WO2015138977A2 - Modification métabolique de clostridium pour la production de biocarburant - Google Patents

Modification métabolique de clostridium pour la production de biocarburant Download PDF

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WO2015138977A2
WO2015138977A2 PCT/US2015/020568 US2015020568W WO2015138977A2 WO 2015138977 A2 WO2015138977 A2 WO 2015138977A2 US 2015020568 W US2015020568 W US 2015020568W WO 2015138977 A2 WO2015138977 A2 WO 2015138977A2
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plasmid
bacterium
clostridium
cellulovorans
methylated
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WO2015138977A3 (fr
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Shang-Tian Yang
Xiaorui YANG
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Ohio State Innovation Foudation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • 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
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01001—Alcohol dehydrogenase (1.1.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/0101—Acetaldehyde dehydrogenase (acetylating) (1.2.1.10)
    • 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

  • CBP bioprocessing
  • saccharobutylicum were isolated with hemicellulosic activity and are capable of producing butanol, none of them have the true cellulase activity to utilize crystalline cellulose (Berezina et al., 2009).
  • saccharoperbutylacetonicum strain Nl-4
  • Minty et al, (2013) also developed a synergistic fungal-bacterial consortia for direct isobutanol production from celluiosic biomass by co-culturing celiulolytic Trichoderma reesei with metabolically engineered E. coli, achieving a low titer of 1.88 g/L in 300 h.
  • these co-cultures also suffered from complicated operation because of different optimal growth conditions for the two very different cultures used in the process.
  • either cellulase production must be introduced into a butanol-producing strain or the pathway for butanol biosynthesis must be introduced into a cellulase producing strain.
  • a celiulolytic acido genie Clostridium bacterium transformed to overexpress aldehyde/alcohol dehydrogenase for biofuel production Also disclosed herein is a method of expressing a heterologous gene sequence in a cellulolytic acidogenic Clostridium bacterium, the method comprising: providing a plasmid containing the heterologous gene sequence, wherein the plasmid is methylated; transforming the bacterium with the methylated plasmid; and culturing the bacterium in order to express the heterologous gene.
  • a method of producing a biofuel from cellulose comprising: transforming a cellulolytic acidogenic Clostridium, bacterium with a plasmid, wherein the plasmid comprises a heterologous gene sequence capable of producing a biofuel; and culturing the transformed cellulolytic acidogenic Clostridium bacterium under conditions sufficient to produce a biofuel.
  • an in vivo method for methyl ating a heterologous gene comprising: introducing a plasmid comprising a methylation system from a Clostridium bacterium into another bacterium that is not sensitive to methylated nucleic acid; introducing a plasmid containing a heterologous gene to be modified into the bacterium containing the plasmid with the methylation system; and culturing the bacterium under conditions sufficient for methylation of the plasmid containing the h eterologous gene, there by methylating the plasmid with the heterologous gene.
  • Figure 1 shows genome analysis of the putative restriction modification (RM) systems in C. cellulovorans by REBASE. Twelve operons were found, consisting of putative restriction enzyme (RE), methyl transferase (MT), and S subunits. The operons marked with stars (*) were the candidates for further analysis.
  • RE putative restriction enzyme
  • MT methyl transferase
  • * stars
  • Figure 2 shows characterization of the res triction profile by restriction assay.
  • the plasmid (pMTL82151 ⁇ adh£2) was restricted by the cell lysate of C. cellulovorans after overnight incubation. Bands around 3 kb were observed. Lane 1, D A marker; Lane 2, the plasmid in the digestion buffer without the addition of cell extract; Lane 3, the plasmid was mcubated with the cell extract for 0 h; Lane 4, the plasmid was incubated with the cell extract for 2 h; Lane 5, the plasmid was incubated with the cell extract for 20 h.
  • Figure 3 shows restriction assay showing the protection effect of in vivo methylation system.
  • the unmethylated and methylated plasmids pMTL llS I -adhEl (left panel) and pMTL83151 -adhEl (right panel) were incubated with the cell lysate of C. cellulovorans for restriction assay. After incubation for 20 h, the unmethylated plasmids were restricted, showing a major band around 3 kb, while the methylated plasmids remained unchanged, compared to the negative control.
  • Lane 1 DN A marker
  • Lane 2 unmethylated plasmid incubated with the cell lysate for 0 h (negative control); Lane 3, unmethylated plasmid incubated with the ceil lysate for 20 h; Lane 4, methylated plasmid incubated with the cell lysate for 0 h (negative control); Lane 5, methylated plasmid incubated with the cell lysate for 20 h.
  • Figure 4 shows overexpressed adhEl was functional in C. cellulovoransl%3151 -adhE2.
  • FIG 4A Enzyme activities of adhE2.
  • the butyraldehyde dehydrogenase activity and butanoi dehydrogenase activity of adhE2 in C. cellulovorans were measured by monitoring NADH consumption at 365 nm.
  • the butyraldehyde dehydrogenase activity of the WT and the mutant strain C. cellulovoransl 3151 -adhEl were 0.006 U/mg and 0.162 U/mg, respectively.
  • the butanoi dehydrogenase activities of th e WT and C, cellulovoransl 3151 -adhEl were 0.011 U/mg and 0.077 U/mg, respectively (n > 3);
  • Figure 5 shows fermentation kinetics of wild-type C. cellulovorans.
  • Figure 5 A shows fermentation kinetics of wild-type C. cellulovorans.
  • Fermentation kinetics of wild-type C. cellulovorans grown on glucose Figure 5B. Fermentation kinetics of wild-type C. cellulovorans grown on celiobiose; Figure 5C. Fermentation kinetics of wild-type C. cellulovorans grown on cellulose (15 g/L). Fermentation was done in the bioreactor, with pH controlled at 6.5.
  • Figure 6 shows fermentation kinetics of C. cellulovorans/%3151 -adbJB2.
  • Figure 7 shows the effects of methyl viologen (MV) on alcohols and acids production from glucose by C. cellulovorans/83151 -adhEl in bioreactor with pH controlled at 6.5.
  • Figure 7A Fermentation kinetics without MV;
  • Figure 7B Fermentation kinetics with 250 mM MV.
