WO2015095298A2 - Utilisation des polyamines et des transporteurs de polyamines pour conférer une tolérance au furfural - Google Patents

Utilisation des polyamines et des transporteurs de polyamines pour conférer une tolérance au furfural Download PDF

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WO2015095298A2
WO2015095298A2 PCT/US2014/070795 US2014070795W WO2015095298A2 WO 2015095298 A2 WO2015095298 A2 WO 2015095298A2 US 2014070795 W US2014070795 W US 2014070795W WO 2015095298 A2 WO2015095298 A2 WO 2015095298A2
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microorganism
furfural
fusarium
polyamines
putrescine
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PCT/US2014/070795
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WO2015095298A3 (fr
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Ryan D. GEDDES
Xuan Wang
Lorraine P. Yomano
Elliot N. MILLER
Huabo ZHENG
Keelnatham T. Shanmugam
Lonnie O. Ingram
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University Of Florida Research Foundation, Incorporated
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/22—Processes using, or culture media containing, cellulose or hydrolysates thereof
    • 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
    • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
    • 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
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
    • 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

  • Lignocellulosic biomass can be used as a renewable carbohydrate feedstock for the production of fuels and chemicals.
  • lignocellulose is a structural component designed to resist microbial enzymes and chemical deconstruction. A pretreatment step is required to increase the accessibility of cellulase enzymes for more efficient carbohydrate hydrolysis. 2
  • Dilute acid pretreatment has been widely investigated and effectively opens the lignocellulose structure by hydro lyzing the pentose sugars in hemicellulose. Dilute acid pretreatments also produce unwanted side products that retard fermentation. 3 ' 4
  • Two of the most abundant side products are furans, namely, furfural and hydroxymethylfurfural (HMF). 5"7
  • the concentration of furans in hydrolysates has been correlated with toxicity. Adding furfural to detoxified hydrolysates has been shown to restore toxicity. 8 ' 9
  • Furfural has been proposed to cause strand breaks in DNA 10 , inhibit glycolytic enzymes, 11 ' 12 damage membranes and react with various cellular components. 13 In yeast, furfural has been shown to elicit a general response to reactive oxygen species. 13 Many different methods have been developed to mitigate or remove inhibitors. 14 However, all of these methods increase costs and process complexity.
  • a genetic solution that provides resistance to hydro lysate inhibitors eliminates additional costs. Genetic methods have been used to increase the resistance of bacterial and fungal biocatalysts to furfural and hydroxymethylfurfural. 5 ' 7 ' 15 19 In yeast, overexpression of the transcription factor Yapl has been shown to increase tolerance to both furfural and HMF. 20 Microarray analysis revealed that the NADPH-dependent oxidoreductase, YqhD, is involved in furan tolerance in E. coli. 21 Native expression of yqhD was found to inhibit the growth of E. coli in media containing furfural. 6 ' 21 Blocking the functional expression of this gene increased furfural tolerance.
  • the current invention provides expression of polyamine transporter genes and/or polyamine supplements to increase furfural tolerance in microorganisms, for example, E. coli.
  • the increase in furfural tolerance may result from polyamine binding to polyanionic constituents, such as nucleic acids and phospholipids, limiting their contact and reactivity with furfural.
  • the present invention provides a genetically modified microorganism comprising genetic modifications involving one or more target genes encoding:
  • genetic modifications cause increased expression and/or activity of the polypeptides produced by said target genes.
  • the genetically modified microorganism of the current invention is resistant to the presence of furfural and/or 5-hydroxymethylfurfural (5-HMF), which is a beneficial characteristic of the microorganism for production of a desired product, for example, a chemical, form biomass. Accordingly, the current invention also provides a method of producing a desired product, the method comprising contacting a biomass with the microorganism and producing the desired product by fermenting the biomass in the presence of the microorganism.
  • furfural and/or 5-hydroxymethylfurfural 5-HMF
  • the current invention also provides a method of increasing furfural and/or 5-HMF resistance in a microorganism by introducing in to the microorganism genetic modifications involving one or more target genes encoding:
  • the genetic modifications increase expression or activity of the polypeptide produced by said target genes.
  • FIGS 1A-1C Plasmid maps showing DNA inserts conferring furfural tolerance. Open arrows represent Sau3AI fragments (surrogate promoters). Solid arrows represent plasmid DNA. Unlabeled upward hash marks represent Sau3AI sites separating unique chromosomal fragments. Downward hash marks provide a scale with 500 bp intervals.
  • Figures 2A-2B Effect of potE plasmids on furfural tolerance to E. coli strain XW092. Growth was used as a measure of furfural tolerance.
  • A Effect of plasmids containing potE, Sau3AI fragments, and the fucO-ucpA cassette on furfural tolerance. Plasmid pACYC184 was included as a control.
  • B Effect of plasmid pLOI5249 containing the potE gene behind the tac promoter on furfural tolerance. IPTG (0.1 mM) was added as indicated. Plasmid pTrc99A was included as a control.
  • FIG. 3 Alignment of sequencing reads from EMFR35 on the puuP region of LY180 (parent). Template coordinates are shown as hatch-marked numbers at the bottom. Sequence coverage of the region containing puuP is increased approximately 5-fold compared to puuR and the flanking genome.
  • Figures 4A-4B Effects of polyamine transporters on furfural tolerance in LY180. Growth was used as a measure of furfural tolerance.
  • Figures 5A-5B Effect of polyamine supplements on furfural (10 mM) tolerance in LY180. Growth was used as a measure of furfural tolerance.
  • IPTG IPTG
  • Figure 7 Effect of polyamine supplements (1.0, 5.0, 10.0 mM) on the growth of LY180 in the absence of furfural.
  • strain designations were deposited with the Agricultural Research Service Culture Collection, 1815 N. University Street, Peoria, Illinois 61604 USA on April 14, 2009: LY180 (strain designation NRRL B-50239); EMFR9 (strain designation NRRL B-50240); and EMFR35 (strain designation NRRL B-50243). These cultures have been deposited under conditions that assure that access to the cultures will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of the deposits does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
  • the phrase "genetic modifications involving one or more target genes encoding”, as used in this application, is to be understood to mean any one target gene or any combination of target genes provided in the list following the phrase.
  • the invention provides microorganisms for production of renewable fuels and other chemicals. Particularly, the invention provides microorganisms that can grow and produce renewable fuels and other chemicals in the presence of increased furfural.
