WO2008052596A1 - Process for the biological production of n-butanol with high yield - Google Patents

Process for the biological production of n-butanol with high yield Download PDF

Info

Publication number
WO2008052596A1
WO2008052596A1 PCT/EP2006/067993 EP2006067993W WO2008052596A1 WO 2008052596 A1 WO2008052596 A1 WO 2008052596A1 EP 2006067993 W EP2006067993 W EP 2006067993W WO 2008052596 A1 WO2008052596 A1 WO 2008052596A1
Authority
WO
WIPO (PCT)
Prior art keywords
butanol
gene
microorganism
erythromycin
thiamphenicol
Prior art date
Application number
PCT/EP2006/067993
Other languages
French (fr)
Inventor
Philippe Soucaille
Original Assignee
Metabolic Explorer
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Metabolic Explorer filed Critical Metabolic Explorer
Priority to PCT/EP2006/067993 priority Critical patent/WO2008052596A1/en
Priority to US12/447,726 priority patent/US20100086982A1/en
Priority to PCT/EP2007/061634 priority patent/WO2008052973A2/en
Priority to BRPI0718142-6A2A priority patent/BRPI0718142A2/en
Priority to CN2007800391785A priority patent/CN101528935B/en
Priority to CA2665102A priority patent/CA2665102C/en
Priority to JP2009533879A priority patent/JP5442441B2/en
Priority to EP07821988A priority patent/EP2084287B1/en
Priority to AU2007316189A priority patent/AU2007316189B2/en
Priority to KR1020097011053A priority patent/KR101444968B1/en
Priority to RU2009118372/10A priority patent/RU2461627C2/en
Priority to DK07821988.8T priority patent/DK2084287T3/en
Priority to MX2009004660A priority patent/MX2009004660A/en
Priority to TW096140669A priority patent/TW200835792A/en
Priority to ARP070104849A priority patent/AR063762A1/en
Publication of WO2008052596A1 publication Critical patent/WO2008052596A1/en
Priority to ZA200902639A priority patent/ZA200902639B/en
Priority to IL198342A priority patent/IL198342A/en
Priority to JP2013172060A priority patent/JP2014000087A/en
Priority to US14/321,173 priority patent/US20140377825A1/en

Links

Classifications

    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C12P7/16—Butanols
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the invention comprises a process for the bioconversion of a fermentable carbon source to n-butanol at high yield by a metabolically engineered microorganism.
  • n-Butanol is a colorless, neutral liquid of medium volatility with restricted miscibility (about 7-8%) in water, but freely miscible with all common solvents such as glycols, ketones, alcohol, aldehydes, ethers, and aromatic and aliphatic hydrocarbons
  • n- Butanol is used i) to make other chemicals, ii) as a solvent and iii) as an ingredient in formulated products such as cosmetics.
  • the major uses of n-butanol as a feed-stock are in the synthesis of acrylate/methacrylate esters, glycol ethers, n-Butyl acetate, amino resins and n-Butylamines.
  • n-butanol is a better bio fuel than ethanol due to lower vapour pressure, higher energy content (closer to that of gasoline) and lesser susceptibility to separation in the presence of water. Furthermore, n-butanol can be blended at higher concentrations than ethanol for use in standard vehicle engines and it does not require automakers to compromise on performance to meet environmental regulations; it is also suitable for transport in pipelines and as a result it has the potential to be introduced into gasoline quickly and avoid the need for additional large-scale supply infrastructures. n-butanol can be produced as an acetone/n-butanol/ethanol (ABE) mixture by the fermentation of carbohydrate by solventogenic Clostridia. The ABE fermentations are biphasic.
  • ABE acetone/n-butanol/ethanol
  • butyryl-CoA as an intermediate which can be reduced, depending on the physiological conditions, by two different bi-functional aldehyde-alcohol dehydrogenases encoded by adhEl and adhE2.
  • Butyryl-CoA can also be converted to butyric acid by a phospho-transbutyrylase and a butyrate kinase encoded respectively by the ptb and buk genes.
  • Acetone is produced from aceto-acetyl-CoA (an intermediate in the production of butyryl-CoA) by a CoA-transferase and an acetoacetate decarboxylase encoded respectively by the ctfAB and adc genes.
  • Hydrogen is produced by an iron only hydrogenase encoded by the hydA gene.
  • a hydrogenase inhibitor When cultures are performed in the presence of carbon monoxide, a hydrogenase inhibitor, n-butanol, ethanol and lactate are the main fermentation products. Lactate is produced from pyruvate by a lactate dehydrogenase encoded by the ldh gene.
  • the problem to be solved by the present invention is to obtain a stable mutant strain with no butyrate kinase activity , that could be cultureD for several generations without any possibility of reversion to the wild type genotype.
  • This strain would be useful for the biological production of n-butanol at high yield, from an inexpensive carbon substrate such as glucose or other sugars, by genetically stable cultures of Clostridia.
  • the number of biochemical steps to inactivate and the complexity of the regulation of the metabolism necessitate, for an industrial feasible process of n-butanol production, the use of a metabolically engineered whole cell catalyst.
  • Applicants have solved the stated problem and the present invention provides a method for bioconverting a fermentable carbon source to n-butanol as a major product by genetically stable cultures of Clostridia.
  • Glucose is used as a model substrate and recombinant Clostridium acetobutylicum is used as the model host.
  • a stable recombinant C. acetobutylicum unable to metabolize butyryl-CoA to butyrate is constructed by deleting the gene coding for the butyrate kinase (buk).
  • a recombinant C is constructed by deleting the gene coding for the butyrate kinase (buk).
  • acetobutylicum unable to produce acetone is constructed by deleting the genes coding for the CoA-transferase (ctfAB).
  • a recombinant strain unable to produce lactate is constructed by deleting the gene coding for the lactate dehydrogenase (Idh).
  • a recombinant C acetobutylicum unable to produce acetate is constructed by deleting the genes coding for the phosphotransacetylase and/or acetate kinase (pta and ack).
  • the flux of hydrogen production is decreased and then the flux of reducing equivalent redirected toward n-butanol production by attenuating the gene encoding the hydrogenase (hydA).
  • the present invention may be generally applied to include any carbon substrate that is readily converted to acetyl-coA.
  • n-butanol comprising: (a) at least deletion of one of the two genes involved in the conversion of butyryl-CoA to butyrate and (b) at least deletion of one of the two genes encoding the CoA-transferase activity.
  • the recombinant organism may comprise i) inactivating mutations in endogenous genes selected from the group consisting of: (a) a gene encoding a polypeptide having lactate dehydrogenase activity (b) a gene encoding a polypeptide having phospho-transacetylase or actate kinase activity and ii) attenuation in a gene encoding a polypeptide having hydrogenase activity.
  • the invention provides a stable process for the production of n-butanol at high yield from a recombinant organism comprising: (a) contacting the recombinant organism of the present invention with at least one carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and single-carbon substrates whereby n-butanol is produced; optionally (b) recovering the n- butanol during the production through a step of gas striping and (c) purifying n-butanol from the condensate by distillation.
  • Figure 1 depicts the genetic engineering of central metabolism in the development of a butanol production system from carbohydrates.
  • microorganism refers to all kind of unicellular organisms, including prokaryotic organisms like bacteria, and eukaryotic organisms like yeasts.
  • appropriate culture medium refers to a culture medium adapted for the used microorganism as it is well known by the man skilled in the art.
  • carbon substrate or “source of carbon” means any carbon source capable of being metabolized by a microorganism wherein the substrate contains at least one carbon atom. In particular it may be glucose, sucrose, mono- or oligosaccharides, starch or its derivatives, glycerol, and their mixtures thereof.
  • Attenuation refers to a decreased expression of a gene or a decreased activity of the protein, product of the gene.
  • the man skilled in the art knows numerous means to obtain this result, and for example:
  • destabilizing the corresponding messenger RNA or the protein Use of elements destabilizing the corresponding messenger RNA or the protein. Deletion of the gene if no expression is needed.
  • the term "deleted gene” means that a substantial part of the coding sequences of said gene was removed. Preferably, at least 50% of the coding sequence was removed, and more preferably at least 80%.
  • enzymes are identified by their specific activities. This definition thus includes all polypeptides that have the defined specific activity also present in other organisms, more particularly in other microorganisms. Often enzymes with similar activities can be identified by their grouping to certain families defined as PFAM or COG.
  • PFAM protein families database of alignments and hidden Markov models; represents a large collection of protein sequence alignments. Each PFAM makes it possible to visualize multiple alignments, see protein domains, evaluate distribution among organisms, gain access to other databases, and visualize known protein structures.
  • COGs clusters of orthologous groups of proteins; hllSl/J ⁇ S. ⁇ JAlhl ⁇ ii ⁇ lh ⁇ B9 ⁇ LQQ ⁇ J) are obtained by comparing protein sequences from 43 fully sequenced genomes representing 30 major phylogenic lines. Each COG is defined from at least three lines, which permits the identification of former conserved domains.
