WO2018127572A1 - Procédé d'utilisation améliorée du glycérol dans une levure - Google Patents

Procédé d'utilisation améliorée du glycérol dans une levure Download PDF

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WO2018127572A1
WO2018127572A1 PCT/EP2018/050291 EP2018050291W WO2018127572A1 WO 2018127572 A1 WO2018127572 A1 WO 2018127572A1 EP 2018050291 W EP2018050291 W EP 2018050291W WO 2018127572 A1 WO2018127572 A1 WO 2018127572A1
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amino acid
yeast
gene
acid sequence
glycerol
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Tomas STRUCKO
Jochen FÖRSTER
Katharina ZIRNGIBL
Kiran Raosaheb PATIL
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Danmarks Tekniske Universitet
European Molecular Biology Laboratory
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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)
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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)
    • C12N9/104—Aminoacyltransferases (2.3.2)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
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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
    • C12P7/06—Ethanol, i.e. non-beverage
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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)
    • C12Y102/04002—Oxoglutarate dehydrogenase (succinyl-transferring) (1.2.4.2), i.e. alpha-ketoglutarat dehydrogenase
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    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/0103—Glycerol kinase (2.7.1.30)
    • 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

  • TITLE A method for improved glycerol utilization in yeast Field of the invention
  • the invention provides GM yeast strains for use as cell factories that are capable of using the waste product, glycerol, as sole carbon source, without the addition of an amino acid supplement.
  • the invention additionally teaches how to modify existing yeast strains to have these advantageous properties.
  • Micro-organisms in particular yeast and fungi, are widely used for the production of valuable compounds, including biofuels, small chemicals, pharmaceuticals and nutraceuticals. This has been facilitated by advances in yeast and fungal genetic engineering, with the successful introduction of genes encoding diverse heterologous single and multi-step metabolic pathways into the cells of the yeast or fungal host, allowing for an increasing diversity of compounds to be produced.
  • yeast or fungi for example Saccharomyces
  • Saccharomyces as a cell factory has raised awareness of the need to reduce production costs, in particular the ongoing costs of nutrients for cell cultivation.
  • Bio-waste products from manufacturing processes can provide an attractive cheap source of carbon.
  • glycerol is an important bio-waste product, since it is a by-product of bio- ethanol, oleo-chemical and bio-diesel production.
  • yeasts such as Saccharomyces cerevisiae are capable of degrading glycerol by purely oxidative metabolism
  • the cells when placed in a medium also comprising sugar, the cells are incapable of consuming the glycerol until the sugar has been consumed, due to catabolic repression.
  • the cells when cultured under oxidative conditions on synthetic media with glycerol as sole carbon source, the cells consume the glycerol extremely slowly, which is not compatible with their use as a cell factory on an industrial scale.
  • Glycerol utilization in for example S. cerevisiae, is facilitated by the uptake of extracellular glycerol via a glycerol/H + -symporter encoded by STL1.
  • glycerol is phosphorylated by a glycerol kinase encoded by GUTl resulting in the formation of L-glycerol-3-phosphate.
  • DHAP then enters directly into the glycolytic pathway via glyceraldehyde-3-phosphate.
  • the three glycerol catabolic pathway genes (STLl, GUTl and GUT2) are reported to be genetic determinants of a glycerol-growth phenotype in yeast (5). Additionally, specific alleles at the UBR2 and SSK1 loci are additionally reported to contribute to a glycerol-growth phenotype in yeast (5).
  • the present invention provides a genetically modified yeast with improved glycerol catabolism, wherein the yeast comprises:
  • transgene gene encoding a polypeptide having glycerol kinase activity (EC: 2.7.1.30), preferably where the amino acid sequence of the polypeptide has at least 80% amino acid sequence identity to SEQ ID No. : 2, or a mutant endogenous gene encoding a mutant polypeptide having enhanced glycerol kinase activity (EC: 2.7.1.30) as compared to a parent endogenous gene from which the mutant gene was derived, wherein the amino acid sequence of said mutant polypeptide has at least 80% amino acid sequence identity to SEQ ID No. : 4, and wherein amino acid residue 572 is Q (where the parent gene encodes an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID No. : 2 wherein amino acid residue 572 is
  • one or more genes in said yeast required for expressing a functional alpha ketoglutarase complex are inactivated, wherein said one or more genes is selected from the group consisting of: a gene encoding a polypeptide having alpha-ketoglutarate a gene encoding dehydrogenase enzyme activity (EC: 1.2.4.2);
  • the invention further provides a method for enhancing glycerol metabolism in a yeast strain comprising :
  • mutant polypeptide having enhanced glycerol kinase activity (EC: 2.7.1.30) as compared to a parent gene from which the mutant gene was derived, wherein the amino acid sequence of said mutant polypeptide has at least 80% amino acid sequence identity to SEQ ID No. : 4, and wherein said sequence comprises an E572Q mutation as compared to the polypeptide encoded by the parent gene (where the parent gene encodes an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID No. : 2 wherein amino acid residue 572 is E);
  • modified gene encodes an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID No. : 8, and wherein said amino acid sequence comprises an A990D mutation;
  • modified gene encodes an amino acid sequence of no more than 71 residues and having at least 80% amino acid sequence identity to amino acid residues 1- 71 of SEQ ID No. : 14 or 16.