  • the present invention provides a genetically modified microorganism comprising genetic modifications involving one or more target genes encoding:
  • genetic modifications cause increased expression and/or activity of the polypeptides produced by the target genes.
  • a microorganism can be a prokaryote, namely a bacterium or an archaea; a eukaryote, for example, a protozoan, a fungus, an alga, a microscopic plant (green algae).
  • the microorganisms of the current invention are typically microscopic, i.e. not visible with the naked eye; however, certain microorganisms, for example, certain filamentous fungi, can be macroscopic and visible to the naked eye.
  • a bacterium is a microscopic, single-celled organisms belonging to Kingdom Monera that possess a prokaryotic type of cell structure, which means their cells are not compartmentalized, and their DNA (usually circular) can be found throughout the cytoplasm rather than within a membrane-bound nucleus.
  • a bacterium typically reproduces by fission or by forming spores.
  • Yeast is typically a microscopic single-celled fungus that reproduces asexually by budding or binary fission, produces ascospores, and is capable of fermenting carbohydrates. Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. 10 M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
  • the fungus can be a filamentous fungus.
  • "Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).
  • a filamentous fungus is multinucleated, filamentous microorganism composed of hyphae.
  • a hypha is a branching tubular structure approximately 2-10 ⁇ in diameter which is usually divided into cell-like units by cross- walls called septa.
  • Filamentous fungus has a cell wall usually composed of chitin, sometimes cellulose, occasionally both chitin and cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Filamentous fungus is obligate aerobe and grows by elongation at apical tips of their hyphae and thus is able to penetrate the surfaces on which they begin growing.
  • Genetic modifications applicable to the current invention include genetic modifications that cause increased expression and/or activity of the polypeptides encoded by the target genes.
  • incrementsing refers to increasing by at least 5%, for example, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 100% or more, a particular activity (e.g., increased PotE activity).
  • decreasing refers to reducing by at least 5%, for example, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 100% or more, a particular activity (e.g., any decreased activity).
  • An increase (or decrease) in activity includes an increase (or decrease) in the rate and/or the level of a particular activity (e.g., furfural tolerance).
  • genetic modifications applicable to current invention include, but are not limited to:
  • the genetic modifications include two or more target genes.
  • a combination of different genetic modifications causing the increased expression and/or activity may be introduced in to the microorganism.
  • a first target gene may be introduced in to the microorganism in an extra-genomic manner, whereas a second target gene may be incorporated into the genomic DNA of the microorganism.
  • a mutation can be introduced in to a first target gene endogenously present in the microorganism, wherein the mutation increases the activity of the polypeptide encoded the target gene, and a mutation can be introduced in the promoter region of the second target gene endogenously present in the microorganism, wherein the mutation increases the expression of the polypeptide.
  • a person of ordinary skill in the art can design permutations and combinations of various genetic modifications that cause increased expression and/or activity of the target genes and such embodiments are within the purview of the current invention.
  • extra-genomic indicates that the gene is present outside the genomic DNA of the microorganism. Expression of a target gene in an extra-genomic manner indicates that a fragment of DNA outside the genomic DNA of the microorganism is transcribed and translated to produce the polypeptide encoded by the target gene.
  • An example of extra-genomic DNA capable of expressing a target gene is a plasmid. Additional examples of extra-genomic DNA are well known to a person of ordinary skill in the art and such embodiments are within the purview of the current invention.
  • a plasmid can be linear or circular DNA.
  • a plasmid typically contains an origin of replication, one or more promoters, and one or more genes under the control of the one or more promoters.
  • a plasmid can also optionally contain one or more genes conferring resistance to one or more antibiotics or other chemicals.
  • a plasmid can be used to express the one or more target genes in microorganisms, including but not limited to, a bacterium, a yeast, and a filamentous fungus.
  • Non- limiting examples of bacterial plasmids that can be used according to the current invention include pACYC177, pASK75, pBAD/His A, pBAD/His B, pBAD/His C, pBAD/MCS, pBADM-11, pBADM-20, pBADM-20(+), pBADM-30, pBADM-30(+), pBADM-41(+), pBADM-52, pBADM-52(+), pBADM-60, pBADM-60(+), pBAT4, pBAT5, pCal-n, pET-3a, pET-3b, pET-3c, pET-3d, pET-12a, pET-14b, pET-15b, pET-16b, pET-19b, pET-20b(+), pET-21d(+), pET-22b(+), pET-24d(+), pET-28a,
  • Fungal plasmids can be of different types.
  • Non- limiting examples of plasmids in fungi include integrative plasmids, episomal plasmids, autonomously replicating plasmids, and cen plasmids. Additional examples of fungal plasmids that can be used for expressing a target gene in fungi are well known to a person of ordinary skill in the art and such plasmids are within the purview of the current invention.
  • the genetic modification applicable to the current invention can be incorporation of the target genes into the genomic DNA of the microorganism. Incorporation of target genes in to the genomic DNA of the microorganisms can be achieved in a site specific manner or in a random manner.
  • Site specific incorporation of a target gene in to the genomic DNA of a microorganism can be achieved through homologous recombination between the genomic DNA of the microorganism comprising a specific DNA sequence and a fragment of DNA introduced in to the microorganism, wherein the fragment comprises the target gene flanked by a DNA sequence homologous to the specific DNA sequence in the genomic DNA of the microorganism.
  • Random incorporation of a target gene in to the genomic DNA of a microorganism involves incorporation of the target gene randomly, i.e. not in a sequence specific manner, in to the genomic DNA.
  • Various methods and techniques of site specific incorporation and random incorporation of a target gene in to the genomic DNA of a microorganism are well known to a person of ordinary skill in the art and such embodiments are within the purview of the current invention.
  • the genetic modifications according to the current invention also include introducing a mutation in the target gene endogenously present in the microorganism and wherein the mutation increases the activity of the polypeptide encoded by the target gene.
  • techniques designed to achieving specific mutations that cause increase in the activity of the polypeptide encoded by the gene include, but are not limited to, site directed mutagenesis of the target gene in the genomic DNA of the microorganism and site specific insertion of a mutant target gene into the genomic DNA of the microorganism.
  • Various methods and techniques of site directed mutagenesis and site specific insertions of mutant target genes are well known to a person of ordinary skill in the art and such embodiments are within the purview of the current invention.