  • the means of identifying homologous sequences and their percentage homologies are well known to those skilled in the art, and include in particular the BLAST programs, which can be used from the website http://www.ncbi.nlm.nih.gov/BLAST/ with the default parameters indicated on that website.
  • the sequences obtained can then be exploited (e.g., aligned) using, for example, the programs CLUSTALW (h ⁇ JMsil ⁇ - ⁇ -yy- ⁇ M3. ⁇ AUkM)i ⁇ 3h * ⁇ ) or MULTALIN (http;//p_ro ⁇ with the default parameters indicated on those websites
  • the present invention provides a method for the fermentative batch or continuous production of n-butanol by culturing a microorganism in an appropriate culture medium comprising a carbon source and the simultaneous recovery of n-butanol from the culture medium wherein at least one gene involved in butyrate formation is deleted in the microorganism.
  • a specific embodiment of the invention provides a method wherein the microorganism is modified to be unable to convert butyryl-CoA to butyrate due to the deletion of at least one gene encoding for phospho-transbutyrylase iptb) or butyrate kinase (buk).
  • Deletion of genes in Clostridia can be done using the method recently described in patent application PCT/EP2006/066997 allowing the i) replacement of the gene to delete with an erythromycin resistance gene and ii) removal of the erythromycin resistance gene with a recombinase.
  • the microorganism is unable to produce acetone due to an attenuation or a deletion of at least one of the gene involved in acetone formation.
  • this gene is encoding for the enzyme CoA-transferase (ctfAB) or acetoacetate decarboxylase (adc). Deletion of one of these genes can be done using the method recently described in patent application PCT/EP2006/066997.
  • the microorganism used in the method of the invention is unable to produce lactate. In particular this can be due to a deletion of the gene ldh encoding for lactate dehydrogenase. Deletion of ldh can be done using the method recently described in patent application PC17EP2006/066997.
  • the microorganism is modified in such a way to be unable to produce acetate.
  • This result can be achieved by deletion of at least one involved in acetate formation.
  • this gene is selected among the group consisting of the genes encoding for phospho-transacetylase (pt ⁇ ) or acetate kinase (ack). Deletion of one of these genes can be done using the method recently described in patent application PCT/EP2006/066997.
  • An embodiment of the invention also provides a microorganism with a decreased flux of hydrogen production and then a redirection of the flux of reducing equivalent toward n-butanol production; this can be done by attenuating the gene encoding the hydrogenase (hydA), an enzyme that provides a sink for reducing equivalent in the form of hydrogen production. Attenuation of hydA can be done by replacing the natural promoter by a low strength promoter or by element destabilizing the corresponding messenger RNA or the protein. If needed, complete attenuation of the gene can also be achieved by a deletion of the corresponding DNA sequence.
  • hydA hydrogenase
  • Attenuation of hydA can be done by replacing the natural promoter by a low strength promoter or by element destabilizing the corresponding messenger RNA or the protein. If needed, complete attenuation of the gene can also be achieved by a deletion of the corresponding DNA sequence.
  • the used microorganism is selected among the group consisting of C. acetobutylicum, C. beijerinckii, C. saccharoperbutylacetonicum or C. saccharobutylicum.
  • the culture is continuous and stable.
  • the method according to the invention comprises the following steps:
  • Clostridia are fermented at a temperature between 20 0 C and 55°C, preferentially between 25°C and 40 0 C, and more specifically about 35°C for C. acetobutylicum.
  • the fermentation is generally conducted in fermentors with an inorganic culture medium of known defined composition adapted to the bacteria used, containing at least one simple carbon source, and if necessary a co-substrate necessary for the production of the metabolite.
  • the invention is also related to the microorganism as described previously.
  • this microorganism is selected among the group consisting of C. acetobutylicum, C. beijerinckii, C. saccharoperbutylacetonicum or C. saccharobutylicum.
  • DNA fragments BUK 1-BUK 2 and BUK 3-BUK 4 were joined in a PCR fusion experiment with primers BUK 1 and BUK 4 and the resulting fragment was cloned in pCR4-TOPO-Blunt to yield pTOPO :buk.
  • pTOPO :buk At the unique Stul site of pTOPO :buk, an antibiotic resistance MLS gene with FRT sequences on both sides was introduced from the Stul fragment of pUC18-FRT-MLS2.
  • the BUK deletion cassette obtained after BamHI digestion of the resulting plasmid was cloned into pCons::upp at the BamHI site to yield the pREP ⁇ BUK::upp plasmid.
  • the pREP ⁇ BUK::upp plasmid was used to transform by electroporation C. acetobutylicum MGCAcacl5Aupp strain. After selection on Petri plate for clones resistant to erythromycin (40 ⁇ g/ml), one colony was cultured for 24 hours in liquid synthetic medium with erythromycin at 40 ⁇ g/ml and 100 ⁇ l of undiluted culture was plated on RCA with erythromycin at 40 ⁇ g/ml and 5-FU at 400 ⁇ M.
  • Colonies resistant to both erythromycin and 5-FU were replica plated on both RCA with erythromycin at 40 ⁇ g/ml and RCA with thiamphenicol at 50 ⁇ g/ml to select clones where 5-FU resistance is also associated with thiamphenicol sensitivity.
  • the genotype of clones resistant to erythromycin and sensitive to thiamphenicol was checked by PCR analysis (with primers BUK 0 and BUK 5 located outside of the buk deletion cassette).
  • the Acacl 5AuppAbuk: :mls R strain which have lost pREP ⁇ buk::upp was isolated.
  • the Acacl 5AuppAbuk: :mls R strain was transformed with pCLFl.l vector expressing the FIp 1 gene encoding the FIp recombinase from S. cerevisiae. After transformation and selection for resistance to thiamphenicol (50 ⁇ g/ml) on Petri plate, one colony was cultured on synthetic liquid medium with thiamphenicol at 50 ⁇ g/ml and appropriate dilutions were plated on RCA with thiamphenicol at 50 ⁇ g/ml. Thiamphenicol resistant clones were replica plated on both RCA with erythromycin at 40 ⁇ g/ml and RCA with thiamphenicol at 50 ⁇ g/ml.
  • the genotype of clones with erythromycin sensitivity and thiamphenicol resistance was checked by PCR analysis with primers BUK 0 and BUK 5. Two successive 24 hours cultures of the Acacl5AuppAbuk strain with erythromycin sensitivity and thiamphenicol resistance were carried out in order to lose pCLFl.l. The Acacl5AuppAbuk strain which has lost pCLFl.l was isolated according to its sensitivity to both erythromycin and thiamphenicol.
  • Ctf 1 SEQ ID N°7 aaaaggGi cxcagacactataatagctttaggtggtacccc Ctf 2 ggggaggcctaaaagggggattataaaaagtagttgaaatatgaaggtttaaggttg Ctf 3 cccccttt ttaggc c tccccatatccaatgaacttagacccatggctg Ctf 4 SEQ ID N 0 IO aaaggai ccgtgttataatgtaaatataaataaataggactagaggcg
  • Two DNA fragments surrounding ctfAB were PCR amplified with the Pwo polymerase with total DNA from C. acetobutylicum as template and two specific couples of oligonucleotides. With the couples of primers CTF 1-CTF 2 and CTF 3-CTF 4, two DNA fragments were respectively obtained. Both primers CTF 1 and CTF 4 introduce a BamHI site while primers CTF 2 and CTF 3 have a complementary region which introduces a Stul site. DNA fragments CTF 1-CTF 2 and CTF 3-CTF 4 were joined in a PCR fusion experiment with primers CTF 1 and CTF 4 and the resulting fragment was cloned in pCR4- TOPO-Blunt to yield pTOPO :CTF.
  • the pREP ⁇ CTF::upp plasmid was used to transform by electroporation C. acetobutylicum MGC Acac 15 Aupp Abuk strain. After selection on Petri plate for clones resistant to erythromycin (40 ⁇ g/ml), one colony was cultured for 24 hours in liquid synthetic medium with erythromycin at 40 ⁇ g/ml and 100 ⁇ l of undiluted culture was plated on RCA with erythromycin at 40 ⁇ g/ml and 5-FU at 400 ⁇ M.
  • Colonies resistant to both erythromycin and 5-FU were replica plated on both RCA with erythromycin at 40 ⁇ g/ml and RCA with thiamphenicol at 50 ⁇ g/ml to select clones where 5-FU resistance is also associated with thiamphenicol sensitivity.
  • the genotype of clones resistant to erythromycin and sensitive to thiamphenicol was checked by PCR analysis (with primers CTF 0 and CTF 5 located outside of the ctfAB deletion cassette).
  • the Acacl 5AuppAbuk ActfAB::mls R strain which have lost pREP ⁇ CTF::upp was isolated.