  • the invention further provides a method for culturing cells of the genetically modified yeast of the invention comprising the steps of:
  • the invention further provides for the use of the genetically modified yeast of the invention, as a cell factory, where for example, the cell factory is provided with a culture medium comprising glycerol as sole carbon source.
  • Figure 1 Graphical presentation of the cell density (measured as OD 600 equivalents) of a parental laboratory yeast strain (CEN.PK (CEN .PK113-1A)) and industrial-yeast strains (L.1528 and CLIB382) over time during cultivation on MG medium comprising a defined mineral (M) media supplemented with 10 mL/L of glycerol as sole carbon source.
  • CEN.PK CEN .PK113-1A
  • M defined mineral
  • FIG. 1 Graphical presentation of the cell density (measured as OD 600 equivalents) of re-engineered yeast strains, derived from a parental laboratory yeast strain (CEN.PK (CEN.PK113-1A)) and comprising two or three of the mutant genes (GUTl-1; KGDl-1 ; UBC13-1 and INO80-1) as compared to Evolved ALE#2 yeast strain, when measured over time during cultivation on MG medium comprising a defined mineral (M) media supplemented with 10 mL/L of glycerol as sole carbon source.
  • CEN.PK CEN.PK113-1A
  • M defined mineral
  • FIG. 4 Graphical presentation of the growth rate the evolved yeast strain ALE#2, and re-engineered yeast strains, derived from a parental laboratory yeast strain (CEN .PK (CEN.PK113-1A)) and comprising two or three of the mutant genes (GUTl-1 ; KGDl-1 ; UBC13-1), measured during cultivation on MG medium comprising a defined mineral (M) media supplemented with 10 mL/L of glycerol as sole carbon source. Growth rates were estimated by finding the linear fit with the highest slope of the log(OD values or CDW). Growth analyses were performed in triplicate with the exception of the strain GUT1_UBC13, which was analysed in duplicate.
  • CEN .PK CEN.PK113-1A
  • M defined mineral
  • Figure 5 Graphical image showing Log2 fold differences (log2FC) in levels of expression and translation of genes in re-engineered yeast strains of the following pairs: (A) GUTl-1 ; KGDl-1 yeast strain (R-GK), compared to re- engineered GUTl-1 ; KGDl-1; UBC13-1 yeast strain (R-GKU) and (B) GUTl-1 ; UBC13-1 yeast strain (R-GU), compared to re-engineered R-GKU yeast strain; based on data derived from transcriptomic and proteomic analysis.
  • A GUTl-1 ; KGDl-1 yeast strain (R-GK), compared to re- engineered GUTl-1 ; KGDl-1; UBC13-1 yeast strain (R-GKU) and
  • B GUTl-1 ; UBC13-1 yeast strain (R-GU), compared to re-engineered R-GKU yeast strain; based on data derived from transcriptomic and proteomic analysis.
  • Pho3 encodes a constitutively expressed acid phosphatase involved in phosphate metabolism; Cit3 encodes a CITrate synthase having dual mitochondrial citrate and methylcitrate synthase activity; Dld3 encodes a D-Lactate Dehydrogenase (that converts D-2- hydroxyglutarate to alpha-ketoglutarate in the presence of FAD, with concomitant reduction of pyruvate to D-lactate).
  • Figure 6 Cartoon showing observed and predicted changes in carbon metabolism in yeast strains of the invention, having gene mutations encoding GUTl-1; KGDl-1 ; UBC13-1 alleles.
  • Black dashed arrows represent predicted flux changes when the carbon source for yeast fermentation is shifted from glucose to glycerol.
  • Black dashed lines with white dots and grey boxes depict observed flux changes estimated by comparing measured metabolite ratios in the R-GKU vs. R-GU mutants grown in MG medium.
  • GABA y-aminobutyric acid
  • SSA succinate semialdehyde.
  • sequence identity indicates a quantitative measure of the degree of homology between two amino acid sequences of substantially equal length. The two sequences to be compared must be aligned to give a best possible fit, by means of the insertion of gaps or alternatively, truncation at the ends of the protein sequences.
  • sequence identity can be calculated as ((Nref- Ndif) 100)/(Nref), wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences. Sequence identity can alternatively be calculated by the BLAST program e.g.
  • the numbers of substitutions, insertions, additions or deletions of one or more amino acid residues in the polypeptide as compared to its comparator polypeptide is limited, i.e. no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additions, and no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 deletions.
  • substitutions are conservative amino acid substitutions: limited to exchanges within members of group 1 : Glycine, Alanine, Valine, Leucine, Isoleucine; group 2 : Serine, Cysteine, Selenocysteine, Threonine, Methionine; group 3 : proline; group 4:
  • CRISPR system is a CJustered R_egularly Lnterspaced S_hort palindromic R epeats is a bacterial immune system that has been modified for genome engineering, including the yeast genome.