  • Figure 8 shows the metabolic pathways in C, cellulovorans with heterologous butanoi and ethanol synthesis pathways (shown in dotted lines) for butanoi and ethanol production by overex pressing aldehyde-alcohol dehydrogenase 2 (adhEl)
  • Figure 9 shows the confirmation of the plasmid in C. cettulovoran$l%3151 -adhEl.
  • the pfasmid was extracted from C. cellulovomnsl%3151 -adhEl, followed by transformation into E, coli DH5a and then plasmid purification from E. coli for confirmation by PCR and enzyme digestion.
  • Lanel positive control of PCR of. adhEl (2,8 kb); Lane2, PCR of adhEl (2.8 kb); Lane3, DNA maker; Lane4, enzyme digestion by BamHl and &cll (4.5 kb+2.6 kb).
  • Figure 10 shows the generation of XY I plasmid by cloning native M . Cee743I and M. ⁇ " ⁇ c743 I l into the pACYC 184 vector. Amplified M. Cce743I and M. Cce743II with the T7 promoter was ligated into pACYC 184 at the distal site of PvuII and Ncoi site to generate pXYl .
  • Figure 11 shows the establishment of the in vivo methylation system with M, Cce l 43 ⁇ . and M. Ccel 3 ⁇ activities.
  • pXYl was transformed into E. coli ⁇ to generate E. coli XY1.
  • Plasmid e.g., pMTL82151 ⁇ a A£ ' 2
  • vector or "construct” refers to a nucleic acid sequ ence capable of transporting into a cell another nucleic acid to which the vector sequence has been linked.
  • expression vector includes any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element).
  • vector e.g., a plasmid, cosmid or phage chromosome
  • vector e.g., a plasmid, cosmid or phage chromosome
  • vector are used interchangeably, as a plasmid is a commonly used form of vector.
  • the invention is intended to include other vectors which serve equivalent functions.
  • operable linkage of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and promoter such that the transcription of such DNA is initiated from the promoter by an RNA. polymerase that specifically recognizes, binds to and transcribes the DNA.
  • nucleic acid e.g., an expression vector
  • transfection mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell including introduction of a nucleic acid to the chromosomal DNA of said ceil.
  • nucleotide sequence is intended to mean a sequence of two or more nucleotides.
  • the nucleotides may be of genomic DNA, cDNA, RNA, semisynthetic or synthetic origin or a mixture thereof.
  • the term includes circular, linear, single and double stranded forms of DNA or RNA.
  • isolated nucleic acid or “purified nucleic acid” is meant DNA that is isolated from the naturally-occurring genome of the organism from whic the DNA of the invention is derived.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, such as an autonomously replicating plasmid or virus; or incorporated into the genomic DNA of a prokaryote or eukaryote (e.g., a transgene); or which exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PGR, restriction endonuclease digestion, or chemical or in vitro synthesis).
  • isolated nucleic acid also refers to RNA, e.g., an mRNA molecule that is encoded by an isolated DNA molecule, or that is chemically synthesized, or that is separated or substantially free from at least some cellular components, for example, other types of RNA molecules or polypeptide molecules.
  • start of replication is intended to mean a nucleotide sequence at, which DNA synthesis for replication of the vector begins. Start of replication may occur at one or more points within the vector dependent on the vector being used, such as at one point in a plasmid vector or at several points in an adenovector.
  • the start of replication is generally termed origin of replication (abbreviated ori site) in a plasm d vector.
  • control sequence or "control sequences” is intended to mean nucleotide sequences involved in control of a response of action. This includes nucleotide sequences and/or proteins invol ved in regulating, controlling or affecting the expression of structural genes, or the replication, selection or maintenance of a plasmid or a viral vec tor. Examples include
  • Attenuators silencers, enhancers, operators, terminators and promoters.
  • methylation is intended to mean that one or more cytosines in a nucleic acid sequence is methylated.
  • One or more methylated cytosines in a nucleotide sequence might result in reduced or eliminated restriction of the nucleic acid sequence by the host cell.
  • resistant against restriction is intended to mean that the methylated nucleic acid is more resistant to restriction than a control which is unmethylated.
  • Exogenous nucleic acids are nucleic acids which originate outside of the microorganism to which the)' are introduced. Exogenous nucleic acids may be derived from any appropriate source, including, but not limited to, the microorganism to which they are to be introduced, strains or species of microorganisms which differ from the organism to which they are to be introduced, or they may be artificially or recombinantly created.
  • the exogenous nucleic acids represent nucleic acid sequences naturally present within the microorganism to which they are to be introduced, and they are introduced to increase expression of or over-express a particular gene (for example, by increasing the copy number of the sequence (for example a gene)).
  • the exogenous nucleic acids represent nucleic acid sequences not naturally present within the microorganism to which they are to be introduced and allow for the expression of a product not naturally present within the microorganism or increased expression of a gene native to the microorganism (for example in the case of introduction of a regulator ⁇ ' element such as a promoter).
  • the exogenous nucleic acid may be adapted to integrate into the genome of the microorganism to which it is to be introduced or to remain in an extra-chromosomal state.
  • aldehyde dehydrogenase designates the aldehyde dehydrogenase (CoA-acyiating) enzyme which catalyzes the reaction of conversion of an acyl- CoA into an aldehyde.
  • alcohol dehydrogenase in this invention designates the enzyme which catalyzes the reaction of conversion of an aldehyde into an alcohol.
  • AdhE enzyme refers to a bifimctionai enzyme having the two activities aldehyde dehydrogenase and alcohol dehydrogenase.
  • activity and “function” refer to a specific catalytic activity or function of an enzyme, i.e. the biochemical reaction(s) that is (are) catalyzed by this enzyme.
  • microorganism refers to a bacterium, yeast or fungus.
  • microorganism or "genetically modified microorganism”, as used herein, refers to a microorganism genetically modified or genetically engineered. It means, according to the usual meaning of these terms, that the microorganism of the invention is not found in nature and is modified either by introduction, by deletion or by modification of genetic elements, it can also be transformed by forcing the development and evolution of new metabolic pathways in combining directed mutagenesis and evolution under specific selection pressure (see for instance WO 2004/076659).
  • a microorganism may be modified to express exogenous genes if these genes are introduced into the microorganism with all the elements a! lowing their expression in the host microorganism.