  • the Acacl5AuppAbukActfAB::mls R strain was transformed with pCLFl.l vector expressing the FIp 1 gene encoding the FIp recombinase from S. cerevisiae. After transformation and selection for resistance to thiamphenicol (50 ⁇ g/ml) on Petri plate, one colony was cultured on synthetic liquid medium with thiamphenicol at 50 ⁇ g/ml and appropriate dilutions were plated on RCA with thiamphenicol at 50 ⁇ g/ml.
  • Thiamphenicol resistant clones were replica plated on both RCA with erythromycin at 40 ⁇ g/ml and RCA with thiamphenicol at 50 ⁇ g/ml.
  • the genotype of clones with erythromycin sensitivity and thiamphenicol resistance was checked by PCR analysis with primers CTF 0 and CTF 5.
  • Two successive 24 hours cultures of the Acacl 5 AuppAbukActfAB strain with erythromycin sensitivity and thiamphenicol resistance were carried out in order to lose pCLFl.l.
  • the Acacl 5 AuppAbukActfAB strain which has lost pCLFl.l was isolated according to its sensitivity to both erythromycin and thiamphenicol.
  • Ldh 2 SEQ ID N°14 GGGG AGGCCJ A AA AAGGGGGTY AG A AAJCTVY A AA AATVYC TCTAT ' AG AGCCC ATC Ldh 3 SEQ ID N°15 CCCCCYTYTY AGGCCYCCCCGG ⁇ AAAAGACCY AAACYCCAAGG G ⁇ GG AGGCY AGG ⁇ C Ldh 4 SEQ ID N°16 AAAAGG ATCCCCCATTGTGG AG AA ⁇ ' ATYCCAAAGAAGAAAA TAATYGC
  • Two DNA fragments surrounding ldh (CAC267) were PCR amplified with the Pwo polymerase with total DNA from C. acetobutylicum as template and two specific couples of oligonucleotides. With the couples of primers LDH 1-LDH 2 and LDH 3-LDH 4, 1135 bp and 1177 bp DNA fragments were respectively obtained. Both primers LDH 1 and LDH 4 introduce a BamHI site while primers LDH 2 and LDH 3 have a complementary region which introduces a Stul site.
  • DNA fragments LDH 1-LDH 2 and LDH 3-LDH 4 were joined in a PCR fusion experiment with primers LDH 1 and LDH 4 and the resulting fragment was cloned in pCR4-TOPO-Blunt to yield pTOPO :LDH.
  • pTOPO :LDH At the unique Stul site of pTOPO :LDH, an antibiotic resistance MLS gene with FRT sequences on both sides was introduced from the 1372 bp Stul fragment of pUC18-FRT-MLS2.
  • the UPP deletion cassette obtained after BamHI digestion of the resulting plasmid was cloned into pCons::upp at the BamHI site to yield the pREP ⁇ LDH::upp plasmid.
  • the pREP ⁇ LDH::upp plasmid was used to transform by electroporation C. acetobutylicum MGCAcacl5AuppAbukActfAB strain. After selection on Petri plate for clones resistant to erythromycin (40 ⁇ g/ml), one colony was cultured for 24 hours in liquid synthetic medium with erythromycin at 40 ⁇ g/ml and 100 ⁇ l of undiluted culture was plated on RCA with erythromycin at 40 ⁇ g/ml and 5-FU at 400 ⁇ M.
  • Colonies resistant to both erythromycin and 5-FU were replica plated on both RCA with erythromycin at 40 ⁇ g/ml and RCA with thiamphenicol at 50 ⁇ g/ml to select clones where 5-FU resistance is also associated with thiamphenicol sensitivity.
  • the genotype of clones resistant to erythromycin and sensitive to thiamphenicol was checked by PCR analysis (with primers LDH 0 and LDH 5 located outside of the ldh deletion cassette).
  • the Acacl 5AuppAbuk ActfAB Aldh::mls R strain which have lost pREP ⁇ LDH::upp was isolated.
  • the Acacl5AuppAbukActfABAldh::mls strain was transformed with pCLFl.l vector expressing the FIp 1 gene encoding the FIp recombinase from S. cerevisiae. After transformation and selection for resistance to thiamphenicol (50 ⁇ g/ml) on Petri plate, one colony was cultured on synthetic liquid medium with thiamphenicol at 50 ⁇ g/ml and appropriate dilutions were plated on RCA with thiamphenicol at 50 ⁇ g/ml.
  • Thiamphenicol resistant clones were replica plated on both RCA with erythromycin at 40 ⁇ g/ml and RCA with thiamphenicol at 50 ⁇ g/ml.
  • the genotype of clones with erythromycin sensitivity and thiamphenicol resistance was checked by PCR analysis with primers LDH 0 and LDH 5.
  • Two successive 24 hours cultures of the Acacl5AuppAbukActfAB Aldh strain with erythromycin sensitivity and thiamphenicol resistance were carried out in order to lose pCLFl.l.
  • the Acacl5AuppAbukActfABAldh strain which has lost pCLFl.l was isolated according to its sensitivity to both erythromycin and thiamphenicol.
  • PA l SEQ ID N°19 aaaaggaicctattataacagtcaaacccaataaaatactggg
  • PA 2 SEQ ID Kf 20 ggggaggcctaaaaagggggttaatccatttgtattttctcccttcataatgcc
  • PA 3 SEQ ID Kf 21 ccccc ttt ttaggc c tccctttattttgcatgcttatataataaattatggctgcg
  • PA 4 SEQ ID N°22 aaaaggatccgcttttcctttttacaagatttaaagcc
  • PA O SEQ ID N°23 cacttttatttatcaagctgtaggcc PA 5 SEQ Ib N°24 tataccttttgaacctaggaaggc
  • Two DNA fragments surrounding pta-ack were PCR amplified with the Pwo polymerase with total DNA from C. acetobutylicum as template and two specific couples of oligonucleotides. With the couples of primers PA 1-PA 2 and PA 3-PA 4, two DNA fragments were respectively obtained. Both primers PA 1 and PA 4 introduce a BamHI site while primers PA 2 and PA 3 have a complementary region which introduces a Stul site. DNA fragments PA 1-PA 2 and PA 3-PA 4 were joined in a PCR fusion experiment with primers PA 1 and PA 4 and the resulting fragment was cloned in pCR4-TOPO-Blunt to yield pTOPO :PA.
  • an antibiotic resistance MLS gene with FRT sequences on both sides was introduced from the Stul fragment of pUC18-FRT- MLS2.
  • the UPP deletion cassette obtained after BamHI digestion of the resulting plasmid was cloned into pCons::upp at the BamHI site to yield the pREP ⁇ PA::upp plasmid.
  • the pREP ⁇ PA::upp plasmid was used to transform by electroporation C. acetobutylicum MGCAcacl5AuppAbukActfABAldh strain.
  • the genotype of clones resistant to erythromycin and sensitive to thiamphenicol was checked by PCR analysis (with primers PA 0 and PA 5 located outside of the pta-ack deletion cassette).
  • the Acacl5AuppAbuk ActfABAldhApta- ack::mls R strain which have lost pREP ⁇ PA::upp was isolated.
  • the Acacl5AuppAbukActfABAldh Apta-ack: :mls R strain was transformed with pCLFl.l vector expressing the FIp 1 gene encoding the FIp recombinase from S. cerevisiae.
  • Hyd l SEQ ID N°25 AAAAGGl TCC GCCTCTTCTGTATTATGCAAGGAAAGC AGCTGC Hyd 2 SEQ ID N°26 GGGG ⁇ GGCC ⁇ AAAAAGGGGGTATATAAAATAAATGTG CCTTAACATC TAAGTTGAGGCC
  • Hyd 3 SEQ ID N°27 CCCCCTTTTT ⁇ GGCC ⁇ CCCCGTTTATCCTCCCAAAATGT AAAATATAA TTAAAATATATT AATAAACTTCGATT AATAAACTVC& Hyd 4 SEQ ID N°28 AAAAGG ⁇ CCCCTTTTAGCGTATAAAGTTTTATATAGC
  • Two DNA fragments surrounding hydA (CAC028) were PCR amplified with the Pwo polymerase with total DNA from C. acetobutylicum as template and two specific couples of oligonucleotides. With the couples of primers HYD 1-HYD 2 and HYD 3-HYD 4, 1269 bp and 1317 bp DNA fragments were respectively obtained. Both primers HYD 1 and HYD 4 introduce a BamHI site while primers HYD 2 and HYD 3 have a complementary region which introduces a Stul site.
  • DNA fragments HYD 1-HYD 2 and HYD 3-HYD 4 were joined in a PCR fusion experiment with primers HYD 1 and HYD 4 and the resulting fragment was cloned in pCR4-TOPO-Blunt to yield pTOPO :HYD.
  • pTOPO :HYD At the unique Stul site of pTOPO :HYD, an antibiotic resistance MLS gene with FRT sequences on both sides was introduced from the 1372 bp Stul fragment of pUC18-FRT-MLS2.
