity of formate dehydrogenase is overexpressed as compared to the yeast's endogenous formate dehydrogenase.
  • the invention provides a yeast having reduced pyruvate decarboxylase activity and increased acetyl-CoA production, comprising (a) a deletion of the endogenous polynucleotide sequences encoding pyruvate
  • PDC decarboxylase
  • PDC6 a deletion of PDC1, PDC5 and PDC6
  • PFL pyruvate formate lyase
  • coli PfIB Chlamydomonas rheinhardtii Pfl; Neocallimastix frontalis PFL; Lactobacillus plantarum PFL, Bifidobacterium longum PFL; Bacteroides thetaiotaomicron PFL; Clostridium pasteurianum PFL; Staphylococcus aureus PFL; Zymomonas mobilis PFL; Archaeoglobus fulgidus PFL; or Methanobacterium thermoautotrophicus PFL or a polypeptide having at least 50 % identity to any of the polypeptides mentioned above and (ii) a polypeptide having the enzyme activity of pyruvate formate lyase-activating enzyme (PFL-AE), which is selected from the group of polypeptides consisting of: E.
  • PFL-AE pyruvate formate lyase-activating enzyme
  • coli PfIA Chlamydomonas rheinhardtii PfIA; Neocallimastix frontalis PFL-AE; Lactobacillus plantarum PFL-AE; Bifidobacterium longum PFL-AE; Bacteroides thetaiotaomicron PFL-AE; Clostridium pasteurianum PFL-AE; Staphylococcus aureus PFL-AE; Zymomonas mobilis PFL-AE;
  • Archaeoglobus fulgidus PFL-AE or Methanobacterium thermoautotrophicus PFL-AE or a polypeptide having at least 50 % identity to any of the polypeptides mentioned above;
  • a polynucleotide encoding a polypeptide having the enzyme activity of formate dehydrogenase e.g., S. cerevisiae FDH1 or FDH2
  • formate dehydrogenase e.g., S. cerevisiae FDH1 or FDH2
  • PDC1, PDC5 and PDC6 were deleted using a bipartite strategy (Erdeniz et al., 1997). Two overlapping fragments of the kanMX resistance marker cassette flanked by loxP sites were amplified via PCR from plasmid pUG6 (Guldener et al., 1996) using primers 13-16. Sequences upstream and downstream of the individual genes were amplified using primers 1 -12. Due to overlapping ends (introduced through the primer sequences) the PDC-upstream fragments could be fused to the 5 ' kanMX fragment and the 3 ' kanMX fragment to the individual PDC-downstream fragments by fusion PCR using the outer primers for amplification.
  • the two overlapping PCR fragments thus generated for each gene deletion were transformed into yeast using the lithium acetate method (Gietz and Woods, 2002). After each gene deletion, the kanMX marker cassette was looped out via Cre recombinase mediated recombination between the two flanking loxP sites using plasmid pUC47 or pUG62 as described previously (Guldener et al., 1996).
  • PDC1, PDC5, and PDC6 were consecutively deleted in two different background strains: CEN.PK 113-5D (MATa ura3-52) and CEN.PK 1 10-10C (MATa his3-A 1). This resulted in construction of strains YMZ-C1 (MATa ura3-52 pdc1A pdcSA pdc6A), and YMZ-A3 (MATa his3-A 1 pdc6A). Strains YMZ-C1 and YMZ-A3 were crossed to generate YMZ-E1 (MATa ura3-52 his3-A 1 pddA pdcSA pdc6A).
  • pfIA encoding PFL-AE
  • pfIB encoding PFL
  • the gene sequences including introduced restriction sites can be found in table 2.
  • pfIB was restricted with Sad/NoA and cloned into vector pSP-GM1 .
  • PfIA was cut with Xma ⁇ /Xho ⁇ and cloned into the same vector generating plasmid pYZ01 (Fig. 3).
  • the pflA-pflB cassette including the bidirectional PTEFI-PPGKI promoter was PCR amplified from pYZ01 using primers 21/22 and cloned into vector pXII-5 (Mikkelsen et al., 2012) using USER cloning.
  • the integration constructs was separated from the vector backbone by Xba ⁇ restriction and integrated into YMZ-E1 from example 1 yielding YMZ-E1 -PFL.
  • the genes fldA and fpr were amplified by PCR using E. coli DH5a genomic DNA as a template and primers 17-20.
  • the NoAISad restricted fldA fragment and Bam VXhol restricted fpr fragment were cloned into plYC04 generating pKB01 (Fig. 4).
  • the E. coli gene coding for fc/ was PCR-amplified using E. coli DH5pgenomic DNA as a template and primers 60/61 . This fragment was then restricted with
  • a fragment containing fpr and either fdxox fldA under P TEFI-PPGKI was PCR-amplified from pKB02 or pKB01 using primers 62/64 or 63/64, respectively, and cloned via CPEC into integrative vector pXI-5HIS (vector pXI-5 described in Mikkelsen et al [2012], in which the KIURA3 marker was replaced by the loxP flanked SphisS cassette of plasmid pUG27 described in Guldener et al [2002]; Jensen et al., submitted).
  • the integration constructs were then PCR-amplified using primers 65/66 and integrated into YMZ-E1 -PFL generating strains YMZ-E1 - PFLfld and YMZ-E1 -PFLfdx, respectively.
  • FDH1 encoding formate dehydrogenase is amplified from yeast genomic DNA using primers 54/55.
  • the P T EFI promoter is amplified from pSP-GM1 using primers 33/53, Both fragments are cloned into pX-2 by USER cloning.
  • the integration construct is separated from the vector backbone by ⁇ ⁇ restriction and integrated in to the genome of YMZ-E1 -PFL, YMZ-E -PFLfld and YMZ-E1 -PFLfdx, respectively, to generate YMZ-E1 -PFLF, YMZ-E1 -PFLfldF and YMZ-E1-PFLfdxF.
  • the coding sequences of pfl (encoding PFL) and pfIA (encoding PFL-AE) are amplified from C. rheinhardtii cDNA (using primers 56-59) and together with the P T EFI-PPGKI promoter (amplified from pSP-GM1 using primers 33/34) cloned into pXII-5 by USER cloning.
  • the integration construct is used to replace the E.
  • PDC deletion strains containing the introduced pyruvate to AcCoA conversion system (YMZ-E1 -PFL, YMZ-E1 -PFLfld and YMZ-E1 -PFLfdx), according to example 2 above, were initially cultivated on yeast extract peptone dextrose (YPD) liquid media in either shake-flasks or tubes and their growth was compared to strains containing the PDC deletion alone to evaluate system function.
  • YPD yeast extract peptone dextrose
  • strains were evolved to increase growth on glucose as the sole carbon source. This involved two phases. In the first phase, strains were cultivated in shake flasks in YP medium (10 g/L yeast extract, 20 g/L peptone)containing 1.4 % glucose and 0.6 % ethanol. Strains were serially transferred every 48 hours or 24 hours, and the ethanol concentration was gradually decreased until glucose became the sole carbon source in the media. The growth rate of the strains was occasionally determined to evaluatetheir adaptation level of glucose tolerance.
  • YP medium 10 g/L yeast extract, 20 g/L peptone
  • glucose tolerant strains were obtained in YPD media, they were further evolved for growth on minimal media.
  • the strains were cultivated in minimal medium (Verduyn et al., 1992) containing 2 % glucose and serially transferred every 24 hours. The growth rate of strains was occasionally determined to evaluate their adaptation level. Growth curves for the evolved strains are shown in figure 6.
  • Giildener U., Heck, S., Fiedler, T., Beinhauer, J., Hegemann, J.H., 1996.