  • a microorganism may be modified to modulate the expression level of an endogenous gene. The modification or "transformation" of microorganisms wi th exogenous DNA is a routine task for those skilled in the art.
  • nucleic acids disclosed herein may have sequences that vary from the sequences specifically exemplified herein provided they perform substantially the same function. For nucleic acid sequences that encode a protein or peptide this means that the encoded protein or peptide has substantially the same function. For nucleic acid sequences that represent promoter sequences, the variant sequence will have the ability to promote expression of one or more genes. Such nucleic acids may be referred to herein as "functionally equivalent variants".
  • functionally equivalent variants of a nucleic acid include allelic variants, fragments of a gene, genes which include mutations (deletion, insertion, nucleotide substitutions and the like) and/or polymorphisms and the like.
  • Homologous genes from other bacteria capable of ethanol or butanol fermentation may also be considered as examples of functionally equivalent variants of the sequences specifically exemplified herein.
  • nucleic acid herein will preferably have at least approximately 70%, preferably approximately 80%, more preferably approximately 85%o, preferably approximately 90%, preferably approximatel 95% or greater nucleic acid sequence identity with the nucleic acid identified.
  • polypeptides disclosed herein may have sequences that vary from the sequences specifically exemplified herein. These variants may be referred to herein as "functionally equivalent variants".
  • a functionally equivalent variant of a protein or a peptide includes those proteins or peptides that share at least 40%, preferably 50%, preferably 60%, preferably 70%, preferably 75%, preferably 80%, preferably 85%, preferably 90%, preferably 95% or greater amino acid identity with the protein or peptide identified and has substantially the same function as the peptide or protein of interest.
  • Such variants include within their scope fragments of a protein or peptide wherein the fragment comprises a truncated form of the polypeptide wherein deletions may be from Ito 5, to 10, to 15, to 20, to 25 amino acids, and may extend from residue 1 through 25 at either terminus of the polypeptide, and wherein deletions may be of any length within the region; or may be at an internal location.
  • Functionally equivalent variants of the specific polypeptides herein should also be taken to include polypeptides expressed by homologous genes in other species of bacteria.
  • substantially the same function is intended to mean that the nucleic acid or polypeptide is able to perform the function of the nucleic acid or polypeptide of which it is a variant.
  • An "appropriate culture medium” designates a medium (e.g., a sterile, liquid media) comprising nutrients essential or beneficial to the maintenance and/or growth of the cell such as carbon sources or carbon substrate, nitrogen sources, for example, peptone, yeast extracts, meat extracts, malt extracts, urea, ammonium sulfate, ammonium chloride, ammonium nitrate and ammonium phosphate; phosphorus sources, for example, monopotassium phosphate or dipotassium phosphate; trace elements (e.g., metal salts), for example magnesium salts, cobalt salts and/or manganese salts; as well as growth factors such as amino acids and vitamins.
  • a medium e.g., a sterile, liquid media
  • nutrients essential or beneficial to the maintenance and/or growth of the cell such as carbon sources or carbon substrate, nitrogen sources, for example, peptone, yeast extracts, meat extracts, malt extracts, urea, ammonium sulfate
  • methylating plasmid refers to a plasmid that comprises genes capable of transforming an intermediate host cell, so that the host cell has the ability to methylate nucleic acids in a site-specific manner.
  • An example of a methylating plasmid is one which comprises a methylating system of a bacterium such as Clostridium cellulovorans .
  • transforming plasmid refers to a plasmid that comprises one or more genes with which the final recipient cell is transformed.
  • the transforming plasmid can be introduced into the intermediate host cell, for example, in order to be methylated.
  • the transforming plasmid can then be extracted from the intermediate host cell and introduced into the final recipient cell, where it can transform the final recipient cell, giving it, for example, the ability to produce an exogenous product.
  • the exogenous product can be produced from a non-native nucleic acid that was introduced via the transforming plasmid.
  • An example of such a gene of a transforming plasmid is adhE2.
  • intermediate host cell refers to a cell which can be used to methylate a nucleic acid.
  • a methylating plasmid can be introduced into an intermediate host cel l, thereby transforming the host ceil into having the ability to methylate nucleic acid in a site- specific manner.
  • a transforming plasmid can then be introduced.
  • the transforming plasmid can comprise a gene of interest, which can be methylated in the intermediate host cell.
  • An example of an intermediate host cell is E. coli.
  • final recipient cell refers to a cell from which a desired product is to be obtained.
  • a transforming plasmid comprising a gene of interest is introduced into the final recipient cell for production of a protein encoded by the gene of interest.
  • the final recipient eel! may comprise a native restriction system, which recognizes non-methylated nucleic acid as being foreign, and digests it accordingly.
  • the transforming plasmid has first been subjected to an intermediate host cell with the ability to methylate it, it can then be introduced into the final recipient cell without being digested/degraded.
  • An example of a final recipient cell is C. cellulovorans.
  • RM systems restriction modification systems
  • type L type II, type III, and type IV consisting of restriction enzyme (RE.) subunits and methyl transferase (MT) subunits
  • S specificity subunits
  • RE. restriction enzyme subunits
  • MT methyl transferase
  • S specificity subunits
  • RM systems are widespread in bacteria. About 95% of the genome-sequeneed bacteria contain RM systems, and about 5000 REs and 8000 MEs have been described in REBASE. Proper DNA methylation prior to transformation can significantly enhance successful transformation by overcoming the restriction barrier.
  • Clostridium celluiovomns 743B is a cellulolytic bacterium producing mainly butyric and acetic acids.
  • Two restriction-modification (RM) systems of Clostridium celluiovomns (Cee743I and Cce743II) were determined.
  • R. Ccel 431 has the same specificity as LlaJl, recognizing 5'- GACGC-3' (SEQ ID NO: 1) and 5'-GCGTC-3' (SEQ ID NO: 2), while M.
  • Ccel 431 methyiates the external cytosine in the strand (5'-GACG m C-3', SEQ ID NO: 1).
  • R. Ccel 4311 has the same specificity as Llal, recognizing 5'-CCAGG-3' (SEQ ID NO: 3) and 5'-CCTGG-3' (SEQ ID NO: 4), while M. Ccel 43H methyiates the external cytosine of both strands.