  • the UPP deletion cassette obtained after BamHI digestion of the resulting plasmid was cloned into pCons::upp at the BamHI site to yield the pREP ⁇ HYD::upp plasmid.
  • the pREP ⁇ HYD::upp plasmid was used to transform by electroporation C. acetobutylicum MGCAcac 15 Aupp AbukActfAB Aldh strain. After selection on Petri plate for clones resistant to erythromycin (40 ⁇ g/ml), one colony was cultured for 24 hours in liquid synthetic medium with erythromycin at 40 ⁇ g/ml and 100 ⁇ l of undiluted culture was plated on RCA with erythromycin at 40 ⁇ g/ml and 5-FU at 400 ⁇ M.
  • Colonies resistant to both erythromycin and 5-FU were replica plated on both RCA with erythromycin at 40 ⁇ g/ml and RCA with thiamphenicol at 50 ⁇ g/ml to select clones where 5-FU resistance is also associated with thiamphenicol sensitivity.
  • the genotype of clones resistant to erythromycin and sensitive to thiamphenicol was checked by PCR analysis (with primers HYD 0 and HYD 5 located outside of the hydA deletion cassette).
  • the Acacl5AuppAbukActfABAldhAhydA::mls R strain which have lost pREP ⁇ HYD::upp was isolated.
  • the Acacl ⁇ AuppAbukActfABAldhAhydA.-.-mls 11 strain was transformed with pCLFl.l vector expressing the FIp 1 gene encoding the FIp recombinase from S. cerevisiae. After transformation and selection for resistance to thiamphenicol (50 ⁇ g/ml) on Petri plate, one colony was cultured on synthetic liquid medium with thiamphenicol at 50 ⁇ g/ml and appropriate dilutions were plated on RCA with thiamphenicol at 50 ⁇ g/ml.
  • Thiamphenicol resistant clones were replica plated on both RCA with erythromycin at 40 ⁇ g/ml and RCA with thiamphenicol at 50 ⁇ g/ml.
  • the genotype of clones with erythromycin sensitivity and thiamphenicol resistance was checked by PCR analysis with primers HYD 0 and HYD 5.
  • Two successive 24 hours cultures of the Acacl 5 AuppAbukActfABAldhAhydA strain with erythromycin sensitivity and thiamphenicol resistance were carried out in order to lose pCLFl.l.
  • the Acacl 5 AuppAbukActfABAldhAhydA strain which has lost pCLFl.l was isolated according to its sensitivity to both erythromycin and thiamphenicol.
  • DASGIP 300 ml fermentors
  • the fermentor was filled with 250 ml of synthetic medium, sparged with nitrogen for 30 min and inoculated with 25 ml of preculture to an optical density (OD600nm) between 0.05 and 0.1.
  • the temperature of the culture was maintained constant at 35 0 C and the pH was permanently adjusted at 5.5 using an NH 4 OH solution.
  • the agitation rate was maintained at 300 rpm during the fermentation.
  • n-butanol producing strain was analyzed in chemostat cultures in the synthetic medium described by Soni et al (Soni et al, 1987, Appl. Microbiol. Biotechnol. 27:1-5).
  • An overnight culture at 35°C was used to inoculate a 300 ml fermentors (DASGIP) using an anaerobic chemostat protocol.
  • DASGIP 300 ml fermentors
  • the fermentor was filled with 250 ml of synthetic medium, sparged with nitrogen for 30 min and inoculated with 25 ml of preculture to an optical density (OD600nm) between 0.05 and 0.1.
  • Metabolic flux analysis elucidates the importance of the acid- formation pathways in regulating solvent production by Clostridium acetobutylicum. Metab Eng. 1999,1:206-13.

Landscapes

  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention provides a method for the biological production of n-butanol at high yield from a fermentable carbon source. In one aspect of the present invention, a process for the conversion of glucose to n-butanol is achieved by the use of a recombinant organism comprising a host C. acetobutlicum transformed i) to eliminate the butyrate pathway ii) to eliminate the acetone pathway iii) to eliminate the lactate pathway and iv) to eliminate the acetate pathway. In another aspect of the present invention, the hydrogen flux is decreased and the reducing power redirected to n-butanol production by attenuating the expression of the hydrogenase gene. Optionally the n-butanol produced can be eliminated during the fermentation by gas striping and further purified by distillation.

Description

Process for the biological production of n-Butanol with high yield
FIELD OF INVENTION
The invention comprises a process for the bioconversion of a fermentable carbon source to n-butanol at high yield by a metabolically engineered microorganism.
BACKGROUND OF THE INVENTION n-Butanol is a colorless, neutral liquid of medium volatility with restricted miscibility (about 7-8%) in water, but freely miscible with all common solvents such as glycols, ketones, alcohol, aldehydes, ethers, and aromatic and aliphatic hydrocarbons, n- Butanol is used i) to make other chemicals, ii) as a solvent and iii) as an ingredient in formulated products such as cosmetics. The major uses of n-butanol as a feed-stock are in the synthesis of acrylate/methacrylate esters, glycol ethers, n-Butyl acetate, amino resins and n-Butylamines. Currently more than 9 millions tons of n-Butanol are consumed annually in the world.
More recently it has been shown that n-butanol is a better bio fuel than ethanol due to lower vapour pressure, higher energy content (closer to that of gasoline) and lesser susceptibility to separation in the presence of water. Furthermore, n-butanol can be blended at higher concentrations than ethanol for use in standard vehicle engines and it does not require automakers to compromise on performance to meet environmental regulations; it is also suitable for transport in pipelines and as a result it has the potential to be introduced into gasoline quickly and avoid the need for additional large-scale supply infrastructures. n-butanol can be produced as an acetone/n-butanol/ethanol (ABE) mixture by the fermentation of carbohydrate by solventogenic Clostridia. The ABE fermentations are biphasic. During the first acidogenic phase, high growth rate is accompanied by acetic and butyric acids production. In the second solventogenic phase growth rate decrease and the solvents (ABE) are produced with the concomitant consumption of the organic acids produced in the first phase. Carbon dioxide and hydrogen are produced throughout the fermentation.
The biological production of n-butanol, presented in figure 1, requires the formation of butyryl-CoA as an intermediate which can be reduced, depending on the physiological conditions, by two different bi-functional aldehyde-alcohol dehydrogenases encoded by adhEl and adhE2. Butyryl-CoA can also be converted to butyric acid by a phospho-transbutyrylase and a butyrate kinase encoded respectively by the ptb and buk genes. Acetone is produced from aceto-acetyl-CoA (an intermediate in the production of butyryl-CoA) by a CoA-transferase and an acetoacetate decarboxylase encoded respectively by the ctfAB and adc genes. Hydrogen is produced by an iron only hydrogenase encoded by the hydA gene. When cultures are performed in the presence of carbon monoxide, a hydrogenase inhibitor, n-butanol, ethanol and lactate are the main fermentation products. Lactate is produced from pyruvate by a lactate dehydrogenase encoded by the ldh gene.
Clostridium acetobutylicum strains with an inactivated buk gene (obtained by single crossing over with a non-replicable plasmid) have already been described in the article
(Green et al., 1996). The non-replicable vector pJC4BK, with a 0.8 kb internal buk fragment was integrated into the chromosomal buk gene which leaded to an inactivation of the endogenous gene. The obtained strain was named "mutant PJC4BK"from the name of the plasmid. As precised in this article, this gene integration did not completely eliminate enzyme activity nor butyrate formation due to the instability of this type of gene inactivation that can reverse to wild type by plasmid excision. This mutant strain was then used in several studies (Green and Bennett, 1998; Desai and Harris, 1999; Harris et al.,
2000).
Traditionally, the commercial ABE fermentation was conducted only in a batch mode due to continuous cultures instability of the producing Clostridia. Several solvent yielding fermentation processes have been described. These processes yield n-butanol, acetone and ethanol in a ratio of 6:3:1. Solvent yields of 29-34% (18-25% for n-butanol only) of fermentable carbon source have been reported in the literature. A total solvent concentration of 16-24 g/1 and a n-butanol concentration of 10-14 g/1 is generally the limit due to toxicity of n-butanol produced. However, these low titers of solvent no longer seem to be an economical limitation to the process as it has recently been demonstrated that solvents can be recovered during fermentation by the use of the "low cost" gas striping technology.
The problem to be solved by the present invention is to obtain a stable mutant strain with no butyrate kinase activity , that could be cultureD for several generations without any possibility of reversion to the wild type genotype. This strain would be useful for the biological production of n-butanol at high yield, from an inexpensive carbon substrate such as glucose or other sugars, by genetically stable cultures of Clostridia. The number of biochemical steps to inactivate and the complexity of the regulation of the metabolism necessitate, for an industrial feasible process of n-butanol production, the use of a metabolically engineered whole cell catalyst.