  • Inactivated gene the inactivation of a gene from the genome of a microbial cell leads to a loss of function (knockout) of the gene and hence where the gene encodes a polypeptide the inactivation results in a loss of expression of the encoded polypeptide or a failure to express a functional polypeptide. Where the encoded polypeptide is an enzyme, the gene inactivation leads to a loss of detectable enzymatic activity of the respective polypeptide in the microbial cell.
  • An inactivation gene in the genome of a microbial cell is characterized by a loss of function due to the deletion of, or substitution of, or addition of, at least one nucleotide leading to a loss of expression of a polypeptide or failure to express a functional polypeptide encoded by the gene.
  • gi number (genlnfo identifier) is a unique integer which identifies a particular sequence, independent of the database source, which is assigned by NCBI to all sequences processed into Entrez, including nucleotide sequences from DDBJ/EMBL/GenBank, protein sequences from SWISS-PROT, PIR and many others.
  • Endogenous gene is a gene that originates from within the genome of a micro-organism, and is considered to be a native gene in said microorganism.
  • the present invention provides a genetically modified yeast with improved glycerol catabolism, having the ability to grown on a medium comprising glycerol as sole carbon source and without an added source of amino nitrogen.
  • the yeast strains of the invention are genetically engineered such as to be capable of expressing elevated levels of glycerol kinase activity (EC: 2.7.1.30); while being unable to express functional enzymes conferring alpha ketoglutarase complex activity and E2 ubiquitin-conjugating activity.
  • these genetically engineered yeast strains exhibit a growth rate of up to 0.23.1V 1 when grown on minimal medium, with 50g/L glycerol as the sole carbon source, and they exhibited a lag time of only 4 hours (when the starting inoculum had an OD600 of >0.3); thereby representing a significantly improved glycerol-growth phenotype over reported genetically-engineered yeast strains.
  • the genetically modified yeast is capable of expressing enhanced levels of glycerol kinase activity (EC: 2.7.1.30).
  • the genetically modified yeast comprises a transgene, integrated into the genome, that encodes a polypeptide having glycerol kinase activity (EC: 2.7.1.30), thereby enhancing the level of glycerol kinase activity detectable in the cell during growth on glycerol.
  • the amino acid sequence of the polypeptide has at least 80%, 82, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98% or 100% amino acid sequence identity to SEQ ID No. : 2.
  • the yeast comprises a mutant gene encoding a mutant polypeptide having enhanced glycerol kinase activity (EC: 2.7.1.30) as compared to the polypeptide encoded by the parent gene from which the mutant was derived.
  • the amino acid sequence of said glycerol kinase has at least 80%, 82, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98% or 100% amino acid sequence identity to SEQ ID No. : 4, and wherein said sequence comprises an E572Q mutation with respect to the polypeptide encoded by the parent gene.
  • the mutant gene encoding the glycerol kinase may be a transgene integrated into the genetically modified yeast; or the mutant gene may be a genetically edited endogenous GUT1 gene.
  • the genetically modified yeast is devoid of genes capable of expressing a functional alpha ketoglutarase complex, as well as being devoid of genes capable of expressing a polypeptide having E2 ubiquitin-conjugating enzyme activity (EC: 2.3.2.23) (see Example 5.5).
  • the genetically modified yeast of the invention is not capable of forming a functional alpha ketoglutarase complex due to inactivation of endogenous gene(s) required to express one or more functional polypeptide components of this complex.
  • the complex comprises three subunits: 2-KetoGlutarate Dehydrogenase (Kgd l) (EC: 1.2.4.2); Dihydrolipoyl transsuccinylase (Kgd2) (EC 2.3.1.61); and dihydrolipoamide dehydrogenase (Lpd l) (EC: 1.8.1.4).
  • the complex catalyzes the oxidative decarboxylation of alpha-ketoglutarate to succinyl-CoA in the TCA cycle is a key control point in the citric acid cycle. Inactivation of the alpha ketoglutarase complex in the yeast of the invention is thought to result in an uncoupling of the TCA cycle and oxidative phosphorylation (see Example 5.5).
  • the genetically modified yeast of the invention is devoid of a KGDl gene that is capable of expressing Kgd l; due to the inactivation or deletion of the endogenous KGDl gene.
  • the amino acid sequence of the polypeptide has at least 80%, 82, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98% or 100% amino acid sequence identity to SEQ ID No. : 6.
  • Inactivation of an endogenous KGDl gene may be due to a failure to transcribe and express the KGDl gene; or may be due to a failure to express a functional alpha-ketoglutarate dehydrogenase enzyme.
  • the genetically modified yeast of the invention comprises a mutant KGDl gene encoding an amino acid sequence having at least 80%, 82, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98% or 100% amino acid sequence identity to SEQ ID No. : 8, and wherein said amino acid sequence comprises an A990D mutation when compared to the polypeptide encoded by the parent gene from which the mutant gene was derived.
  • the genetically modified yeast of the invention comprises a mutant KGDl gene may be derived from a parent yeast strain by means of genetically editing the endogenous KGDl gene in cells of the parent yeast, as described in Example 3.
  • the genetically modified yeast of the invention is devoid of a KGD2 gene that is capable of expressing dihydrolipoyl transsuccinylase (Kgd2) (EC 2.3.1.61); due to the absence of this gene or due to the inactivation of the endogenous KGD2 gene.