  • An in vivo methylation system expressing M. Ccel 431 and M. Ccel 4311 in E. coli, was then established to protect plasmids from being degraded.
  • transformants were obtained, harboring the plasmid with pCBI02 replicon from C. butyricum, expressing an aldehyde/alcohol dehydrogenase 2 (adhE2) for biofuel production.
  • AdhE2 aldehyde/alcohol dehydrogenase 2
  • adhE2 1.42 g/L butanol and 1.60 g/L ethanol was produced from crystalline cellulose by the new strain of C. celluiovomns. Therefore, an effective transformation method was developed for metabolic engineering of C. celluiovomns, providing a platform for biofuel production from cellulosic biomass.
  • the aldehyde/alcohol dehydrogenase gene can be methylated before introduction into the Clostridium bacterium, which can reduce or eliminate restriction/digestion of the heterologous gene by the Clostridium bacterium.
  • This can be done via an in vivo methylation system.
  • a methylating plasmid can be introduced into a host cell, thereby transforming a host cell, so that it has the ability to methylate DNA in a certain pattern.
  • the methylation system can be native to C.
  • celluiovomns can comprise M, Ccel 431 and M, Ccel 4311 (SEQ ID NO: 22 and 23, respectively).
  • a transforming plasmid can be introduced into the intermediate host cell.
  • the transforming plasmid can comprise a gene to be methylated, such as adhE2.
  • the transforming plasmid Once the transforming plasmid has been methylated, it can be extracted from the intermediate host ceil, and transferred to the final recipient cell, such as Clostridium. Methylation systems are discussed in more detail below.
  • Clostridium bacterium which as capable of producing ;?-butanol when properly transformed.
  • Examples of cellulolytic, acidogenic Clostridium bacterium which as capable of producing ;?-butanol when properly transformed.
  • Clostridium bacterium include Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium aceticum, Clostridium formicoaceticum, Clostridium magnum, Clostridium acetohutylicum, Clostridium beijerinckii, Clostridium sacharoperbutylacetonicum, Clostridium saccharobutylicum, Clostridium
  • thermocellum Clostridium cellulolyticum, Clostridium phytofermentans, Clostridium
  • Clostridium cellulovorans Clostridium pasterianum, Clostridium kluyveri,
  • Clostridium butyricum Clostridium carboxidivorans, Clostridium phytofermentans, Clostridium propionicum, Clostridium saccharobutylicum, Clostridium saccharolyticum, Clostridium saccharoperbutylacetonicum, Clostridium kluyveri, Clostridium iyrobutyricum, .
  • the bacterium can be Clostridium cellulovorans.
  • bifunctional aldehyde/alcohol dehydrogenase can be identified by the gene symbol adhE2.
  • the adhEl gene is exogenous to one or more particular organisms.
  • the adhEl gene is a Clostridium acetohutylicum gene.
  • An example of the adhEl gene can be found in SEQ ID NO: 26.
  • the adhEl can be introduced into the bacterium in a transforming plasmid, A
  • microorganism can be transformed with a plasmid of the invention using any number of techniques known in the art for producing recombinant microorganisms.
  • transformation including transduction or transfection
  • transformation may be achieved by electroporation, electrofusioii, ultrasonication, polyethylene glycol-mediated transformation, chemical or natural competence, protoplast transformation, prophage induction or conjugation. Suitable
  • inactivation of the competing acetate and butyrate biosynthesis pathways in Clostridium can be accomplished by knocking out or knocking down genes native to Clostridium. Examples include, but are not limited to, phosphotraiisacetylase (pta) or acetate kinase (ack), and phosphotransbutyrylase (ptb) or butyrate kinase (buk), respectively.
  • pta phosphotraiisacetylase
  • ack acetate kinase
  • ptb phosphotransbutyrylase
  • buk butyrate kinase
  • Homologous recombination has been widely used to knock out, such as, pta and ack in C. iyrobutyricum and ack and buk in C. acetohutylicum.
  • Z selection marker are cloned into a non-replicative vector, which is then introduced into the host to inactivate the target gene via homologous integration either by single crossover or double crossover recombination.
  • Targetron/Clostron knockout system based on intron target specific integration can be used to knock out these genes in Clostridium, Compared to homologous integration system, the Targetron/Clostron system may give a higher specificity and integration efficiency. Partial interested knockout gene is amplified by PGR and cloned into MCS sites and then the recombinant knockout plasmid is transferred into Clostridium by conjugation or electroporation.
  • colonies are first selected on plasmid marker plates for plasmid-containing colony selection and then intron marker resistant clones are restreaked on plates with intron marker antibiotics for integrant clone selection.
  • the obtained intron mar ker resistant clones are confirmed by PCR verification of the presence of the desired insert in chromosome.
  • Butano! (also butyl alcohol) refers to a four-carbon alcohol with a formula of C4H9OH.
  • butanol f om a straight-chain primary alcohol to a branched-chain tertiary alcohol , it is primarily used as a solvent, as an intermediate in chemical synthesis, and as a fuel. It is sometimes also called biobutanol when produced biologically.
  • the unmodified term butanol usually refers to the straight chain isomer with the alcohol functional group at the terminal carbon, which is also known as »-butanol or 1 -butanol.
  • the straight chain isomer with the alcohol at an internal carbon is sec-butanol or 2-butanoi.
  • the branched isomer with the alcohol at a terminal carbon is isobutanol or 2-methyl- 1 -propanol
  • the branched isomer with the alcohol at the interna] carbon is tert-butanol. or 2 -methyl-2 -propanol.
  • Etbanol can also be produced from the bacterium disclosed herein.
  • lignocellulosic material means any type of biomass comprising cellulose, hemicellulose, lignin, or combinations thereof, such as but not limited to woody biomass, forage grasses, herbaceous energy crops, non- woody-plant biomass, agricultural wastes and/or agricultural residues, forestry residues and/or forestry wastes, paper-production sludge and/or waste paper sludge, waste -water-treatment sludge, municipal solid waste, corn fiber from wet and dry mil l corn ethanol plants, and sugar- processing residues.