SUMMARY OF THE INVENTION
Applicants have solved the stated problem and the present invention provides a method for bioconverting a fermentable carbon source to n-butanol as a major product by genetically stable cultures of Clostridia. Glucose is used as a model substrate and recombinant Clostridium acetobutylicum is used as the model host. In one aspect of this invention, a stable recombinant C. acetobutylicum unable to metabolize butyryl-CoA to butyrate is constructed by deleting the gene coding for the butyrate kinase (buk). In another aspect of this invention, a recombinant C. acetobutylicum unable to produce acetone is constructed by deleting the genes coding for the CoA-transferase (ctfAB). In a further aspect of this invention a recombinant strain unable to produce lactate is constructed by deleting the gene coding for the lactate dehydrogenase (Idh). Furthermore, a recombinant C acetobutylicum unable to produce acetate is constructed by deleting the genes coding for the phosphotransacetylase and/or acetate kinase (pta and ack). In a final aspect of this invention, the flux of hydrogen production is decreased and then the flux of reducing equivalent redirected toward n-butanol production by attenuating the gene encoding the hydrogenase (hydA).
The present invention may be generally applied to include any carbon substrate that is readily converted to acetyl-coA.
Accordingly it is an object of the present invention to provide a recombinant organism, useful for the production of n-butanol comprising: (a) at least deletion of one of the two genes involved in the conversion of butyryl-CoA to butyrate and (b) at least deletion of one of the two genes encoding the CoA-transferase activity. Optionally the recombinant organism may comprise i) inactivating mutations in endogenous genes selected from the group consisting of: (a) a gene encoding a polypeptide having lactate dehydrogenase activity (b) a gene encoding a polypeptide having phospho-transacetylase or actate kinase activity and ii) attenuation in a gene encoding a polypeptide having hydrogenase activity. In another embodiment the invention provides a stable process for the production of n-butanol at high yield from a recombinant organism comprising: (a) contacting the recombinant organism of the present invention with at least one carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and single-carbon substrates whereby n-butanol is produced; optionally (b) recovering the n- butanol during the production through a step of gas striping and (c) purifying n-butanol from the condensate by distillation.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawing which is incorporated in and constitutes a part of this specification exemplifies the invention and together with the description, serve to explain the principles of this invention. Figure 1 depicts the genetic engineering of central metabolism in the development of a butanol production system from carbohydrates.
1 : Pyruvate-ferredoxin oxydoreductase ; 2 : Thiolase ; 3 : β-Hydroxybutyryl-CoA dehydrogenase ; 4 : Crotonase ; 5 : Butyryl-CoA dehydrogenase ; 6 : Lactate dehydrogenase ; 7 : Phospho-transacetylase ; 8 : Acetate kinase ; 9 : Acetaldehyde deshydrogenase ; 10 : Ethanol dehydrogenase ; 11 : CoA transferase (Acetoacetyl- CoA :acetate/butyrate : CoA transferase) ; 12 : Acetoacetate decarboxylase ; 13 : Phospho- transbutyrylase ; 14 : Butyrate kinase ; 15 : Butyraldehyde-Butanol dehydrogenase ; 16 : hydrogenase.
DETAILED DESCRIPTION OF THE INVENTION
As used herein the following terms may be used for interpretation of the claims and specification.
The term "microorganism" refers to all kind of unicellular organisms, including prokaryotic organisms like bacteria, and eukaryotic organisms like yeasts. The expression "appropriate culture medium" refers to a culture medium adapted for the used microorganism as it is well known by the man skilled in the art. The term "carbon substrate" or "source of carbon" means any carbon source capable of being metabolized by a microorganism wherein the substrate contains at least one carbon atom. In particular it may be glucose, sucrose, mono- or oligosaccharides, starch or its derivatives, glycerol, and their mixtures thereof.
The term "attenuation" refers to a decreased expression of a gene or a decreased activity of the protein, product of the gene. The man skilled in the art knows numerous means to obtain this result, and for example:
Introduction of a mutation into the gene, decreasing the expression level of this gene, or the level of activity of the encoded protein. - Replacement of the natural promoter of the gene by a low strength promoter, resulting in a lower expression.
Use of elements destabilizing the corresponding messenger RNA or the protein. Deletion of the gene if no expression is needed. The term "deleted gene" means that a substantial part of the coding sequences of said gene was removed. Preferably, at least 50% of the coding sequence was removed, and more preferably at least 80%.
In the description of the present invention, enzymes are identified by their specific activities. This definition thus includes all polypeptides that have the defined specific activity also present in other organisms, more particularly in other microorganisms. Often enzymes with similar activities can be identified by their grouping to certain families defined as PFAM or COG. PFAM (protein families database of alignments and hidden Markov models;
Figure imgf000006_0001
represents a large collection of protein sequence alignments. Each PFAM makes it possible to visualize multiple alignments, see protein domains, evaluate distribution among organisms, gain access to other databases, and visualize known protein structures. COGs (clusters of orthologous groups of proteins; hllSl/J^Η}^S.ΦJAlhl}iiΩlhΛB9ΥLQQΩJ) are obtained by comparing protein sequences from 43 fully sequenced genomes representing 30 major phylogenic lines. Each COG is defined from at least three lines, which permits the identification of former conserved domains.
The means of identifying homologous sequences and their percentage homologies are well known to those skilled in the art, and include in particular the BLAST programs, which can be used from the website http://www.ncbi.nlm.nih.gov/BLAST/ with the default parameters indicated on that website. The sequences obtained can then be exploited (e.g., aligned) using, for example, the programs CLUSTALW (h}JMsil^-^-yy-^M3.^AUkM)i^3h*ϊ) or MULTALIN (http;//p_ro^^ with the default parameters indicated on those websites
Using the references given on GenBank for known genes, those skilled in the art are able to determine the equivalent genes in other organisms, bacterial strains, yeasts, fungi, mammals, plants, etc. This routine work is advantageously done using consensus sequences that can be determined by carrying out sequence alignments with genes derived from other microorganisms, and designing degenerate probes to clone the corresponding gene in another organism. These routine methods of molecular biology are well known to those skilled in the art, and are described, for example, in Sambrook et al. (Molecular Cloning: a Laboratory Manual. 2nd ed. Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1989.).
The present invention provides a method for the fermentative batch or continuous production of n-butanol by culturing a microorganism in an appropriate culture medium comprising a carbon source and the simultaneous recovery of n-butanol from the culture medium wherein at least one gene involved in butyrate formation is deleted in the microorganism.
A specific embodiment of the invention provides a method wherein the microorganism is modified to be unable to convert butyryl-CoA to butyrate due to the deletion of at least one gene encoding for phospho-transbutyrylase iptb) or butyrate kinase (buk). Deletion of genes in Clostridia can be done using the method recently described in patent application PCT/EP2006/066997 allowing the i) replacement of the gene to delete with an erythromycin resistance gene and ii) removal of the erythromycin resistance gene with a recombinase. In another embodiment of the invention, the microorganism is unable to produce acetone due to an attenuation or a deletion of at least one of the gene involved in acetone formation. Preferably, this gene is encoding for the enzyme CoA-transferase (ctfAB) or acetoacetate decarboxylase (adc). Deletion of one of these genes can be done using the method recently described in patent application PCT/EP2006/066997. In a further embodiment of the invention, the microorganism used in the method of the invention is unable to produce lactate. In particular this can be due to a deletion of the gene ldh encoding for lactate dehydrogenase. Deletion of ldh can be done using the method recently described in patent application PC17EP2006/066997.
In another embodiment, the microorganism is modified in such a way to be unable to produce acetate. This result can be achieved by deletion of at least one involved in acetate formation. Preferably this gene is selected among the group consisting of the genes encoding for phospho-transacetylase (ptά) or acetate kinase (ack). Deletion of one of these genes can be done using the method recently described in patent application PCT/EP2006/066997. An embodiment of the invention also provides a microorganism with a decreased flux of hydrogen production and then a redirection of the flux of reducing equivalent toward n-butanol production; this can be done by attenuating the gene encoding the hydrogenase (hydA), an enzyme that provides a sink for reducing equivalent in the form of hydrogen production. Attenuation of hydA can be done by replacing the natural promoter by a low strength promoter or by element destabilizing the corresponding messenger RNA or the protein. If needed, complete attenuation of the gene can also be achieved by a deletion of the corresponding DNA sequence.
Preferably, the used microorganism is selected among the group consisting of C. acetobutylicum, C. beijerinckii, C. saccharoperbutylacetonicum or C. saccharobutylicum. In another embodiment of the invention, the culture is continuous and stable. In another embodiment, the method according to the invention comprises the following steps:
(a) contacting a microorganism with at least one carbon source selected from the group consisting of glucose, xylose, arabinose, sucrose, monosaccharides, oligosaccharides, polysaccharides, cellulose, xylan, starch or its derivatives and glycerol, whereby n-butanol is produced
(b) Recovering the n-butanol during the fermentation by gas striping and
(c) Isolation of n-butanol from the condensate by distillation.