  • Kgd2 dihydrolipoyl transsuccinylase
  • the amino acid sequence of the polypeptide has at least 80%, 82, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98% or 100% amino acid sequence identity to SEQ ID No. : 10.
  • the genetically modified yeast of the invention is devoid of a LPD1 gene that is capable of expressing
  • dihydrolipoamide dehydrogenase (EC: 1.8.1.4); due to the absence of this gene or due to the inactivation of the endogenous LPD1 gene.
  • the amino acid sequence of the polypeptide has at least 80%, 82, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98% or 100% amino acid sequence identity to SEQ ID No. : 12 .
  • the genetically modified yeast of the invention is devoid of a UBC13 gene that is capable of expressing a polypeptide having E2 ubiquitin- conjugating enzyme activity; due to the absence of this gene or due to the inactivation of the endogenous UBC13 gene.
  • amino acid sequence of the polypeptide has at least 80%, 82, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98% or 100% amino acid sequence identity to SEQ ID No. : 14.
  • Inactivation of an endogenous UBC13 gene may be due to a failure to transcribe and express the UBC13 gene; or may be due to a failure to express a functional E2 ubiquitin-conjugating enzyme.
  • the genetically modified yeast of the invention comprises a mutant UBC13 gene encoding an amino acid sequence of no more than 71 residues having at least 80%, 82, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98% or 100% amino acid sequence identity to SEQ ID No. : 16.
  • the genetically modified yeast of the invention comprises a mutant UBC13 gene may be derived from a parent yeast strain by means of genetically edited endogenous UBC13 gene in cells of the parent yeast, as described in Example 3.
  • the genetically modified yeast of the invention may be selected from a member of the genus Saccharomyces (e.g. S. cerevisiae), Kluyveromyces (e.g. K. lactis, K.
  • a genetically modified yeast of the invention may be derived from a parent strain selected from any member of the above genera.
  • the invention provides a method for culturing cells of the genetically modified 5 yeast comprising the steps of:
  • the cultivation medium may comprise 10 - 60 g/L glycerol, for example at least 15, 20, 25, 30, 35, 40, 45, 50, 55 g/L glycerol.
  • the defined cultivation medium may be a synthetic medium.
  • the medium comprises a source of minerals, inorganic nitrogen, trace metals and vitamins, as illustrated in Example 1.
  • Additional supplementary carbon sources may include: methanol, molasses (sugar cane of sugar beet), corn syrup and hydrolyzed biomass (e.g. lignocellulose).
  • the cells may be culture by batch fermentation, or in a chemostat under continuous aeration.
  • the invention provides for the use of a genetically modified yeast of the genus Saccharomyces (e.g. S. cerevisiae), Kluyveromyces (e.g. K. lactis, K. marxianus and Lachancea thermotolerans); Komagataella (e.g. Komagataella phaffii/ Komagataella pastoris/ Pichia pastoris), Scheffersomyces (e.g. S. stipites/ Pichia stipitis); Torulaspora (T. delbrueckii) and Zygosaccharomyces (e.g. Z. bailii and Z.
  • Saccharomyces e.g. S. cerevisiae
  • Kluyveromyces e.g. K. lactis, K. marxianus and Lachancea thermotolerans
  • Komagataella e.g. Komagataella phaffii/ Komagataella pastoris/ Pi
  • a genetically modified micro-organism of the invention may be a strain of yeast that is suitable for use as a cell factory for the production of valuable compounds, including biofuels, small chemicals, pharmaceuticals and nutraceuticals.
  • the genetically modified micro-organism of the invention may be derived from a parent yeast strain, where the parent yeast strain itself comprises one or more genetic modification resulting in the expression of genes encoding heterologous single and multi-step metabolic pathways, allowing the production of these compounds.
  • a parent yeast strain may first be genetically modified to produce the micro-organism of the invention; which thereafter is subjected to further genetic modified to introduce metabolic pathways required for the production of valuable compounds.
  • the present invention provides a method for enhancing glycerol metabolism in a parent yeast strain by genetic modification of cells of the parent strain to produce a genetically modified yeast of the invention.
  • a nucleic acid molecule comprising a gene encoding a polypeptide having glycerol kinase activity may be introduced into an integration or self-replicating vector suitable for cloning and introducing into cells of a parent micro-organism using methods and techniques for transformation that are well known to those skilled in the art (see, e.g., Sambrook et al., Molecular Cloning : A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989).
  • the existing endogenous GUTl and UBC13 genes and at least one the alpha ketoglutarase complex genes (KGD1, LPD1 and KGD2), in a cell of a selected parent yeast may be genetically modified to produce a yeast of the invention.
  • the nucleic acid sequence of an endogenous gene can be modified by means of gene editing using CRISPR-Cas9 techniques (1, 2) optimized for the S. cerevisiae, as illustrated in Example 3.
  • the endogenous UBC13 gene and at least one of the endogenous KGD1, LPD1 and KGD2 genes in a cell of a selected parent yeast may be inactivated by deletion of, or substitution of, or addition of, at least one nucleotide within the gene leading to a loss of expression of a functional polypeptide encoded by the gene.