  • the lignocellulosic material can include, but is not limited to, woody biomass, such as recycled wood pulp fiber, sawdust, hardwood, softwood, and combinations thereof; grasses, such as switch grass, cord grass, rye grass, reed canary grass, miscanthus, or a combination thereof; sugar-processing residues, such as but not limited to sugar ⁇ cane bagasse; agricultural wastes, such as but not limited to rice straw, rice hulls, barley straw r , com cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, and com fiber; stover, such as but not limited to soybean stover, corn stover; succulent plants, such as but not limited to agave; and forestry wastes, such as but not limited to recycled wood pulp fiber, sawdust, hardwood (e.g., poplar, oak, maple, birch, willow), softwood, or any combination thereof, Lignocelhilosic
  • Particularly advantageous lignoceliulosic materials are agricultural wastes, such as cereal straw's, including wheat straw, barley straw, canola straw and oat straw; com fiber; stovers, such as corn stover and soybean stover; grasses, such as switch grass, reed canary grass, cord grass, and miscanthus; or combinations thereof.
  • Biofueis can be produced from cellulose, ce!iodextrms, or glucose.
  • Biofuels can be produced from the ceilulolytic acidogenic Clostridium bacterium transformed to overexpress aldehyde/alcohol dehydrogenase, as disclosed herein.
  • the amount of biofuel produced can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 or more times greater than the amount of biofuel produced from a ceilulolytic acidogenic Clostridium bacterium that has not been transformed to overexpress aldehyde/alcohol dehydrogenase.
  • the bacterium can produce n-butaiiol at a titer greater than 0.1, 0.5, 1.0, 1.5, 2,0, 2,5, 3.0, 3.5, or 4.0 g/L.
  • the yield (g/g) of «-butanol can be greater than .01 , .05, .10, .15, .20, .25, .30, .35, or .40 g/g.
  • the bacterium can produce ethanoi at a titer greater than 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, or 4.0 g/L.
  • the yield (g/g) of ethanoi can be greater than .01, .05, .10, .15, .20, .25, .30, .35, .40, .45, or .50 g/g.
  • a plasmid comprising a methylated adhE2 gene.
  • the plasmid can be methylated in a manner such that the methylation pattern is recognized by C. cellulovorans. Being methylated can result in reduced or eliminated restriction/digestion in a C cellulovorans bacterium, compared to a non-methylated plasmid. More detail on the methylation system is given below. Also disclosed are cell lines comprising such plasmids.
  • ceilulolytic acidogenic Clostridium bacterium comprising: providing a plasmid containing the heterologous gene sequence, wherein the plasmid has been methylated; transforming the bacterium with the methylated plasmid; and cul taring the bacterium in order to express the heterologous gene.
  • the method can comprise introducing a methylating plasmid into an intermediate bacterial host cell.
  • the methylating plasmid can comprise genetic material from C, cettulovorans, specifically those genes which encode rnethylation systems, such as those found in M. Ccel 431 and M. Ccel 4311 (SEQ ID NO: 22 and 23, respectively).
  • Also disclosed is a method of producing a biofuel from cellulose comprising: transforming a celluloiytic acidogenic Clostridium bacterium with a plasmid, wherein the plasmid comprises a heterologous gene sequence capable of producing a biofuel; and culturing the transformed a celluloiytic acidogenic Clostridium bacterium under conditions sufficient to produce a biofuel.
  • a plasmid comprising a rnethylation system (“a methylating plasmid") from a Clostridium bacterium into another bacterium that is not sensitive to methylated nucleic acid, such as E. coli DH10 ⁇ ; introducing a plasmid containing a
  • heterologous gene to be modified into the bacterium containing the plasmid with the rnethylation system ("a transforming plasmid"); and culturing the bacterium under conditions sufficient for rnethylation of the transforming plasmid containing the heterologous gene, thereby methylating the transforming plasmid.
  • a transforming plasmid the gene(s) of interest encoded by the transforming plasmid can be any gene for which an exogenous product is desired.
  • a methylating plasmid can comprise the genetic sequence necessary to confer the ability on the intermediate host cell to methylate nucleic acid.
  • rnethylation is typically site- specific, in that rnethylation systems of different organisms recognize different motifs, and methylate them in a site-specific manner. More detail on this is given in Example 1.
  • Suitable intermediate bacterial host cells include, but are not limited to Gram negative bacteria such as E. coli and closely related Enter oh acteriaceae including Salmonella spp., Yersinia spp., Klebsiella spp. Shigella spp. Enterobacter spp., Serratia spp. and Citrohacter spp. Specific examples include E. coli methylation-minus strains, such as E. coli ⁇ and E. coli TOP 10
  • a transforming plasmid such as a plasmid comprising the adhEl gene, can be introduced into the intermediate bacterial host cell, thereby allowing for the transforming plasmid to be methylated by the rnethylation plasmid.
  • Co-residing plasmids in this host environment can be methylated only to the extent of sequence recognition by the methylation system. More deta regarding how methylation occurs can be found in Example 1 .
  • the transforming vector After the transforming vector has been properly methylated, it can be extracted from the intermediate host cell, and used to transform the final recipient cell, such as Clostridium,
  • the transforming DNA can be integrated into the final bacterial recipient cell chromosome.
  • the methylated DNA from the transforming factor can be purified from the intermediate bacterial host cell and transferred to a final recipient cell that can degrade the plasmid DNA but which cannot degrade the methylated DNA.
  • the purified DNA can be transferred to the final recipient cell by methods of transformation, such as transduction, conjugation, and electroporation, which are discussed in more detail herein.
  • a final recipient cell is a bacterial cell into which exogenous methylated DNA is introduced, such as a Clostridium bacterium.
  • Electroporation is a significant increase in the electrical conducti vity and permeability of the cell plasma membrane caused by an externally applied electrical field. It is usually used in molecular biology as a way of introducing some substance into a cell, such as loading it with a molecular probe, a drug that can change the cell's function, or a piece of coding DNA. such, as an expression vector.
  • the transforming plasmids disclosed herein can contain an antibiotic resistance marker for selection in both the methylating bacterial host cell and the recipient bacterial cell along with a Gram-negative and aGram positive replicon.
  • the transforming plasmid can also include a transposon expressed in the bacterial recipient cell.