Those skilled in the art are able to define the culture conditions for the microorganisms according to the invention. In particular the Clostridia are fermented at a temperature between 200C and 55°C, preferentially between 25°C and 400C, and more specifically about 35°C for C. acetobutylicum.
The fermentation is generally conducted in fermentors with an inorganic culture medium of known defined composition adapted to the bacteria used, containing at least one simple carbon source, and if necessary a co-substrate necessary for the production of the metabolite.
The invention is also related to the microorganism as described previously. Preferably, this microorganism is selected among the group consisting of C. acetobutylicum, C. beijerinckii, C. saccharoperbutylacetonicum or C. saccharobutylicum.
EXAMPLE 1
Construction of strains unable to produce butyrate: Clostridium acetobutylicum
Acacl515 Aupp Abuk
To delete the buk gene, the homologous recombination strategy described by Croux & Soucaille (2006) in patent application PCT/EP2006/066997 is used. This strategy allows the insertion of an erythromycin resistance cassette, while deleting most of the gene concerned. The buk deletion cassette in pCons::upp was constructed as follows. Table 1 : primers sequences
Name Primer sequences
Buk l SEQ ID N0I aaaaggatcctagtaaaagggagtgtacgaccagtg Buk 2 SEQ ID N°2 QQQQtcgcgaaaaaaggggggattattagtaatctatacatgttaacattcctccac Buk 3 SEQ 15 N°3 c c c c c tttt TtcQCQoe cccacttcttgcacttgcagaaggtggac Buk 4 SEQ ID N°4 aaaaggotcctctaaattctgcaatatatgccccccc
Buk O SEQ Ib N°5 ataacaggatatatgctctctgacgcgg Buk 5 SEQ Ib N°6 gatcatcactcattttaaacatggggcc Two DNA fragments surrounding buk were PCR amplified with the Pwo polymerase with total DNA from C. acetobutylicum as template and two specific couples of oligonucleotides. With the couples of primers BUK 1-BUK 2 and BUK 3-BUK 4, two DNA fragments were respectively obtained. Both primers BUK 1 and BUK 4 introduce a BamHI site while primers BUK 2 and BUK 3 have a complementary region which introduces a Nrul site. DNA fragments BUK 1-BUK 2 and BUK 3-BUK 4 were joined in a PCR fusion experiment with primers BUK 1 and BUK 4 and the resulting fragment was cloned in pCR4-TOPO-Blunt to yield pTOPO :buk. At the unique Stul site of pTOPO :buk, an antibiotic resistance MLS gene with FRT sequences on both sides was introduced from the Stul fragment of pUC18-FRT-MLS2. The BUK deletion cassette obtained after BamHI digestion of the resulting plasmid was cloned into pCons::upp at the BamHI site to yield the pREPΔBUK::upp plasmid.
The pREPΔBUK::upp plasmid was used to transform by electroporation C. acetobutylicum MGCAcacl5Aupp strain. After selection on Petri plate for clones resistant to erythromycin (40 μg/ml), one colony was cultured for 24 hours in liquid synthetic medium with erythromycin at 40 μg/ml and 100 μl of undiluted culture was plated on RCA with erythromycin at 40 μg/ml and 5-FU at 400 μM. Colonies resistant to both erythromycin and 5-FU were replica plated on both RCA with erythromycin at 40 μg/ml and RCA with thiamphenicol at 50 μg/ml to select clones where 5-FU resistance is also associated with thiamphenicol sensitivity. The genotype of clones resistant to erythromycin and sensitive to thiamphenicol was checked by PCR analysis (with primers BUK 0 and BUK 5 located outside of the buk deletion cassette). The Acacl 5AuppAbuk: :mlsR strain which have lost pREPΔbuk::upp was isolated. The Acacl 5AuppAbuk: :mlsR strain was transformed with pCLFl.l vector expressing the FIp 1 gene encoding the FIp recombinase from S. cerevisiae. After transformation and selection for resistance to thiamphenicol (50 μg/ml) on Petri plate, one colony was cultured on synthetic liquid medium with thiamphenicol at 50 μg/ml and appropriate dilutions were plated on RCA with thiamphenicol at 50 μg/ml. Thiamphenicol resistant clones were replica plated on both RCA with erythromycin at 40 μg/ml and RCA with thiamphenicol at 50 μg/ml. The genotype of clones with erythromycin sensitivity and thiamphenicol resistance was checked by PCR analysis with primers BUK 0 and BUK 5. Two successive 24 hours cultures of the Acacl5AuppAbuk strain with erythromycin sensitivity and thiamphenicol resistance were carried out in order to lose pCLFl.l. The Acacl5AuppAbuk strain which has lost pCLFl.l was isolated according to its sensitivity to both erythromycin and thiamphenicol.
EXAMPLE 2
Construction of strains unable to produce butyrate and acetone: C. acetobutylicum
Acacl515 Aupp Abuk ActfAB
To delete the ctfAB genes, the homologous recombination strategy described by
Croux & Soucaille (2006) in patent application PCT/EP2006/066997 is used. This strategy allows the insertion of an erythromycin resistance cassette, while deleting most of the genes concerned. The ctfAB deletion cassette in pCons::upp was constructed as follows. Table 2 : primers sequences
Name Primer sequences
Ctf 1 SEQ ID N°7 aaaaggGi cxcagacactataatagctttaggtggtacccc Ctf 2 ggggaggcctaaaaagggggattataaaaagtagttgaaatatgaaggtttaaggttg Ctf 3 cccccttt ttaggc c tccccatatccaatgaacttagacccatggctg Ctf 4 SEQ ID N0IO aaaaggai ccgtgttataatgtaaatataaataaataggactagaggcg
Ctf 0 SEQ ID N0I l taccaccttctttcacgcttggctgcgg Ctf 5 SEQ ID N°12 tatttaaagaggcattatcaccagagcg
Two DNA fragments surrounding ctfAB were PCR amplified with the Pwo polymerase with total DNA from C. acetobutylicum as template and two specific couples of oligonucleotides. With the couples of primers CTF 1-CTF 2 and CTF 3-CTF 4, two DNA fragments were respectively obtained. Both primers CTF 1 and CTF 4 introduce a BamHI site while primers CTF 2 and CTF 3 have a complementary region which introduces a Stul site. DNA fragments CTF 1-CTF 2 and CTF 3-CTF 4 were joined in a PCR fusion experiment with primers CTF 1 and CTF 4 and the resulting fragment was cloned in pCR4- TOPO-Blunt to yield pTOPO :CTF. At the unique Stul site of pTOPO :CTF, an antibiotic resistance MLS gene with FRT sequences on both sides was introduced from the Stul fragment of pUC18-FRT-MLS2. The UPP deletion cassette obtained after BamHI digestion of the resulting plasmid was cloned into pCons::upp at the BamHI site to yield the pREPΔCTF::upp plasmid.
The pREPΔCTF::upp plasmid was used to transform by electroporation C. acetobutylicum MGC Acac 15 Aupp Abuk strain. After selection on Petri plate for clones resistant to erythromycin (40 μg/ml), one colony was cultured for 24 hours in liquid synthetic medium with erythromycin at 40 μg/ml and 100 μl of undiluted culture was plated on RCA with erythromycin at 40 μg/ml and 5-FU at 400 μM. Colonies resistant to both erythromycin and 5-FU were replica plated on both RCA with erythromycin at 40 μg/ml and RCA with thiamphenicol at 50 μg/ml to select clones where 5-FU resistance is also associated with thiamphenicol sensitivity. The genotype of clones resistant to erythromycin and sensitive to thiamphenicol was checked by PCR analysis (with primers CTF 0 and CTF 5 located outside of the ctfAB deletion cassette). The Acacl 5AuppAbuk ActfAB::mlsR strain which have lost pREPΔCTF::upp was isolated. The Acacl5AuppAbukActfAB::mlsR strain was transformed with pCLFl.l vector expressing the FIp 1 gene encoding the FIp recombinase from S. cerevisiae. After transformation and selection for resistance to thiamphenicol (50 μg/ml) on Petri plate, one colony was cultured on synthetic liquid medium with thiamphenicol at 50 μg/ml and appropriate dilutions were plated on RCA with thiamphenicol at 50 μg/ml. Thiamphenicol resistant clones were replica plated on both RCA with erythromycin at 40 μg/ml and RCA with thiamphenicol at 50 μg/ml. The genotype of clones with erythromycin sensitivity and thiamphenicol resistance was checked by PCR analysis with primers CTF 0 and CTF 5. Two successive 24 hours cultures of the Acacl 5 AuppAbukActfAB strain with erythromycin sensitivity and thiamphenicol resistance were carried out in order to lose pCLFl.l. The Acacl 5 AuppAbukActfAB strain which has lost pCLFl.l was isolated according to its sensitivity to both erythromycin and thiamphenicol.