  • Suitable techniques for gene inactivation (knock-out) can be achieved by gene targeting via homologous recombination, techniques that are standard in the art; or by means of gene editing using CRISPR-Cas9 techniques (1, 2).
  • a nucleic acid molecule, that leads to one or more genetic modifications of GUTl, KGD1, LPD1, KGD2 or UBC13 can be introduced into a yeast of interest via well-established genetic transformation techniques (3).
  • a genetically modified yeast of the invention is characterised by a lack of both alpha ketoglutarase complex activity (e.g. 2-ketoglutarate dehydrogenase enzyme activity (EC: 1.2.4.2)) and E2 ubiquitin-conjugating enzyme activity (EC: 2.3.2.23).
  • alpha ketoglutarase complex activity e.g. 2-ketoglutarate dehydrogenase enzyme activity (EC: 1.2.4.2)
  • E2 ubiquitin-conjugating enzyme activity EC: 2.3.2.23
  • a commercially available assay for example, BioVision, http://www.biovision.com/alpha-ketoqlutarate-dehvdroqenase-activitv- colorimetric-assav-kit-7786.html
  • a well-established assay as described in (4) may be used to measure 2-ketoglutarate dehydrogenase enzyme activity (EC: 1.2.4.2) to identify a micro-organism of the invention lacking this enzyme activity.
  • Microorganisms lacking the KGD1 activity can also be identified by analysing growth curves of mutant strains cultured on a minimal glucose media and observing the lack of growth after diauxic shift (deficiency in respiratory growth) (4).
  • An enzyme assay for measuring E2 ubiquitin-conjugating enzyme activity (EC: 2.3.2.23) to identify a micro-organism of the invention lacking this enzyme activity is described in the literature (7).
  • a genetically modified yeast of the invention lacking polypeptides having alpha-ketoglutarate dehydrogenase enzyme activity (EC: 1.2.4.2) and polypeptides having E2 ubiquitin-conjugating enzyme activity (EC: 2.3.2.23) can be detected by immunodetection. Preparation of cell extracts and immunodetection methods for detecting the presence of these specific polypeptides are well known in the art.
  • Example 1 Isolation and characterization of a mutant yeast strain adapted for growth on glycerol
  • the parent yeast strain was the wild-type laboratory strain CEN.PK113-7D [CBS 8340:
  • the cultivation media were based on a defined mineral (M) media described by Verduyn et al., (8) containing 5 g/L (NH 4 ) 2 S0 4 , 3 g/L KH 2 P0 4 , 0.75 g/L Mg 2 S0 4 , 1.5 mL/L trace metal solution and 1.5 mL/L vitamins solution.
  • M defined mineral
  • the composition of the trace metal solution was 3 g/L FeS0 4 -7H 2 0, 4.5 g/L ZnS0 4 -7H 2 0, 4.5 g/L CaCI 2 -6H 2 0, 0.84 g/L MnCI 2 -2H20, 0.3 g/L CoCI 2 -6H 2 0, 0.3 g/L CuS0 4 -5H 2 0, 0.4 g/L NaMo0 4 -2H 2 0, 1 g/L H 3 B0 3 , 0.1 g/L KI and 15 g/L Na 2 EDTA-2H 2 0.
  • the vitamin solution includes 50 mg/L d-biotin, 200 mg/L para-amino benzoic acid, 1.0 g/L nicotinic acid, 1.0 g/L Ca-pantothenate, 1.0 g/L pyridoxine-HCI, 1.0 g/L thiamine-HCI and 25 mg/L inositol.
  • the mineral medium when supplemented with 10 mL/L of glycerol as sole carbon source, was designated "MG" medium.
  • the pH was adjusted with KOH/H 2 S0 4 to 4.2.
  • the "MG” medium was additionally supplemented with 1.92 g/L of Y1501 amino acid mix (Sigma), it was designated "MG+” medium.
  • the cultivation media were filter-sterilized using bottle-top (0.45 ⁇ pore size) filters
  • the parent strain was pre-cultured in 500 mL shake flask with 50 mL of MG+ medium to provide a starter culture, which was used to inoculate five flat- bottom plastic tubes with 15 mL of MG+ medium at a starting cell density of 0.3 OD 6 oo-
  • the five tubes were cultured at 30°C with constant agitation using a magnetic stirrer at 1000 rpm. Growth in each tube was monitored, and once the cell culture reached early exponential growth phase, an aliquot of 900 ⁇ was then serially passaged to another tube comprising fresh medium.
  • the composition of the growth medium was gradually changed from MG+ to the MG medium (by providing a mixture of the MG+ and MG medium starting with a ratio of 100: 0 and gradually transferring to a ratio of 0 : 100).
  • Yeast cultures were evolved over at least 300 cell generations, resulting in strains capable of growing on a minimal medium supplemented with glycerol as carbon source.
  • One of the best performing strains was designated, ALE#2.
  • ALE#2 was analysed by whole-genome sequencing that revealed the genetic mutations in relation to the parental strain CEN.PK113-7D. Further genetic characterization, using re-engineering, was performed to pinpoint three causative mutations, located in the genes: GUTl, KGD1 and UBC13, that were found responsible for the observed superior glycerol growth phenotype of ALE#2.