  • the plasmids When methylating plasmids are transformed with general replication and/or conjugative plasmids, the plasmids can be purified and transformed together into the bacterial recipient ceil for additional protection upon entry into the recipient cell. Alternatively, the co-residing plasmids may first be separated and then only the modified transforming plasmid transferred to the bacterial recipient cell.
  • Example 1 Metabolic engineering of Clostridium ceMulovomm for biof sel production from cellulose: Restriction-modification system analysis and in vivo methylation protection for enhanced transformation and adhE2 expression
  • C. cellulovorans was chosen as the host to incorporate the heterologous n-butanol synthesis pathway from C. acetobutylicum.
  • C. cellulovorans, isolated from a wood chip pile was capable of utilizing various substrates, including cellulose, xylan, pectin, celiobiose, glucose, maltose, galactose, sucrose, lactose, and mannose. It was found to encode 57 celluiosomal genes (Tamatu et al., 2010). Compared to C. cellulolyticum. and C. Ihermocellum, C.
  • C. cellulovorans has the ad vantage of the simpler metabolic engineering process for butanol production, since it already encodes the butyric acid synthesis pathway ( Figure 8). Therefore, only one gene, aldehyde/alcohol dehydrogenase 2 (adhE2), needs to be overexpressed in C. cellulovorans for n- butanol production. Therefore, C. cellulovorans can be used for /i-butanol production from celiulosic biomass by metabolic engineering. This is due to its efficient utilization of celiulosic biomass and its existing metabolic pathway.
  • E. coli was grown in Luria--Bertani (LB) medium (Fisher Scientific, US). C.
  • DSMZ modified medium 520
  • lOOOx trace elements contained: HC1 (25%; 7.7 M), 10 ml; FeCl 2 -4H 2 0, 1.5 g; ZnCk, 70 mg; MnCl 2 -4H 2 0, 100 mg; H3BO3, 6 mg; Cod;- ! ⁇ ). 190 mg; Cai i.. 2 ⁇ 0. 2 mg; ⁇ l b-M ⁇ ). 24 mg;
  • the cell debris was removed by centrifugation at 13,200 rpm for 30 min at 4 °C.
  • the lysate was adjusted to contain 50 mM NaCl. After 10 min on ice, the lysate was centrifuged at 13,200 rpm for 10 min at 4 °C. The same volume of glycerol was mixed with the supernatant, stored at -20 °C.
  • 0.5-1 ⁇ g piasmid substrate was incubated with 3 ⁇ cell extract at 37 °C in the digestion buffer ( 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCk, 1 mM dithiothreitol, 0.01% BSA) in 25 ⁇ reaction volume. All the manipulations were handled in anaerobic condition.
  • coli pXYl with M. Cce743I and M. Cce743II activities.
  • pMTL 80000 series shuttle plasmids for transformation of C. cellulovorans were then transformed into competent E. coli pXYl for in vivo methylation. Plasmids prepared from these strains were subjected to restriction assay as previously described to test its protection effect or used for transformation.
  • cytosine The methylation status on cytosine was determined by using Epimark bisulfite conversion kit (NEB, US). Genomic DNA of C. cellulovorans prepared as described above was treated by bisulfite reagent. Unmethylated cytosine was converted to uracil, while methylated cytosine remained unchanged. The converted genomic DNA was then subjected to PCR to amplify the DNA pieces covering Ccel Jl site and Cce743II site. Two pairs of specific primers were designed, shown in Table 3. Cytosines in the forward primers were replaced by thymines. Similarly, guanines base-paired with cytosines were replaced by adenines in the reverse primers. By comparing the sequencing results of the PCR products with their original sequences, the methylation pattern at single-nucleotide resolution was determined.
  • the mixture was then transferred to a pre-chilled electroporation cuvette (0.2 cm inter-electrode distance) and incubated on ice for 10 min.
  • Cells were pulsed once at 1.8 kV, 25 ⁇ , 800 ⁇ (Bio-Rad Gene Pulser). The resulting pulse duration was 4-6 ms.
  • the cuvette was moved to ice immediately after pulse, incubating for 10-15 min.
  • the cell suspension was then transferred to a tube with 2-3 ml medium 520, incubating at 37 °C for recovery. After overnight incubation, 15-20 ml medium 520 was added, supplemented with 15 ⁇ ' ⁇ ].
  • thiamphenicol thiamphenicol.
  • plasmid from C. cellulovorans was prepared. 25 ml ceils were collected, and treated by 20 mg ml lysozyme from chicken egg (Sigma, US) in PI buffer (miniprep kit, QIAGEN, US) for 5 h prior to plasmid purification. The prepared plasmid was then transformed into competent E. coli DH5a for further replication. The plasmid was prepared from E. coli DH5a for verification.
  • the plasmid was verified by PCR using specific primers flanking adhE2 (M 13f: TGTAAAACGACGGCCAGT, SEQ ID NO: 7; M13r: GGAAACAGCTATGACCGC, SEQ ID NO: 8). Secondly, the plasmid was verified by enzyme digestion by BamHl and SacJl.
  • Exponential-phase (OD600 0.5-0,6) cells (100 mi), harvested by centrifugation, were washed once and resuspended in 1 mi Tris-HCl buffer (0.1 M Tris-HCl, pH 7.5, 1 mM dithiothrei
  • NADH consumption was monitored every 12 s for 5 min at 365 nm.
  • Enzyme activity was calculated on the basis of a molar NADH extinction coefficient of 3.4 cm 'mM One unit of enzyme activity was defined as the amount of enzyme converting 1 ⁇ NADH per minute under the reaction conditions. Protein concentration in cell extract was determined using the Bio-Rad protein assay kit with bovine serum albumin as standard.
  • batch fermentations with C. cellulo vorans/% > 151 -adhE2 were earned out in serum bottles containing 50 ml medium 520 supplemented with 30 iig/mi thiarnphenicol.
  • the medium pH was maintained between 6.0 and 7.0 by adding NaOH solution twice a day.
  • Batch fermentations with wild-type C. cellulovorans and C. cellulovorans/ ⁇ 151- adhE2 with methyl viologen (MV) were earned out in a bioreactor containing 500 ml medium 520. pH was control led at 6.5. Samples were taken twice per day to monitor cell growth, substrate consumption and production of ethanol, butanoi, acetic acid and butyric acid during the fermentation.