  • Additional techniques of achieving specific mutations that cause increase in the activity of the polypeptide encoded by a target gene are well known to a person of ordinary skill in the art and such techniques are within the purview of the current invention.
  • the genetic modifications according to the current invention further include introducing a mutation in the promoter region of the target gene endogenously present in the microorganism wherein the mutation increases the expression of the polypeptide.
  • Examples of technologies designed to achieving specific mutations that cause increase in the promoter activity of the target gene include, but are not limited to, site directed mutagenesis of the promoter of the target gene in the genomic DNA of the microorganism and site specific insertion of a mutant promoter for the target gene into the genomic DNA of the microorganism.
  • site directed mutagenesis and site specific insertions of mutant promoter for the target genes are well known to a person of ordinary skill in the art and such embodiments are within the purview of the current invention.
  • Other techniques of achieving specific mutations that cause increase in the promoter activity of the target gene are well known to a person of ordinary skill in the art and such techniques are within the purview of the current invention.
  • a target gene is endogenous or exogenous to the microorganism.
  • An endogenous gene is naturally present in the microorganism; whereas, an exogenous gene is not naturally present in the microorganism but is present in an exogenous source, i.e. from an organism other than the microorganism.
  • certain strains of E. coli naturally contain potE gene.
  • a plasmid containing a potE gene can be introduced into a cell of E. coli endogenously containing the same potE gene, thereby expressing higher amounts of PotE protein than endogenously expressed in the cell of E. coli.
  • a cell of Saccharomyces cerevisiae does not contain a potE gene.
  • a plasmid containing a potE gene can be introduced into the cell of S. cerevisiae thereby expressing PotE protein exclusively from the exogenous potE gene.
  • all of the one or more target genes introduced in to the microorganism are present on one or more extra-genomic DNAs.
  • all of the one or more target genes introduced in to the microorganism are incorporated in to the genomic DNA of the microorganism.
  • the genetically modified microorganism contains some of the one or more target genes on the one or more extra-genomic DNAs introduced in to the microorganism and some of the one or more target genes are incorporated in to the genomic DNA of the microorganism.
  • Non-limiting examples of methods of bacterial transformation include heat-shock method or electroporation. Additional methods and techniques of introducing DNA into bacterial cells are well known to a person of ordinary skill in the art and such methods are within the purview of the current invention.
  • Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al, 1984, Proc. Natl. Acad. Sci. USA 81 : 1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al, 1989, Gene 78: 147-156, and WO96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, 15 In Abelson, J. N.
  • the target genes for the current invention are selected from genes encoding putrescine-ornithine antiporter (PotE), putrescine importer (PuuP), ATP-dependent polyamine transporter (PotABCD), and low affinity putrescine-importer (PlaP).
  • Proteins putrescine-ornithine antiporter is also referred to as Putrescine/proton symporter. Both these names refer to the same gene, potE.
  • Various PotE proteins present in a number of microorganisms, their corresponding entry numbers, entry names, protein names, and gene names are available in the UniProt Knowledge Base (UniProtKB) (see world wide website: uniprot.org).
  • UniProtKB UniProt Knowledge Base
  • a non-exhaustive list of PotE proteins represented by their corresponding Entry Numbers in the UniProt Knowledge Base is provided below. A person of ordinary skill in the art can obtained additional information about these proteins, for example, amino acid sequences of the proteins and nucleotide sequences of the corresponding genes, based on the Entry numbers provided herein to practice the current invention.
  • N2M4E2 N3J0W1; N2M2C9; N1SNA9; N2ME87; M9DZ98; N2FCK5; I2X7T8; I2Y210;
  • M8SWN6 M8RP98; N2QZJ8; I2YRJ8; I2TKD5; I2XUR9; K5F2F5; L9IQB6; I2YT51;
  • K3JXZ6 I2UM11; I2WQE5; I2S884; I2VWD6; K5G568; B2N7S4; I4TQA8; I4SMJ6; K3HVT2; K3HAV8; K5FY87; L9H3A8; K5KH62; K5H918; K5GZN2; K5I851; K5IIY5;
  • L1F2E6 L1GDW1; L1RXP4; I2RQ71; L1GD88; L9J5Q5; L1HIW6; L1HD70; L1HBE3; L8Z2K1; L8ZMD2; L9AUC3; L9ART5; L9B5J4; L9C044; L9HJ64; L9C5N7; L9H9A6;
  • E1I640 D7XXE6; E6BKW7; G0D138; E0QVU2; K3DXT1; K3SX11; K3CHR2; A1A8V6;
  • N3SJS6 N3TTH3; M9EY22; N3GCI1; N3G8C2; N4QVF7; N4QSX9; N4QSG6; N3HFC1;
  • I5QQZ4 I5UQS2; I5V1G0; K3HH65; F4M911; G2F846; F9R4B3; I1ZRS3; B7LKS3;
  • L9RLS2 J1W7N9; L6Q7Q1; L5W7K7; L6XLM8; L9SCJ4; J1IHW1; J2I495; L7A3R2;
  • L6DEP9 L6HKM7; L6IPH3; L6ESP1; L6AG48; L6GA54; L5WWD3; L5XLF0; L6PGD5;
  • L6L000 L5XVQ0; L9RXF2; L9QWG8; L6N263; L6N5Y7; L9Q6Z8; F2FPW5; I0ME47;
  • K8SN38 K8TNL0; K8U024; K8W6Q1; K8V4P0; K8STF4; K8TX32; M9XNS2; S5HF62;
  • I6DKK2 Q0T6T7; D2A9W7; Q3Z4B1; I6ES03; I6EVG9; F4NLT3; I2BDF4; I2B3S9;
  • G6ZMS3 G6ZY56; G7ACG2; G7AIZ1; G7B2K0; G7BD15; K5S2T7; F9CD04; K2UBU3;
  • H8K2A3 A3GIP7; M7G3S2; M7FY20; G7TVE6; M7FRI3; M7GAC1; M7H0S7; M7G8B2;
  • D2YQY4 D2YHA0; F3S0T7; S5J9B2; S5J6S7; F9RS74; H2ILM1; K5SV86; K5VD32;
  • Putrescine importer (PuuP) protein is encoded by the gene puuP.
  • Various PuuP proteins present in a number of microorganisms, their corresponding entry numbers, entry names, protein names, and gene names are available in the UniProt Knowledge Base.
  • a non- exhaustive list of PuuP proteins represented by their corresponding Entry Numbers in the UniProt Knowledge Base is provided below.