Abstract

Cette invention concerne le développement de l'ingénierie métabolique chez la levure. Un procédé de production d'une levure caractérisée par une production accrue de coenzyme A cytosolique et qui élimine l'activité pyruvate décarboxylase pour donner une levure productrice d'acétyl-coenzyme A cytosolique efficace qui ne peut pas produire d'éthanol est décrit.
PCT/EP2013/071102 2012-10-09 2013-10-09 Ingénierie métabolique de l'acétyl-coenzyme a chez la levure WO2014057008A1 (fr)

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WO2015057154A3 (fr) * 2013-10-18 2015-07-02 Biopetrolia Ab Modification du métabolisme de l'acétyl-coa dans la levure
WO2016193540A1 (fr) * 2015-05-29 2016-12-08 Teknologian Tutkimuskeskus Vtt Oy Conversion directe de sucres en acide glycolique
WO2022020748A1 (fr) * 2020-07-24 2022-01-27 Duke University Procédés et compositions pour la production de produits dérivés d'acétyl-coa

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015057154A3 (fr) * 2013-10-18 2015-07-02 Biopetrolia Ab Modification du métabolisme de l'acétyl-coa dans la levure
US10704050B2 (en) 2013-10-18 2020-07-07 Biopetrolia Ab Engineering of acetyl-CoA metabolism in yeast
US11104907B2 (en) 2013-10-18 2021-08-31 Biopetrolia Ab Engineering of acetyl-CoA metabolism in yeast
WO2016193540A1 (fr) * 2015-05-29 2016-12-08 Teknologian Tutkimuskeskus Vtt Oy Conversion directe de sucres en acide glycolique
WO2022020748A1 (fr) * 2020-07-24 2022-01-27 Duke University Procédés et compositions pour la production de produits dérivés d'acétyl-coa

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