  • RM restriction-modification
  • C. cellulovorans The genome of C. cellulovorans published in 2010 consists of 4254 ORFs, By analyzing its genome sequences in siiico on REBASE (rebase.neb.com), 12 operons were found, consisting of putative ME subunits, RE subunits, and S subunits (shown in Figure 1). The proteins in the same operon are generally transcriptionally coupled and functionally related. Among these operons, three candidates were selected (marked with * in Figure 1), containing at least a RE and a MT working in pairs. They are type I, type II, and type III RM systems, respectively. The details of gene locus, functions, and putative target sequences were summarized in Table 2.
  • the type I RM system consisted of a RE, a MT, and an 8 subunit, possibly recognizing the EcoKl sites (5'-AACGTGC-3 ' (SEQ ID NO: 9); 5'-GCACGTT-3', (SEQ ID NO: 10)) based on protein sequence homology of the RE subunit with other organisms. Since the RE, MT, and S subunit were encoded in the same operon, it was hypothesized that they recognized the same site and work coordinately.
  • the type II RM system contained two REs and two MTs in the same operon. The first RE subunit, namely R.
  • Cee7 3I may have the same specificity as Lla l, recognizing 5'- GACGC-3' (SEQ ID NO: 1 ) and 5'-GCGTC-3' (SEQ ID NO: 2), based on the protein sequence homology with other organisms.
  • the second RE subunit namely R. ( Vi'?43H. possibly has the same specificity as ⁇ ,/ ⁇ , recognizing 5'-CCAGG ⁇ 3' (SEQ ID NO: 4) and 5'-CCTGG-3' (SEQ ID NO: 3 ), based on the protein sequence blast.
  • the MT subunit at the end of the operon (M. Cce743I!) seemed to work in pairs with R.
  • the type III RM system contained a RE, a MT and a SAM (the substrate to offer methyl group during methylation) protein. However, there was no clear knowledge of their specificity. Among these three RM systems, we narrowed down our candidates to the type II operon, since type II RM systems are the most common and dominant.
  • restriction assay was performed.
  • the cell !ysate of C. cellulovorans was prepared and incubated with plasmid pMTL82151 -adhE2, the plasmid used for transformation.
  • the DNA digestion pattern was subjected to analysis. If only R. Cce7431 worked, it was predicted to see a band around 7 kb. If only R. Cce743II played a role of restriction, it was predicted to see two bands around 3 kb overlapped, which was a similar pattern when both R. Cee743I and R. Cce743 I I worked.
  • pMTL82I51-aii 2 harbors pBPl replicon from C. hotulinwn; pMTL83151-ai/M'2 harbors pCB102 replicon from C. butyricum; pMTL84151 -a ⁇ sfAE2 harbors pCD6 replicon from C.
  • Electroporation and conjugation were employed for transformations. No matter what conditions were tested, transformations without proper methylation of the plasmids were never successful.
  • a transformation method combining plasmid methylation to overcome the restriction barrier was then established based on the RM systems identified above.
  • M. Cce743I and M. Cce743II from the genomic DNA of C. cellulovoram were cloned into a vector (pXYl) and were further expressed in E. coli, which was suitable for methylation.
  • the pMTL80000 series plasmids were transformed into the new strain E. coli XY 1 for methylation in vivo, which were then prepared for tests and electrotransformation, respectively.
  • restriction assay was performed.
  • the methylated pMTL80000 series plasmids were used for electroporation.
  • transformants harboring p. ITl.8 15 i ⁇ : ⁇ //;/: (C. cellulovorans/S3 l5 l-adhE2) were obtained. Since the plasmids prepared directly from C. cellulovoram transformant was invisible in the agarose gel electrophoresis, due to the low copy number of the replicon, it was therefore transformed into E. coli for replication, followed by plasmid purification and plasmid
  • adhE2 is a bifimctional enzyme
  • the butyraldehyde dehydrogenase activity and butanol dehydrogenase activity were measured by monitoring NADH consumption at 365nm.
  • the butyraldehyde dehydrogenase activity (0.162 U/mg) of adhE2 increased about 27-fold
  • butanol dehydrogenase activity (0.077 U/mg) of adhE2 increased about 7- fold in the strain C. cellulovoransl%3151 -adhE2, compared to wild-type control.
  • C. celMovorans/ 15 l-adhE2 was able to produce n-butanol and ethanol with the adhE2 activity, utilizing crystalline cel lulose as carbon source. Fermentation kinetics of wild-type C. cellulovorans and C. cellulovoransl%3151 -adhE2 when grown on glucose, celiobiose, and crystalline celluiose were analyzed (shown in Figure 5 and Figure 6). The results showed that 1.42 g/L butanol and 1.60 g/L ethanol were produced by C. cellulovoransl 3151 -adhE2 when grown on 15 g/L crystalline celluiose (shown in Figure 4B and Figure 6C).
  • butanol titer from crystalline cellulose by this strain was the highest, compared to other known engineered or wild-type celluioiytic strains. A much higher butanol titer and yield can be achieved with farther metabolic engineering and process optimization.
  • RM systems existing in the bacteria as defensive machineries is the most dominant factor in affecting the transformation efficiency (Roberts et al., 2010).
  • RM systems became better understood.
  • Putative RM systems in various frequently-used Clostridia species were analyzed in silico on REBASE (shown in Table 6). The analysis suggested that RM systems were common defensive machineries in Clostridia, resulting in low transformation efficiency. To achieve successful transformation of Clostridia, circumvention of their restri ction barriers is the key.
  • plasmid methylation was not absolutely necessary. However, problems arose when using piasmids with either no restriction sites, or few restriction sites. 3), Temporarily inactivating the RM system by heat to facilitate transformation. However, the method was only effective to certain strains. 4), Methylation of piasmids prior to transformation to enhance the transformation efficiency. The key to success of this strategy was the utilization proper MTs, recognizing the same restriction sites as the REs in the strain. Successful transformation was shown to be facilitated significantly, or be absolutely dependent, on plasmid methylation in several Clostridia species, since no transformants could be obtained without methylation (shown in the Table 7). Similarly, plasmid methylation facilitated transformation in C, cellulovorans.