  • a person of ordinary skill in the art can obtained additional information about these proteins, for example, amino acid sequences of the proteins and nucleotide sequences of the corresponding genes, based on the Entry numbers provided herein to practice the current invention.
  • G7GIC4 J7LT56; K4M6E9; R1BRB6; C0Z898; D2TK07; C9Y2T6; L8Z7Z7;
  • N2LUN8 N1SHZ9; N2M1M7; M9E0Z9; N2ETZ0; N2INX5; M9L620; M9KM14; N2FFH5; N1TFD2; N2L2H5; N2KWM8; N2NH90; N2N3Z1; M9CSN7; N3JG51; N2DVC8;
  • M8ZV30 M9A1T9; M8YZK2; N2ECZ8; M8Y7C1; M8YA57; M8X7W6; M8WI59;
  • N1T2L7 N3QU15; N3S036; N3S6H2; N3PGR9; N3SEF6; N3T909; M9FV09; N3GBK2;
  • ATP-dependent polyamine transporter is encoded by the gene operon potABCD.
  • Operon potABCD consists of four genes, namely, potA, potB, potC, and potD. These four genes encode for four subunits of ATP-dependent polyamine transporter.
  • potA gene encodes PotA protein which forms the ATPase subunit
  • potB and potC genes encode for PotB and PotC proteins, which contain multiple a-helical hydrophobic domains and form a channel for transport of polyamines
  • potD gene encodes for PotD protein which is a surface- associated spermidine- and putrescine -binding protein.
  • PotA, PotB, PotC, and PotD proteins present in a number of microorganisms, their corresponding entry numbers, entry names, protein names, and gene names are available in the UniProt Knowledge Base.
  • a non-exhaustive list of PotA, PotB, PotC, and PotD proteins represented by their corresponding Entry Numbers in the UniProt Knowledge Base is provided below.
  • a person of ordinary skill in the art can obtained additional information about these proteins, for example, amino acid sequences of the proteins and nucleotide sequences of the corresponding genes, based on the Entry numbers provided herein to practice the current invention.
  • H6LF97 D4XH98; D4X5J6; D4XDT4; D4X3Q6; A0LUE6; B9CXM3; C5RZQ0; B0BT28; A3MZ02; B3H0C2; D9P4Y3; D9P8T5; K0G6D6; E8KIM5; N9VHC2; 085818;
  • E3PY06 A3DDF6; M1ZFS6; M1Z4U8; G0AFT6; G0A8H0; G0AEJ5; Q483J9; C3RKH2;
  • R9VT04 F0EIW8; F2MTU1; Q830W6; C2JME6; E1ERA3; C0X814; C2DAB7; H8LDC2;
  • K3RZW9 K3S225; K3S0T1; K3T7H1; K3TIU8; I5VYL5; I5TE64; I5T6J1; I5WLQ4;
  • E6BT33 K3KWU8; G0D6U5; E0QZR4; K3BYA1; K3U8L5; K3C3Q7; M2P5C7; A1AA20; C8U5Z0; I4P1D0; I4NCY1; J9ZN27; K0BLK8; K0APC7; F9CGE3; M7WDA5; M7V9S1;
  • K4V5C3 K4UX72; M7WNG9; B7UQ35; A7ZKR6; E8HSQ0; E8I5M7; N6VGX5; P69876;
  • H6MEE9 E8IY54; P69875; Q0TIU8; B7NKF3; G4PRN7; B7LX60; B7MTQ6; E4PDV1;
  • I5RVN6 I5RD98; I5TAM0; I5RZ31; I5Q0E0; I5UKE4; I5UN96; K3I6E8; G2F4G9;
  • I3D606 F0ET96; F0EQM6; I3BJG6; I2J3G6; I2NC52; N1VEB6; B8F532; R9XRE5;
  • F9QB42 Q0I3Y9; J4KDH7; Q2SJY7; Q18K92; G0LK30; D8J1H7; A4G8X8; F8J9G4; A6T2P9; F5S9P5; G8WM88; K6K5H4; G0GK54; B5XSP3; S2BF59; S2D8H7; S2CA47;
  • S2GE72 S1TRN5; S1XLL1; S2G654; S1TXZ1; S1WD49; R9BC30; S1WAW7; S1VZJ7;
  • S1VVU6 S2H243; S2JEX4; S1U1Y5; S2GTA4; S2I4N2; S2EH54; S1UHR9; S1X2C5;
  • I7KD72 I7KB86; C6J1J3; E1SJS3; F8KXX9; I3DAK3; C9PRB7; H8IDI5; Q9CP06;
  • K1CSR9 J6MLA7; K1DIW0; G2L062; G4LSL3; G2UA93; Q3KBH4; Q4K681; G8QB53;
  • F7ZLM4 F7ZHG2; F7ZHU0; D5RIL4; I0HUZ7; Q1AS06; Q5LT05; Q1GIE5; F4XA30;
  • N0TNZ3 N0TAX2; N1HG67; N0SGN6; N0SXQ8; N0S2X9; N0RV77; N0RBE1; N0R103;
  • L6M1V6 L6PM90; L9S9G4; L9S0E6; J2GGE6; L5Y544; L6XLY6; L9THY4; J1IFM2;
  • L6ETU4 L6EM44; L5Y8L3; L6GLS3; L5YRF3; L5XY78; L6H2R3; L6BNB6; L6GQR3;
  • L6D134 L6HZ85; L6IFE7; L6FGR8; L6A7F1; L6FVD9; L5X0X8; L5XHV6; L6PMC9;
  • K8SEI2 K8UKF0; K8VBX8; K8VEW7; K8SGJ5; K8TK42; K8TTE0; K8W1G1; K8UNQ4;
  • H4BPG6 H4HF61; H3RWX3; H4B0X8; H4ABW3; H4BEP2; H4AK80; H4C5S7;
  • E6K 89 F8DI37; E8K5E6; I1ZMJ0; F5ZHV1; M3IZT5; M5PJ08; F5X6V6; E8KB69;
  • K5QM90 G6ZEW3; J1DS44; G6ZSF9; G7A2Y8; G7AA54; G7ANB6; G7AWU6;
  • G7B7D4 K5REQ8; F9C6T7; K2TR43; F8YYE6; K2UD62; J1Z1U5; G7BI74; J1DZZ8; K5TTV1; J1XQN7; K5SLW7; J1FX31; F8Z9C4; G7BVX7; F8ZJT1; K2UZE2; K2XRB4;
  • F8LD90 H1X6P6; C5R7V7; D3UWB1; D3VAW1; N1NUX1; B0HHN7; B0HQX4;
  • K3GW86 K5G078; K5II97; K5H677; I5FI60; L1H6H8; K3JN70; K3H868; I5YWW2;
  • I5WVQ9 I5YLJ5; K3GP34; K3AWW6; I5E6X6; K2Z1L2; K3A856; I5E5I5; I5FMY0;
  • I5IDJ2 I5IH68; I5IFF5; I5JCG4; K3F727; I5K9Z3; I5KAL0; I5KEL8; I5GZD2; I5LNM0;
  • M7GQ94 M7GUK4; M7HGL6; M7HD80; M7I505; M7IUY0; M7INC1; M7J119; M7J2I2;
  • B7NB02 C8TPJ4; B7MJB1; G4PRN5; B7NKF5; B7LX58; B7MTQ4; M7US57; H0Q9U9;
  • H6LF94 G4Q3U7; C8KXG5; B0BTC0; A3MZ86; B3H0N0; D9P576; D9P9M4;