  • the identified mutant GUTl gene [SEQ ID No. : 3] had a positive strand G1711C substitution as compared to wild-type GUTl, and encoded a mutant Glycerol UTilization polypeptide, Gutlp (>GUT1_1 ; [SEQ ID No. :4]) having a (E572Q) substitution.
  • the identified mutant KGD1 gene [SEQ ID No. : 7] had a positive strand C2969A substitution as compared to wild-type KGD1, and encoded a mutant 2-KetoGlutarate Dehydrogenase polypeptide, Kgd lp (>KGD1_1; [SEQ ID No. : 8]) having a (A990D) substitution.
  • the identified mutant UBC13 gene [SEQ ID No. : 15] had a positive strand 209AG deletion as compared to wild-type UBC13, creating a frame shift, and encoded a C-terminally truncated mutant UBiquitin-Conjugating polypeptide, Ubcl3 (>UBC13; [SEQ ID No. : 16]) having an R70L substitution and a stop codon after the 71 st a. a. residue.
  • ALE#2 was found to contain a point mutation in the INO80 gene.
  • the mutant INO80 gene [SEQ ID No. : 17] had a positive strand G1076A substitution encoding a mutant ATPase and nucleosome spacing factor (EC: 3.6.4.12), INO80_l [SEQ ID No. : 18]) having a C359Y substitution.
  • Example 2 Functional properties of the polypeptides encoded by the mutant genes in the adapted yeast strain, ALE#2
  • the presence of the Gutl enzyme in yeast is indispensable for its utilization of glycerol, and hence deletion of the GUTl gene in yeast completely abolishes growth on glycerol.
  • the mutant GUTl gene comprising the SNP (G1711C), encodes a mutant glycerol kinase Gutl having an E572Q substitution.
  • the mutation confers a gain-of-function phenotype which improves flux in the glycerol uptake pathway resulting in an increased efficiency of glycerol catabolism (figure 2).
  • KGD1 is a component of a mitochondrial multiprotein alpha ketoglutarase complex, comprising three subunits: 2-KetoGlutarate Dehydrogenase (Kgd l); Dihydrolipoyl transsuccinylase (Kgd2); and dihydrolipoamide dehydrogenase (Lpd l).
  • the complex catalyzes the oxidative decarboxylation of alpha- ketoglutarate to succinyl-CoA in the TCA cycle, and is a key control point in the citric acid cycle.
  • the mutant KGD1 gene comprising the SNP (C2969A), encodes a mutant Kgdlp having an A990D substitution.
  • UBC13 is an E2 ubiquitin-conjugating polypeptide, performing the second step in the ubiquitination reaction that targets a protein for degradation via the proteasome; and involved in the error-free DNA post-replication repair pathway.
  • the mutant UBC13 gene comprising a deletion (209AG) that creates a frame shift, encodes a C-terminally truncated mutant UBiquitin- Conjugating polypeptide of 71 amino acids, having a R70L substitution.
  • the truncated polypeptide lacks the active site cysteine residue required to form a thiol-ester with ubiquitin or ubiquitin-like proteins. While not wishing to be bound by theory, it is predicted that inactivation of the UBC polypeptide due to its truncation, confers a gain-of-function phenotype which increases the efficiency of glycerol catabolism.
  • Example 3 Re-engineering yeast strains adapted for growth on glycerol 2.1 Reengineering mutations in wild-type yeast strains
  • the mutations identified in the three genes (GUT1_1, KGD1_1 and UBC13_1) were introduced into selected wild type yeast strains by means of CRISPR- Cas9 techniques (1, 2) optimized for S. cerevisiae.
  • a specific synthetic guide RNA (gRNA) sequence (see Table 2) was designed for targeting the Cas9 nuclease to the appropriate genetic locus at which each genetic modification was to be introduced.
  • the quality and specificity of gRNA was assessed using a CRISPRdirect online tool developed by Naito et al., (9).
  • Repair templates (90-bp dsOligo flanking 45 bp up- and 45 bp downstream of the specific cut site) were designed to repair the DNA double strand break introduced by the Cas9 nuclease at each genetic locus.
  • Each repair template contained a specific mutation and a silent mutation that would disturb the Protospacer Adjacent Motif site, PAM (5'-NGG-3') (see Table 2).
  • GUT1_1 CA 1 CCAC 1 GCCAGAACCGACG 1 1 1 1 AGAGCTAGAA 20
  • KGD1_1 CaGCAGCAACAaCACCAC I I G I 1 1 1 AGAGCTAGAA 21
  • INO80_l GGAATCGATTGGATTGTAGTG 1 1 1 1 AGAGCTAGAA 23
  • Double stranded repair templates (only
  • GUT1_1 AGACAGGGAC 1 1 1 1 1 1 1 AGAGGAAA 1 1 1 CCGACG 1 CA 24
  • Cells of four parental strains (Laboratory strain CEN.PK113-1A (isogenic to the CEN.PK113-7D) and industrial strains (CLIB328 and L1528) listed in Table 3) were each transformed with the Cas9 expressing plasmid pCfB2312 (2) resulting in the respective transformed strains (e.g. reconstructed laboratory strain lA_Cas9).
  • the yeast transformants were selected on yeast extract peptone dextrose (YPD) agar plates supplemented with the selective antibiotic, Geneticin (G418).