EXAMPLE 3
Construction of strains unable to produce butyrate, acetone and lactate: C. acetobutylicum Acacl515 Aupp Abuk ActfΛB Aldh
To delete the ldh gene, the homologous recombination strategy described by Croux
& Soucaille (2006) in patent application PCT/EP2006/066997 is used. This strategy allows the insertion of an erythromycin resistance cassette, while deleting most of the genes concerned. The ldh deletion cassette in pCons::upp was constructed as follows. Table 3 : primers sequences
Name Primer sequences
Ldh l SEQ ID N°13 AAAAGGAΎCCGCΎTYAAAAΎTYGGAAAGAGGAAGTYGTG
Ldh 2 SEQ ID N°14 GGGG AGGCCJ A AA AAGGGGGTY AG A AAJCTVY A AA AATVYC TCTAT 'AG AGCCC ATC Ldh 3 SEQ ID N°15 CCCCCYTYTY AGGCCYCCCCGGΥ AAAAGACCY AAACYCCAAGG GΥGG AGGCY AGGΥC Ldh 4 SEQ ID N°16 AAAAGG ATCCCCCATTGTGG AG AAΛ 'ATYCCAAAGAAGAAAA TAATYGC
Ldh O SEQ ID N°17 C AG A AGGC A AG A ATGJ ATI A AGCGG A AATGC Ldh 5 SEQ ID N°18 CTTCCCATTATAGCTCTTATTCACATTAAGC
Two DNA fragments surrounding ldh (CAC267) were PCR amplified with the Pwo polymerase with total DNA from C. acetobutylicum as template and two specific couples of oligonucleotides. With the couples of primers LDH 1-LDH 2 and LDH 3-LDH 4, 1135 bp and 1177 bp DNA fragments were respectively obtained. Both primers LDH 1 and LDH 4 introduce a BamHI site while primers LDH 2 and LDH 3 have a complementary region which introduces a Stul site. DNA fragments LDH 1-LDH 2 and LDH 3-LDH 4 were joined in a PCR fusion experiment with primers LDH 1 and LDH 4 and the resulting fragment was cloned in pCR4-TOPO-Blunt to yield pTOPO :LDH. At the unique Stul site of pTOPO :LDH, an antibiotic resistance MLS gene with FRT sequences on both sides was introduced from the 1372 bp Stul fragment of pUC18-FRT-MLS2. The UPP deletion cassette obtained after BamHI digestion of the resulting plasmid was cloned into pCons::upp at the BamHI site to yield the pREPΔLDH::upp plasmid. The pREPΔLDH::upp plasmid was used to transform by electroporation C. acetobutylicum MGCAcacl5AuppAbukActfAB strain. After selection on Petri plate for clones resistant to erythromycin (40 μg/ml), one colony was cultured for 24 hours in liquid synthetic medium with erythromycin at 40 μg/ml and 100 μl of undiluted culture was plated on RCA with erythromycin at 40 μg/ml and 5-FU at 400 μM. Colonies resistant to both erythromycin and 5-FU were replica plated on both RCA with erythromycin at 40 μg/ml and RCA with thiamphenicol at 50 μg/ml to select clones where 5-FU resistance is also associated with thiamphenicol sensitivity. The genotype of clones resistant to erythromycin and sensitive to thiamphenicol was checked by PCR analysis (with primers LDH 0 and LDH 5 located outside of the ldh deletion cassette). The Acacl 5AuppAbuk ActfAB Aldh::mlsR strain which have lost pREPΔLDH::upp was isolated. The Acacl5AuppAbukActfABAldh::mls strain was transformed with pCLFl.l vector expressing the FIp 1 gene encoding the FIp recombinase from S. cerevisiae. After transformation and selection for resistance to thiamphenicol (50 μg/ml) on Petri plate, one colony was cultured on synthetic liquid medium with thiamphenicol at 50 μg/ml and appropriate dilutions were plated on RCA with thiamphenicol at 50 μg/ml. Thiamphenicol resistant clones were replica plated on both RCA with erythromycin at 40 μg/ml and RCA with thiamphenicol at 50 μg/ml. The genotype of clones with erythromycin sensitivity and thiamphenicol resistance was checked by PCR analysis with primers LDH 0 and LDH 5. Two successive 24 hours cultures of the Acacl5AuppAbukActfAB Aldh strain with erythromycin sensitivity and thiamphenicol resistance were carried out in order to lose pCLFl.l. The Acacl5AuppAbukActfABAldh strain which has lost pCLFl.l was isolated according to its sensitivity to both erythromycin and thiamphenicol.
EXAMPLE 4
Construction of strains unable to produce butyrate, acetone, lactate and acetate: C. acetobutylicum Acacl515 Aupp Abuk ActfAB Aldh Apta-ack
To delete the pta and ack genes, the homologous recombination strategy described by Croux & Soucaille (2006) in patent application PCT/EP2006/066997 is used. This strategy allows the insertion of an erythromycin resistance cassette, while deleting most of the genes concerned. The pta-ack deletion cassette in pCons::upp was constructed as follows.
Table 4 : primers sequences
Name Primer sequences
PA l SEQ ID N°19 aaaaggaicctattataacagtcaaacccaataaaatactggg PA 2 SEQ ID Kf 20 ggggaggcctaaaaagggggttaatccatttgtattttctcccttcataatgcc PA 3 SEQ ID Kf 21 ccccc ttt ttaggc c tcccctttattttgcatgcttatataataaattatggctgcg PA 4 SEQ ID N°22 aaaaggatccgcttttccttcttttacaagatttaaagcc
PA O SEQ ID N°23 cacttttatttatcaagctgtaggcc PA 5 SEQ Ib N°24 tataccttttgaacctaggaaaggc
Two DNA fragments surrounding pta-ack were PCR amplified with the Pwo polymerase with total DNA from C. acetobutylicum as template and two specific couples of oligonucleotides. With the couples of primers PA 1-PA 2 and PA 3-PA 4, two DNA fragments were respectively obtained. Both primers PA 1 and PA 4 introduce a BamHI site while primers PA 2 and PA 3 have a complementary region which introduces a Stul site. DNA fragments PA 1-PA 2 and PA 3-PA 4 were joined in a PCR fusion experiment with primers PA 1 and PA 4 and the resulting fragment was cloned in pCR4-TOPO-Blunt to yield pTOPO :PA. At the unique Stul site of pTOPO :PA, an antibiotic resistance MLS gene with FRT sequences on both sides was introduced from the Stul fragment of pUC18-FRT- MLS2. The UPP deletion cassette obtained after BamHI digestion of the resulting plasmid was cloned into pCons::upp at the BamHI site to yield the pREPΔPA::upp plasmid. The pREPΔPA::upp plasmid was used to transform by electroporation C. acetobutylicum MGCAcacl5AuppAbukActfABAldh strain. After selection on Petri plate for clones resistant to erythromycin (40 μg/ml), one colony was cultured for 24 hours in liquid synthetic medium with erythromycin at 40 μg/ml and 100 μl of undiluted culture was plated on RCA with erythromycin at 40 μg/ml and 5-FU at 400 μM. Colonies resistant to both erythromycin and 5-FU were replica plated on both RCA with erythromycin at 40 μg/ml and RCA with thiamphenicol at 50 μg/ml to select clones where 5-FU resistance is also associated with thiamphenicol sensitivity. The genotype of clones resistant to erythromycin and sensitive to thiamphenicol was checked by PCR analysis (with primers PA 0 and PA 5 located outside of the pta-ack deletion cassette). The Acacl5AuppAbuk ActfABAldhApta- ack::mlsR strain which have lost pREPΔPA::upp was isolated. The Acacl5AuppAbukActfABAldh Apta-ack: :mlsR strain was transformed with pCLFl.l vector expressing the FIp 1 gene encoding the FIp recombinase from S. cerevisiae. After transformation and selection for resistance to thiamphenicol (50 μg/ml) on Petri plate, one colony was cultured on synthetic liquid medium with thiamphenicol at 50 μg/ml and appropriate dilutions were plated on RCA with thiamphenicol at 50 μg/ml. Thiamphenicol resistant clones were replica plated on both RCA with erythromycin at 40 μg/ml and RCA with thiamphenicol at 50 μg/ml. The genotype of clones with erythromycin sensitivity and thiamphenicol resistance was checked by PCR analysis with primers PA 0 and PA 5. Two successive 24 hours cultures of the Acacl 5 AuppAbukActf AB Aldh Apta-ack strain with erythromycin sensitivity and thiamphenicol resistance were carried out in order to lose pCLFl.l. The Acacl 5 ' AuppAbukActf AB Aldh Apta-ack strain which has lost pCLFl.l was isolated according to its sensitivity to both erythromycin and thiamphenicol. EXAMPLE 5
Construction of strains with lower hydrogen production: C. acetobutylicum Acacl515 Aupp Abuk ActfAB Aldh AhydA
To delete the hydA gene, the homologous recombination strategy described by Croux & Soucaille (2006) in patent application PCT/EP2006/066997 is used. This strategy allows the insertion of an erythromycin resistance cassette, while deleting most of the genes concerned. The hydA deletion cassette in pCons::upp was constructed as follow.