  • B3ZC73 C1ELZ6; B7I0J5; B7JEY6; Q73BL7; B7HH44; B7IMB9; B9IU94; Q63E81;
  • I5IJB1 I5IHH3; I5JD42; K3EKZ0; I5KBL6; I5KCG6; I5KIB5; I5GZD0; I5LNT6; I5LQG2;
  • M7QCI3 M7Q0A7; S2GU49; M2AJ05; S2BHT5; S1TQ14; M5QA37; B5XSP6; S3L078;
  • S2A6N6 S2GM07; S1ZZL3; S1ZIE5; S2B247; J4WS48; E6LR47; C2HNF5; Q5FL38;
  • E6LTD6 C8PD11; D0DX47; C7XYR6; D6S615; D0DLQ4; D0R514; C2FGU3; I8R8Q0;
  • F9UTR9 C6VK15; E1TS55; D7V893; H3P0T1; R4PPR2; M4KL28; E9RQR8; C7TB74;
  • H5SZ03 F2HKP4; Q9CGD1; A9QSB4; D2BQR8; C1DCN5; M1GJJ4; Q1MQ41; D1RK60; D3HKP0; D5TCD3; A5IB34; Q5WXF3; Q5X630; I7HR40; I7HXU7; R9SDB0; D0GJP1;
  • G7SSS2 H8IGJ0; H8IDI8; H8IDI9; Q9CP09; Q9CL58; Q9CP10; S3LRJ1; S3M2T2; S3LLB9; M7P4J1; EONMPO; G4D5B0; D1VUI0; F2P830; F2P831; F2PG05; Q4ZHQ9;
  • L6K1L6 J2BY85; L6JAY1; L6JHL2; L6KFR1; L6ZL49; J1HMH2; L6KAN2; J1JUA3;
  • L6V8Z9 L6WRT1; JIWUGO; L6VM05; L6X3F2; J2BKI3; J2FA78; J1LN47; J1MDK2;
  • L6Z207 J1MMI1; L6BHH2; L6CHA6; L6FNP9; L6DND3; L6B7Y1; L6ASW3; L5ZGW4;
  • K5AR20 K5B3P1; K4ZTI2; K5B6V4; M4LPP7; E7YJ13; F0CX24; E8EBF1; G9USI3;
  • E9CPD4 B2U501; Q31ZH8; B3X2V9; Q32EY1; POAFLO; K0X8M2; F5QWW2; I0VBY8;
  • B2D6S9 A3GLG7; A5F8B1; A5F8B2; C3LMC1; C3LMC0; A1EMA5; A1EMA6; Q5E582;
  • any combinations of potA, potB, potC, and potD genes present in different organisms can be selected to produce a hybrid potABCD operon.
  • Various methods and techniques of combining various potA, potB, potC, and potD genes to produce the hybrid potABCD operon are well known to a person of ordinary skill in the art and such embodiments are within the purview of the current invention.
  • PlaP Low affinity putrescine importer
  • Various PlaP proteins present in a number of microorganisms, their corresponding entry numbers, entry names, protein names, and gene names are available in the UniProt Knowledge Base.
  • the genetically modified microorganism of the current invention is capable of growing in the presence furfural and/or 5-HMF, i.e. the genetically modified microorganism is resistant to the presence of furfural and/or 5-HMF.
  • growth means an increase in the number or mass of the microorganism over time.
  • the characteristic of resistance to furfural and/or 5-HMF is beneficial for use of the genetically modified microorganisms for production of a desired product, for example a chemical, from biomass. Therefore, the genetically modified microorganism produces higher amounts of the desired product in the presence of furfural and/or 5-HMF as compared to the amount of the desired product produced by a reference microorganism in the presence of furfural and/or 5-HMF. Accordingly, the current invention provides a method of producing a desired product, the method comprising contacting a biomass with the microorganism and producing the desired product by fermenting the biomass in the presence of said microorganism.
  • the biomass which can be utilized to produce a desired product can be a hemicellulosic biomass, a lignocellulosic biomass, a cellulosic biomass, or an oligosaccharide containing biomass.
  • Non-limiting examples of the desired product include ethanol, lactic acid, succinic acid, malic acid, acetic acid, 1,3-propanediol, 2,3-propanediol, 1 ,4-butanediol, 2,3- butanediol, butanol, pyruvate, dicarboxylic acids, adipic acid, and amino acids. Additional products that can be produced from a biomass are well known to a person of ordinary skill in the art and such products are within the purview of the current invention.
  • the genetically modified microorganism of the current invention is further engineered to produce the desired product.
  • Non-limiting methods and techniques of engineering a microorganism to produce a desired product include genetic engineering, metabolic engineering, and/or metabolic evolution.
  • Methodabolic engineering is the practice of optimizing genetic and regulatory processes of a cell to increase the cell's ability to produce a desired product. The selection process for strains with improved production of a desired product is referred to as "metabolic evolution”. Additional methods of engineering a microorganism to produce a desired product are well known to a person of ordinary skill in the art and such embodiments are within the purview of the current invention.
  • the characteristic of the genetically modified microorganism of the current invention to grow in the presence of furfural and/or 5- HMF is further enhanced by addition of polyamines in the media in which the microorganisms are grown.