  • yeast strains were replica-plated on YPD+G418+CloNat and YPD+G418 media in order to select for the mutants that have lost the respective gRNA expressing plasmid.
  • Yeast cells with a single gene mutation (lacking the corresponding gRNA plasmid) were then transformed with an additional gRNA expressing plasmid targeting a different locus.
  • gRNA plasmids were "kicked out" from cells of the selected strain harboring two gene mutations.
  • the CRISPR- Cas9 gene mutagenesis procedure was then repeated on the selected strains, in order to generate yeast strains containing three gene mutations (see Table 3). Table 3. List of the yeast strains
  • a 200 ⁇ _ aliquot of each re-suspended pre-culture was transferred to an appropriate volume of fresh MG medium such as to give a pre-inoculum suspension having a cell density of 4.5 OD 6 oo-
  • 50 ⁇ _ of each pre- inoculum suspension was inoculated into a separate well of a Krystal 24-well clear bottom white microplate (Porvair Sciences) prefilled with 700 ⁇ _ MG media per well; and incubated for minimum of 80 hours at 30°C with 225 rpm shaking. Cell growth (green color intensity, G-value) was monitored by scanning the bottom of the plates at 30 minute intervals.
  • the re-engineered yeast strains derived from a parental laboratory yeast strain (CEN .PK (CEN.PK113-1A)), and comprising at least two mutant gene (GUTl-1; KGDl-1 ; UBC13-1 and INO80- 1), showed measurable growth on MG medium comprising a defined mineral (M) media supplemented with 10 mL/L of glycerol as sole carbon source (Figure 2). Growth of the engineered strains was detected after a shorter lag phase, and the cell density reached within lOOh was significantly increased. Growth of re-engineered strains comprising three mutant genes was greater than for any combination of two mutant genes.
  • the re-engineered yeast strains derived from the industrial yeast strains (L.1528 and CLIB382), and comprising at least one mutant gene (UBC13-1) showed improved growth on MG medium ( Figure 3).
  • the batch cultures were sampled at regular intervals to determine cell density (OD 600 ), cell dry weight (CDW).
  • CDW Cell dry weight sampling was performed to determine the biomass concentration of each yeast culture over time.
  • the growth rate of the re-engineered yeast strain comprising the three mutant genes: GUTl-1 ; KGDl-1 and UBC13-1, was 0.130 ⁇ 0.007.h _1 , which was not significantly less than the evolved strain ALE#2 ( Figure 4). This indicates that the combination of GUTl-1 ; KGDl-1 and UBC13-1 mutations represent essential genetic modifications required for conferring the glycerol-growth phenotype of the ALE#2 yeast strain.
  • Example 5 Molecular characterization of the re-engineering yeast strains adapted for growth on glycerol Transcriptomic and proteomic profiles of the re-engineered strains (as described in Example 3) grown in well-controlled reactors, were used to determine the impact of the causal mutant genes (KGDl-1 and UBC13-1) at a molecular level. These data were complemented with an analysis of changes in metabolic flux in the re-engineered strains; in particular to determine the impact of the KGDl-1 mutant gene.
  • RNA samples were prepared as follows, 10 ml_ of fermentation broth, derived from cultivation of each respective yeast strain, was sprayed into 50 mL 25 Falcon® tube filled with ice and immediately centrifuged at lOOOOxg for 5 min at 4°C. After centrifugation, the supernatant was discarded and the cell pellet was frozen; and kept at -80°C until extraction. Total RNA in each frozen cell pellet was isolated using RNAeasy kit (Qiagen) by following manufacturer's recommendations.
  • RNA was then eluted with 60 ⁇ _ of RNase- free water; digested with Turbo DNAse (Invitrogen Ambion) accordingly to manufacture instructions followed by RNA clean-up (RNAeasy kit, Qiagen).
  • RNA library was prepared using the Illumina TruSeq Stranded mRNA LT sample prep kit starting with 500ng of total RNA, following manufactures instructions using Beckman Biomek FX Laboratory automation station. Samples were sequences using Hiseq2000 instruments in the 50 bp single read mode and loaded 8pM onto the flowcell at the Genomics Core Facility of the EMBL (Heidelberg, Germany).
  • the quality of the raw RNA sequencing reads was assessed using FastQC (version 0.11.3). Prior to the alignment, adapter trimming was performed using cutadapt (version 1.9.1) with default options providing the standard Illumina TrueSeq Index adapters. Subsequent quality trimming and filtering was performed with FaQCs (version 1.34) using the following parameters: -q 20 -min_L 25 -n 5 -discard 1. The total reads per sample after trimming and filtering ranged from 17.5 to 27 million. The sequencing reads were aligned to the reference genome of S.
  • the sample was cooled to 24 °C and alkylated with iodacetamide (room temperature, in the dark, 30 minutes, 10 mM). Proteins were TCA precipitated; TCA pellet was washed by acetone and dried. The proteins were digested in 50 mM HEPES (pH 8.5) using LysC (Wako) with an enzyme to protein ration 1 : 50 at 37°C for 4 hours, followed by trypsin (Promega) with an enzyme to protein ratio 1 : 50 at 37°C overnight. TMTlOplexTM Isobaric Label Reagent (ThermoFisher) was added to the samples according the manufacturer's instructions. Labeled peptides were cleaned up using OASIS® HLB ⁇ Plate (Waters).