  • Example 2 The Effects of Methyl Viologeii on Biofuel Production by Fermentation with Engineered Clostridium cellulovorans
  • methyl viologen used to increase NADH availability. Since «.-butanol and ethanol production was usually limited by NADH availability, increasin g NADH by th e addition of m ethyl viologen showed great impact on metabolism of the engineered C. cellulovorans, increasing production of /i-butanol and ethanol as well as inhibiting production of acetic acid and butyric acid. Particularly, the optimum timing and concentration of methyl viologen were determined. In general, with the addition of methyl viologen, biofuel production by the engineered C, cellulovorans was greatly enhanced, showing high level biofuel production directly from cell losic biomass is achievable.
  • C. cellulovoransi%S 151 -adhE2 was cultured in modified media 520 (DSMZ), containing
  • GC gas chrornatograph
  • FID flame ionization detector
  • the GC was operated at an injection temperature of 200 °C with 1 of sample injected with an auto injector (AOC-20i, Shimadzu).
  • the column temperature was initially held at 80 °C for 3 min, then increased at a constant rate of 30 °C per min to 150 °C, and held at 150 °C for 3.7 min.
  • Samples were centrifuged at 1 ,3200 rpm for 5 min in 1.5 ml microcentrifuge tubes and the supernant was subjected to GC.
  • Glucose and cellobiose were quantified by high performance liquid chromatography (HPLC, LC-20AD, Shimadzu, Columbia, MD) with an organic acid column (Bio-Rad HP.X- 87H, ion exclusion organic acid column, 300*7.8 mm).
  • HPLC high performance liquid chromatography
  • LC-20AD LC-20AD
  • Shimadzu Columbia, MD
  • organic acid column Bio-Rad HP.X- 87H, ion exclusion organic acid column, 300*7.8 mm.
  • Cellulose sample 1 ml was washed by distilled water prior to autoclaving. After autoclaving, the cellulose pellet was washed again by distilled water.
  • ceUulovorans was unknown. Particularly, when different carbon substrates were used, the effect of methyl viologen might vary due to their different reducing power. Therefore, the
  • methyl viologen The inhibitory effect of methyl viologen on cell growth limited the application of methyl viologen for higher alcohol production. Therefore, whether the inhibitory effect on cell growth could be relieved was tested, by adding methyl viologen at a later growth stage. After overnight incubation (18 h), cell growth was entering the stationary phase from the exponential phase, with OD 600 at - 2-3. Then, various concentrations of methyl viologen were added to the
  • methyl viologen Since different sugars can require different amounts of methyl viologen to achieve the best effect, different concentrations of methyl viologen were added, when cellobiose served as the carbon source. As shown in Table 13, ethanol and butanol titers were significantly increased with the addition of 100-500 ⁇ . ⁇ methyl viologen. Specifically, the addition of 100 ⁇ or 250 ⁇ had similar effects on alcohol and acid production, producing 1.9 g/L ethanol, 2.6-2.8 g/L butanol, 0.3-0.5 g/L acetic acid, and 0.1-0.2 g L butyric acid.
  • butanol and ethanol production by the engineered C. cellulovorans can be greatly enhanced by manipulating the fermentation conditions such as adding the artificial electron carrier (e.g., methyl viologen). It is conceivable that other means, including but not limited to, such as inhibiting hydrogen production, can also be used to increase butanol and ethanol production by the engineered C. cellulovorans.
  • the artificial electron carrier e.g., methyl viologen
  • cytosine was converted to uracil. Therefore, cytosine was replaced by thymine in the forward primers, while guanine was replaced by adenine in the reverse primers.
  • the small "t” in the primers originally was cytosine in the sense strand.
  • the small "a” in the primers originally was guanine in the anti sense strand.
  • AACGTGC (SEQ ID NO: 16)
  • Acetate kinase (ack, SEQ ID NO: 18)
  • Butyrate kinase (huk, SEQ ID NO: 20)
  • ClosTron a universal gene knock-out system for the genus Clostridium. J Microbiol Methods. 70(3):452-64. Heap JT, Pennington OJ, Cartman ST, Minton NP. 2009, A modular system for
  • Mingardon F Ferret S, Belai ' ch A, Tardif C, Belai ' ch JP, Fierobe HP. 2005. Heterologous production, assembly, and secretion of a mmicellulosome by Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol. 71 (3): 1215-22.
  • CipA scaffolding protein Characterization of the CipA scaffolding protein and in vivo production of a minicellulosome in Clostridium acetobutylicum. .1 Bacterioi. 185(3 ): 1092-6.
  • Argyros DA Tripathi SA, Barrett TF, Rogers SR., Feinberg LF, Olson DG, Foden JM, Miller BB, Lynd LR, Hogsett DA, Caiazza NC. 2011. High ethanol titers from cellulose by using metabolically engineered thermophilic, anaerobic microbes. Appl Environ Microbiol.
  • Bokinsky G Peralta-Yahya PP. George A, Holmes BM, Steen EJ, Dietrich J, Lee TS, Tullman-Ercek D, Voigt CA, Simmons BA, easfing JD. 201 1 . Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli. Proc Natl Acad Sci U S A. 108(50): 19949-54.
  • CBP Consolidated bioprocessing
  • Tashiro Y Takeda K, Kobayashi G, Sonomoto K. 2005. High production of acetone- butanol-ethanol with high ceil density culture by cell -recycling and bleeding. J BiotechnoL 120(2): 197-206.
  • Yamada R, Taniguchi N, Tanaka T, Ogino C, Fukuda H, Kondo A. 2011 Direct ethanol production from ceilulosic materials using a diploid strain of Saccharomyces cerevisiae with optimized cellulase expression. Biotechnol Biofueis. 4:8.

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Abstract

L'invention concerne une bactérie Clostridium acidogène cellulolytique transformée pour qu'elle surexprime une aldéhyde/alcool déshydrogénase pour la production de biocarburant. L'invention concerne également des procédés permettant l'expression d'une séquence de gène hétérologue dans une bactérie Clostridium acidogène cellulolytique. L'invention concerne également des procédés de production d'un biocarburant à partir de cellulose. L'invention concerne également un procédé in vivo pour méthyler un plasmide contenant des gènes hétérologues pour leur expression dans une bactérie Clostridium.
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