  • polyamines that can enhance furfural and/or 5-HMF resistance in the microorganism of the current invention include agmantine, putrescine, cadaverine, spermidine, spermine, homospermidine, norspermidine, homospermine, norspermine, thermospermine, cadaverine, or a combination thereof. Additional examples of polyamines are well known to a person of ordinary skill in the art and such embodiments are within the purview of the current invention.
  • the current invention provides a method of producing a desired product, the method comprising contacting a mixture of a biomass and one or more polyamines with the genetically modified microorganism and producing the desired product by fermenting the biomass in the presence of the microorganism.
  • the one or more polyamines that can be added to the biomass include, but are not limited to, agmantine, putrescine, cadaverine, spermidine, spermine, homospermidine, norspermidine, homospermine, norspermine and thermospermine, cadaverine, or a combination thereof. Additional examples of polyamines are well known to a person of ordinary skill in the art and such embodiments are within the purview of the current invention.
  • Polyamines can be added to the biomass at a concentration of about 0.5 mM to about 20 mM, about 5 mM to about 15 mM, about 8 mM to about 12 mM, or about 10 mM. Methods and techniques of determining the optimal concentration of polyamine to obtain the highest production of the desired product are well known to a person of ordinary skill in the art and such embodiments are within the purview of the current invention.
  • An embodiment of the current invention provides a method of increasing furfural and/or 5-HMF resistance in a microorganism.
  • the method comprises introducing in to the microorganism genetic modifications involving one or more target genes encoding:
  • ATP-dependent polyamine transporter PotABCD
  • PlaP low affinity putrescine-importer
  • the method of increasing furfural and/or 5- HMF resistance in the microorganism further comprises culturing the microorganism in the presence of one or more polyamines, for example, in a medium containing the one or more polyamines.
  • the one or more polyamines that can be added to the medium include, but are not limited to, agmantine, putrescine, cadaverine, spermidine, spermine, homospermidine, norspermidine, homospermine, norspermine and thermospermine, cadaverine, or a combination thereof. Additional examples of polyamines are well known to a person of ordinary skill in the art and such embodiments are within the purview of the current invention.
  • Polyamines can be added at a concentration of about 0.5 mM to about 20 mM, about 5 mM to about 15 mM, about 8 mM to about 12 mM, or about 10 mM. Methods and techniques of determining the optimal concentration of polyamine to obtain the highest resistance to furfural and/or 5-HMF are well known to a person of ordinary skill in the art and such embodiments are within the purview of the current invention.
  • microorganism can be a wild type microorganism, a mutant microorganism, an engineered microorganism, or the genetically modified microorganism of the current invention.
  • Microorganisms can be cultured in the presence of polyamines at a concentration of about 0.5 mM to about 20 mM, about 5 mM to about 15 mM, about 8 mM to about 12 mM, or about 10 mM.
  • Methods and techniques of determining the optimal concentration of polyamine to obtain the highest resistance to furfural and/or 5-HMF are well known to a person of ordinary skill in the art and such embodiments are within the purview of the current invention.
  • a further aspect of the invention provides a method of producing a desired product, the method comprising contacting a mixture of a biomass and one or more polyamines with a microorganism capable of producing the desired product.
  • Non-limiting examples of the biomass include a hemicellulosic biomass, a lignocellulosic biomass, a cellulosic biomass, or an oligosaccharide containing biomass.
  • Polyamines can be added to the biomass at a concentration of about 0.5 mM to about 20 mM, about 5 mM to about 15 mM, about 8 mM to about 12 mM, or about 10 mM. Methods and techniques of determining the optimal concentration of polyamine to obtain the highest production of the desired product are well known to a person of ordinary skill in the art and such embodiments are within the purview of the current invention.
  • Non-limiting examples of the desired products that can be produced from a biomass include ethanol, lactic acid, succinic acid, malic acid, acetic acid, 1,3 -propanediol, 2,3- propanediol, 1 ,4-butanediol, 2,3-butanediol, butanol, pyruvate, dicarboxylic acids, adipic acid, and amino acids.
  • microorganisms are useful for producing genetically modified microorganism of the current invention and carrying out various methods of the current invention.
  • the microorganism can be a prokaryotic microorganism or a eukaryotic microorganism.
  • the prokaryotic microorganism can be a bacterium, which can be a Gram-negative bacterium or a Gram-positive bacterium.
  • the Gram-positive bacterium include a bacterium from the genera of Bacillus, Clostridium, Corymb acterial cell, Lactobacillus, Lactococcus, Oenococcus, Streptococcus, or Eubacterial cell; whereas, non- limiting examples of the Gram-negative bacterium include a bacterium from the genera of Escherichia, Zymomonas, Acinetobacter, Gluconobacter, Geobacter, Shewanella, Salmonella, Enterobacter, or Klebsiella.
  • the bacterium is Escherichia coli or Klebsiella oxytoca. In certain other embodiments of the invention, the bacterium is Thermoanaerobes spp., Bacillus spp., Paenibacillus spp., or Geobacillus spp. In certain other embodiments, the bacterium is Thermoanaerobacterium saccharolyticum, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bacillus amyloliquifaciens, Bacillus megaterium, Bacillus macerans, Paenibacillus spp. or Geobacillus stearothermophilus.
  • the eukaryotic microorganism can be a yeast or a filamentous fungus.
  • yeast include a yeast from the genera of Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia.
  • the yeast is Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica.
  • Non-limiting examples of the filamentous fungus include a filamentous fungus from the genera of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma.
  • a filamentous fungus from the genera of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus,
  • the filamentous fungus is Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysospor
  • Table 1 contains a list of strains, plasmids, and primers used in this study.
  • Table 2 lists primers used to sequence genes cloned into plasmids.
  • Ethanologenic E. coli strain LY180 (E. coli W derivative) 21 and a furfural-resistant derivative XW092 (LY180, AyqhD) 17 were used in growth-based tests of furfural tolerance.
  • Furfural-resistant mutants of strain LY180 were isolated after serial transfer of cultures in the presence of increasing concentrations of furfural.
  • Strain EMFR9 was isolated after 54 transfers.
  • 21 Strain EMFR35 was isolated in a similar manner after 108 transfers in AMI medium containing furfural.