  • Offline high pH reverse phase fractionation was performed using an Agilent 1200 Infinity high-25 performance liquid chromatography (HPLC) system, equipped with a Gemini C18 column (3 ⁇ , 110 A, 100 x 1.0 mm, Phenomenex).
  • the solvent system consisted of 20 mM ammonium formate (pH 10.0) as mobile phase (A) and 100% acetonitrile as mobile phase (B).
  • Peptides were separated using the UltiMate 3000 RSLC nano LC system (Dionex) fitted with a trapping cartridge ( ⁇ -Precolumn C18 PepMap 100, 5 ⁇ , 300 ⁇ i.d.
  • cells of each respective yeast strain, cultured on MG medium were harvested using a modified fast filtration protocol (13), as follows. Briefly, 5ml of culture were sampled at mid- exponential growth phase and were vacuum-filtered through nylon membrane filters (0.45 ⁇ , WhatmanTM), followed by three rapid washing steps with 5 ml of PBS to ensure no contamination from extracellular metabolites.
  • the polar metabolites were extracted by adding the cell-containing filter in 5 ml of cold (-20°C) HPLC-grade 2 methanol (Biosolve Chimie, France)/MilliQ water (1 : 1, v/v) and incubating for lh at -20°C.
  • the mixture of metabolites and cell debris was centrifuged at 10000 rpm and 0°C for 10 min, and the supernatants were collected and dried with speed-vac.
  • the dried metabolites were derivatized to their (MeOx)TMS-derivatives through reaction with 100 ⁇ _ of 20 mg/mL methoxyamine hydrochloride (Alfa Aesar, UK) solution in pyridine (Sigma-Aldrich) for 90 min at 40°C, followed by reaction with 200 ⁇ _ N-methyl-trimethylsilyl-trifluoroacetamide (MSTFA) (Alfa Aesar, UK) for 10 hours at room temperature.
  • MSTFA N-methyl-trimethylsilyl-trifluoroacetamide
  • the metabolic profile of each sample was measured thrice using a Shimadzu TQ8040 GC-(triple quadrupole) MS system (Shimadzu Corp.).
  • the metabolite quantification was carried out by calculating the peak areas of the identified marker ions of each metabolite. For glucose, the smaller of the two derivative peaks was used for quantification.
  • Metabolic reactions in S. cerevisiae that might facilitate glycerol utilization were investigated by means of model simulations. Specifically, a mixed- integer linear programming routine was used to identify a minimum number of reactions, in the genome-scale metabolic model of S. cerevisiae (iFF70816) that might be required for optimal glycerol utilization when provided with glycerol as carbon source. The metabolic flux during respiratory growth on glucose was used as a reference metabolic state.
  • R-GU UBC13-1 yeast strain
  • Dld3 Dld3 (2-hydroxyglutarate transhydrogenase

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Abstract

L'invention concerne des souches de levure GM pour utilisation en tant qu'usines cellulaires qui sont capables d'utiliser un déchet, le glycérol, comme unique source de carbone, sans addition du moindre supplément en acides aminés. L'invention enseigne en outre comment modifier des souches de levure existantes pour obtenir ces propriétés avantageuses, qui font que le niveau d'activité de la glycérol kinase (EC :2.7.1.30) est renforcé et que l'expression des gènes codant pour l'activité du complexe fonctionnel alpha-cétoglutarase (EC : 2.7.1.30) et pour l'activité enzymatique de conjugaison de l'ubiquitine E2 (EC : 2.3.2.23) est inactivée.
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CN112280762A (zh) * 2020-11-13 2021-01-29 中山俊凯生物技术开发有限公司 一种烟酰胺核糖激酶突变体及其编码基因和应用
GR1010622B (el) * 2022-12-29 2024-01-30 Εθνικο Κεντρο Ερευνας Και Τεχνολογικης Αναπτυξης (Ε.Κ.Ε.Τα), Νεα στελεχη yarrowia lipolytica με ικανοτητα αναπτυξης σε υψηλα ποσοστα ακατεργαστης γλυκερολης

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020109371A1 (fr) * 2018-11-28 2020-06-04 Rheinisch-Westfälische Technische Hochschule (Rwth) Aachen Extraits de levure riches en polyphosphates et procédé de fabrication y relatif
CN112280762A (zh) * 2020-11-13 2021-01-29 中山俊凯生物技术开发有限公司 一种烟酰胺核糖激酶突变体及其编码基因和应用
CN112280762B (zh) * 2020-11-13 2022-11-01 中山俊凯生物技术开发有限公司 一种烟酰胺核糖激酶突变体及其编码基因和应用
GR1010622B (el) * 2022-12-29 2024-01-30 Εθνικο Κεντρο Ερευνας Και Τεχνολογικης Αναπτυξης (Ε.Κ.Ε.Τα), Νεα στελεχη yarrowia lipolytica με ικανοτητα αναπτυξης σε υψηλα ποσοστα ακατεργαστης γλυκερολης

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