  • AfrdBC :(frg Zm celY Ec )
  • AldhA ::(frg Zm casAB Ko )
  • Strain, Relevant characteristic(s) Reference or Strains and plasmids were constructed using Luria-Bertani medium. After construction, cultures were grown in AMI mineral salts medium. 23 All media were supplemented with xylose (20 g/liter for solid medium and 50 g/liter for liquid medium). Ampicillin (50 mg/liter), kanamycin (50 mg/liter) and chloramphenicol (40 mg/liter) were added as needed for selections.
  • Furfural tolerance was examined by measuring growth after 48 h (37°C) in tube cultures (13 mm by 100 mm) containing 4 ml of AMI medium. Ampicillin (12.5 mg/liter), furfural, HMF, isopropyl-P-D-thiogalactopyranoside (IPTG) and other supplements were added as indicated. Inocula were grown overnight on AMI xylose. Colonies were scraped, resuspended in AMI media and adjusted to OD 55 o nm of 1.0. Tube cultures were inoculated to an initial OD 55 onm Of 0.1 (43 mg dry cell weight (dcw)/liter). Construction of promoter library for expression of fucO-ucpA cassette
  • a genome-wide promoter library was constructed previously in plasmid pLOI4870 (derivative of pACYC184). 17 DNA from E. coli W was digested with excess Sau3AI and ligated into the unique BamHl site upstream from a promoterless cassette containing two genes that increase furfural tolerance, fucO and ucpA (Fig. 1A). More than 10,000 transformants (colonies) were pooled and used to prepare a plasmid library containing inserted Sau3AI fragments.
  • the promoter library and control vectors (pACYC184 and pLOI4870) were transformed into XW092 (LY180, AyqhD). Immediately after heat shock and 30 min incubation for recovery, the mixture of transformants was resuspended in AMI medium (OD 55 onm of 0.05) containing 50 g/liter xylose, 10 mM furfural and 40 mg/liter chloramphenicol. Cultures (100 ml) were incubated in 250 ml shake flasks at 37°C, 100 rpm for 48 h. No significant growth (OD 55 onm of lower than 0.1) was observed for transformants containing empty vectors (pACYC184 or pLOI4870).
  • transformants containing plasmids with promoter fragments grew to densities over OD 55 onm of 1.0.
  • cultures containing the promoter library were serially transferred three times (inoculum at OD 55 onm of 0.05, 24 h incubation) in fresh AMI medium containing 12.5 mM furfural and 40 mg/liter chloramphenicol. After final incubation, plasmids were extracted, back-transformed, and screened to identify clones with an increase in furfural tolerance. Construction of polyamine transporter plasmids
  • the potE gene was amplified (including ribosomal-binding site and terminator region) from strain E. coli W (ATCC 9637) chromosomal DNA by using polymerase chain reaction (PCR). This fragment was cloned into the Ncol and BamHI sites of pTrc99A to produce pLOI5249 (Fig. 1C). Seven other polyamine transporter genes were cloned in a similar manner using primers with flanking restriction sites (Table 1). After ligation, plasmids were transformed into E. coli Topi OF . Plasmids were purified using a QiaPrep spin miniprep kit (Qiagen Valencia, CA). Clones were verified by digestion with restriction enzymes, size of PCR products and sequencing. Data and analyses
  • Genomic DNA samples from LY180, EMFR9 and EMFR35 were purified according to the bacterial genomic DNA isolation protocol from the DOE Joint Genome Institute (see world wide website: jgi.doe.gov). Next generation sequencing was performed using Illumina paired-end short read technology provided by the Tufts University Core facility (Boston, MA). Sequence data for LY180 was assembled using Geneious software (Auckland, New Zealand). Sequence assembly of EMFR9 and EMFR35 is currently ongoing. Genome annotation of LY180 was provided by the Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) of the National Center for Biotechnology Information (NCBI). The fully assembled genome of LY180 has been deposited in NCBI's GenBank (see world wide website: ncbi.nlm.nih.gov/genbank) under the accession number CP006584.
  • NCBI Prokaryotic Genomes Automatic Annotation Pipeline
  • Microarray analysis Cultures were grown in 500 ml fermenters (350 ml working volume) and sampled at an OD 55 onm Of 1.5 (650 mg dew/liter). Furfural was added to EMFR9 at 5 mM and to the more tolerant EMFR35 at 15 mM. Corresponding concentrations of furfural were added to LY180 with empty vector as a control. Samples were prepared after 15 min of incubation with furfural. Expression of mRNA was analyzed as previously described. 21 Expression data are reported for each gene as the arithmetic ratio mutant transcripts divided by parent (LY180).
  • Microarray data for gene expression in LY180, EMFR9, and EMFR35 has been deposited in NCBFs Gene Expression Omnibus (see world wide website: ncbi.nlm.nih.gov/geo) with GEO series accession number GSE46442.
  • EXAMPLE 1 - PLASMIDS CONTAINING potE CONFER FURFURAL RESISTANCE The plasmid library from a population enriched for furfural tolerance was transformed into XW092. Using a BioScreen C growth curve analyzer (Piscataway, NJ), 64 colonies were picked randomly and tested for growth in AMI medium containing 12.5 mM furfural. The 7 clones that grew best were isolated, designated pLOI5241, pLOI5242, pLOI5243 (4 siblings recovered) and pLOI5244 (Fig. IB). After back transformation, all of these plasmids increased growth in the presence of 12.5 mM furfural (Fig.
  • Plasmids pLOI5241-pLOI5244 were initially assumed to contain only small Sau3AI fragments that increased furfural tolerance by serving as transcriptional promoters, restoring expression of the fucO-ucpA cassette.
  • sequencing revealed that all seven of these plasmids contained unique combinations of small Sau3AI fragments from different regions of the E. coli W chromosome together with a single larger fragment encoding a full length potE gene, a statistically unlikely event. (Note that potE from E. coli W lacks internal Sau3AI sites.)
  • potE gene was cloned into pTrc99A (Fig. 1C) and tested for furfural tolerance using E. coli XW092 with the empty vector as a control (Fig. 2B).
  • the combination of potE and the fucO-ucpA cassette provided more furfural resistance than potE alone (Figs. 2 A and 2B).
  • EXAMPLE 2 - EXPRESSION LEVELS OF POLYAMINE TRANSPORTERS (potE AND puuP) ARE ELEVATED IN FURFURAL-RESISTANT MUTANTS PotE is one of 8 polyamine transporters that have been identified in E. coli.
  • 24 Expression levels for the 18 genes encoding polyamine transporters in the parent (LY180) were compared to two furfural-resistant mutants (EMFR9 and EMFR35) isolated after serial transfers in AMI medium containing furfural. Comparisons were made in the absence of added furfural, in the presence of 5 mM furfural and in the presence of 15 mM furfural (Table 3). Changes were remarkably small for most polyamine transporter genes.
  • expression of potE was more than one hundred times higher in EMFR9 than in LY180 (parent). In EMFR35, expression of potE was unchanged but the expression of puuP was more than five hundred times higher than in LY180.
  • Strains LY180, EMFR9, and EMFR35 were sequenced using Illumina technology by Tufts University Core Facility (Boston, MA). The transporter genes (as well as upstream and downstream regions) were compared between the parent and mutant strains. No mutations were present in these regions, or in the puuR gene (putative repressor of puuP). Mapping sequence reads for EMFR35 on the LY180 sequence as a template revealed that an 8.8 kbp region including the puuP gene was amplified 5 -fold in EMFR35, in comparison to adjacent chromosomal DNA (Fig. 3). The puuR (repressor) was outside of the amplified region, present only as a single copy. The increase in copy number of puuP without concurrent repressor amplification is proposed as a basis for the high level expression of puuP in EMFR35.
  • the intracellular levels of polyamines in E. coli are regulated by biosynthesis, degradation, and expression of transporter genes. 25
  • the importance of the native puuP and potE genes was examined by constructing deletions in LY180 (Fig. 4B), designated RG100 ( ⁇ ), RG101 ( ⁇ ) and RG102 (ApuuP, ⁇ ). Deletion of puuP decreased furfural tolerance as evidenced by a 70% decrease in growth in 10 mM furfural. Deletion of potE also decreased furfural tolerance in comparison to LY180, but to a lesser extent. Deletion of both puuP and potE from LY180 decreased growth in 10 mM furfural by 80%. Neither the control strain LY180 nor any of the mutants were able to grow in the presence of 12.5 mM furfural. These results demonstrate that native levels of the polyamine transporters PuuP and PotE contribute to furfural tolerance in LY180.
  • FURFURAL TOLERANCE E. coli contains 3 polyamines (in order of abundance): putrescine, > spermidine, > cadaverine. 26 The effect of each polyamine was tested as a supplement for LY180 in AMI medium containing 10 mM furfural (Fig. 5 A). Agmatine, a precursor of putrescine, was also included. All polyamines except spermidine were beneficial for furfural tolerance. The addition of spermidine decreased growth in the presence of furfural and to a lesser extent in the absence of furfural (Fig. 7). The beneficial activity of plasmid-borne polyamine transporters for furfural tolerance can be partially replaced by supplementing with putrescine, agmantine or cadaverine.
  • EXAMPLE 7 - PUTRESCINE TRANSPORTER PLASMIDS AND PUTRESCINE The combination of suboptimal levels of putrescine (1.0 mM) and plasmid pLOI5249 (potE) or plasmid pLOI5412 (puuP) increased the growth of LY180 in the presence of 10 mM furfural (Fig. 5B). In both cases, combinations were more effective than putrescine or transporter plasmid alone. These results are consistent with the uptake of polyamines as a beneficial activity that increases resistance to furfural.
  • Inhibitors such as furfural are formed during dilute acid pretreatment of lignocellulosic biomass. These pose a significant challenge for the fermentation of resulting sugars into fuels and chemicals.
  • 9 ' 14 Furfural-resistant mutants of E. coif' 18 ' 22 and yeasts 27 have been described that express oxidoreductases which reduce furfural to the less toxic furfuryl alcohol. 28 Increasing concentrations of furfural have been shown to correlate with a rise in single strand breaks and DNA mutagenesis, primarily in AT -rich regions.
  • 10 Allen et al. 13 have reported reactive oxygen species (ROS) accumulate in yeast cells during growth in the presence of furfural. ROS compounds are known to damage DNA, proteins and phospholipid membranes. Polyamines have been reported to protect DNA from damage by oxidative stress agents. 29 ' 30 In E. coli, overexpression of thy A, thymidylate synthase, has been demonstrated to increase tolerance to furfural. 18
  • Polyamines are essential for cell division. 24 ' 31 Concentrations are maintained within a specific range 32 and elevated during exponential growth. 33 Anionic cellular structures such as plasma membranes, ribosomes and nucleic acids bind polyamines. 30 ' 34 Binding can directly protect DNA from damage by reactive compounds 29 as well as regulate gene expression and modulate translational fidelity. 32 ' 35 However, excess polyamines are toxic, displacing magnesium from ribosomes and inhibiting protein synthesis. 36 ' 37
  • Polyamine binding may be the basis for the increase in furfural tolerance observed during transporter expression from plasmids.
  • Polyamine transporters such as PotABCD, PlaP, PotE and PuuP and polyamine supplements in the medium could increase intracellular levels, restoring growth and increasing furfural tolerance. Stabilization and protection of DNA, R A and other cellular macromolecules by polyamines provides a novel approach to mitigate the inhibitory actions of furfural.

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Abstract

Cette invention concerne des micro-organismes comportant des modifications génétiques qui impliquent un ou plusieurs gènes cibles codant pour : l'antiport de putrescine-ornithine (PotE), l'import de putrescine (PuuP), le transport de polyamine ATP-dépendant (PotABCD), et l'import de putrescine à basse affinité (PlaP), les modifications génétiques provoquant une expression et/ou une activité accrue des polypeptides produits par les gènes cibles. Les procédés de production d'un produit recherché par fermentation d'une biomasse en présence du micro-organisme selon l'invention sont également décrits. En plus, cette invention concerne des procédés destinés à accroître la résistance au furfural et/ou au 5-hydroxyméthylfurfural (5-HMF) chez un micro-organisme par introduction chez ledit micro-organisme de modifications génétiques impliquant un ou plusieurs gènes cibles codant pour : PotE, PuuP, PotABCD, et PlaP, lesdites modifications génétiques provoquant une expression et/ou une activité accrue des polypeptides produits par les gènes cibles et/ou culture du micro-organisme en présence d'une ou de plusieurs polyamines.
PCT/US2014/070795 2013-12-17 2014-12-17 Utilisation des polyamines et des transporteurs de polyamines pour conférer une tolérance au furfural WO2015095298A2 (fr)

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CN108185352B (zh) * 2017-12-20 2021-04-23 四川东坡中国泡菜产业技术研究院 一种低生物胺发酵泡菜的制作方法

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