Table 5 : primers sequences
Name Primer sequences
Hyd l SEQ ID N°25 AAAAGGl TCC GCCTCTTCTGTATTATGCAAGGAAAGC AGCTGC Hyd 2 SEQ ID N°26 GGGG^GGCCΓAAAAAGGGGGTATATAAAATAAATGTG CCTTAACATC TAAGTTGAGGCC
Hyd 3 SEQ ID N°27 CCCCCTTTTT^GGCCΓCCCCGTTTATCCTCCCAAAATGT AAAATATAA TTAAAATATATT AATAAACTTCGATT AATAAACTVC& Hyd 4 SEQ ID N°28 AAAAGGΛΓCCCCTTTTAGCGTATAAAGTTTTATATAGC
TATTG
Hyd O SEQ ID N°29 CATGTTCTATTGTTACTATGGAAGAGGTAGTAG Hyd 5 SEQ ID N°30 GCAGTTATTATAAATGCTGCTACTAGAGC
Two DNA fragments surrounding hydA (CAC028) were PCR amplified with the Pwo polymerase with total DNA from C. acetobutylicum as template and two specific couples of oligonucleotides. With the couples of primers HYD 1-HYD 2 and HYD 3-HYD 4, 1269 bp and 1317 bp DNA fragments were respectively obtained. Both primers HYD 1 and HYD 4 introduce a BamHI site while primers HYD 2 and HYD 3 have a complementary region which introduces a Stul site. DNA fragments HYD 1-HYD 2 and HYD 3-HYD 4 were joined in a PCR fusion experiment with primers HYD 1 and HYD 4 and the resulting fragment was cloned in pCR4-TOPO-Blunt to yield pTOPO :HYD. At the unique Stul site of pTOPO :HYD, an antibiotic resistance MLS gene with FRT sequences on both sides was introduced from the 1372 bp Stul fragment of pUC18-FRT-MLS2. The UPP deletion cassette obtained after BamHI digestion of the resulting plasmid was cloned into pCons::upp at the BamHI site to yield the pREPΔHYD::upp plasmid. The pREPΔHYD::upp plasmid was used to transform by electroporation C. acetobutylicum MGCAcac 15 Aupp AbukActfAB Aldh strain. After selection on Petri plate for clones resistant to erythromycin (40 μg/ml), one colony was cultured for 24 hours in liquid synthetic medium with erythromycin at 40 μg/ml and 100 μl of undiluted culture was plated on RCA with erythromycin at 40 μg/ml and 5-FU at 400 μM. Colonies resistant to both erythromycin and 5-FU were replica plated on both RCA with erythromycin at 40 μg/ml and RCA with thiamphenicol at 50 μg/ml to select clones where 5-FU resistance is also associated with thiamphenicol sensitivity. The genotype of clones resistant to erythromycin and sensitive to thiamphenicol was checked by PCR analysis (with primers HYD 0 and HYD 5 located outside of the hydA deletion cassette). The Acacl5AuppAbukActfABAldhAhydA::mlsR strain which have lost pREPΔHYD::upp was isolated.
The AcaclδAuppAbukActfABAldhAhydA.-.-mls11 strain was transformed with pCLFl.l vector expressing the FIp 1 gene encoding the FIp recombinase from S. cerevisiae. After transformation and selection for resistance to thiamphenicol (50 μg/ml) on Petri plate, one colony was cultured on synthetic liquid medium with thiamphenicol at 50 μg/ml and appropriate dilutions were plated on RCA with thiamphenicol at 50 μg/ml. Thiamphenicol resistant clones were replica plated on both RCA with erythromycin at 40 μg/ml and RCA with thiamphenicol at 50 μg/ml. The genotype of clones with erythromycin sensitivity and thiamphenicol resistance was checked by PCR analysis with primers HYD 0 and HYD 5. Two successive 24 hours cultures of the Acacl 5 AuppAbukActfABAldhAhydA strain with erythromycin sensitivity and thiamphenicol resistance were carried out in order to lose pCLFl.l. The Acacl 5 AuppAbukActfABAldhAhydA strain which has lost pCLFl.l was isolated according to its sensitivity to both erythromycin and thiamphenicol.
EXAMPLE 6
Batch fermentation of n-butanol producing strains.
Strains were initially analyzed in anaerobic flask cultures in the synthetic medium described by Soni et al (Soni et al, 1987, Appl. Microbiol. Biotechnol. 27:1-5) supplemented with 2.5 g/1 of ammonium acetate. An overnight culture at 35°C was used to inoculate a 30 ml culture to an OD600 of 0.05. After incubation of the culture for 3 days at 35°C, glucose, organic acids and solvents were analyzed by HPLC using a Biorad HPX 97H column for the separation and a refractometer for the detection.
Strains with the correct phenotype were subsequently tested under production conditions in 300 ml fermentors (DASGIP) using an anaerobic batch protocol. For this purpose the fermentor was filled with 250 ml of synthetic medium, sparged with nitrogen for 30 min and inoculated with 25 ml of preculture to an optical density (OD600nm) between 0.05 and 0.1.
The temperature of the culture was maintained constant at 35 0C and the pH was permanently adjusted at 5.5 using an NH4OH solution. The agitation rate was maintained at 300 rpm during the fermentation.
EXAMPLE 7
Continuous fermentation of n-butanol producing strains.
The best n-butanol producing strain was analyzed in chemostat cultures in the synthetic medium described by Soni et al (Soni et al, 1987, Appl. Microbiol. Biotechnol. 27:1-5). An overnight culture at 35°C was used to inoculate a 300 ml fermentors (DASGIP) using an anaerobic chemostat protocol. For this purpose the fermentor was filled with 250 ml of synthetic medium, sparged with nitrogen for 30 min and inoculated with 25 ml of preculture to an optical density (OD600nm) between 0.05 and 0.1. After 12 hours of batch culture at 35 0C, pH 5.5 (regulated using an NH4OH solution) and an agitation rate of 300 rpm, the fermentor was continuously fed with oxygen free synthetic medium at a dilution rate of 0.05 h-1 while the volume was kept constant by sequential removal of fermentated medium. Stability of the culture was followed by products analysis using the HPLC protocol previously described.
REFERENCES
Desai RP, Harris LM, Welker NE, Papoutsakis ET.
Metabolic flux analysis elucidates the importance of the acid- formation pathways in regulating solvent production by Clostridium acetobutylicum. Metab Eng. 1999,1:206-13.
Green EM, Bennett GN.
Genetic manipulation of acid and solvent formation in Clostridium acetobutylicum ATCC 824
Biotechnol Bioeng. 1998, 58:215-21.
Green EM, Bovnton ZL, Harris LM, Rudolph FB, Papoutsakis ET, Bennett GN. Genetic manipulation of acid formation pathways by gene inactivation in Clostridium acetobutylicum ATCC 824.
Microbiology. 1996,142 :2079-86.
Harris LM. Desai RP. Welker NE. Papoutsakis ET.
Characterization of recombinant strains of the Clostridium acetobutylicum butyrate kinase inactivation mutant: need for new phenomenological models for solventogenesis and butanol inhibition? Biotechnol Bioeng. 2000,;67:l-l l.
Soni B. K.. Soucaille P. Goma G. Continuous acetone butanol fermentation : influence of vitamins on the metabolic activity of Clostridium acetobutylicum. Appl. Microbiol. Biotechnol. 1987. 27 : 1-5.

Claims

1) A method for the production of n-butanol by culturing a microorganism in an appropriate culture medium comprising a source of carbon and recovery of n- butanol from the culture medium, wherein at least one gene involved in butyrate formation is deleted in the microorganism.
2) A method according to claim 1 wherein the deleted gene is at least one of the following genes:
• ptb encoding phospho-transbutyrylase • buk encoding butyrate kinase.
3) A method according to claim 1 or 2 wherein at least one gene involved in acetone formation is attenuated in the microorganism.
4) A method according to claim 3 wherein at least one of the following genes is deleted: • ctfAB encoding CoA-transferase
• adc encoding aceto-acetate decarboxylase.
5) A method according to any one of claims 1 to 4 wherein the microorganism is modified to be unable to produce lactate.
6) A method according to claim 5 wherein the ldh gene is deleted. 7) A method as claimed in any one of claims 1 to 6 wherein the microorganism is modified to be unable to produce acetate.
8) A method as claimed in claim 7 in which at least one gene involved in acetate formation is deleted.
9) A method according to claim 10 wherein the deleted gene is selected among the following :
• pta encoding phospho-transacetylase
• ack encoding acetate kinase.
1O)A method as claimed in any one of claims 1 to 9, wherein the hydrogen flux is decreased and the reducing power redirected to butanol production. H)A method as claimed in claim 10 wherein the hydA gene is attenuated.
12) A method according to anyone of claims 1 to 11 wherein the microorganism is selected among the group consisting of C. acetobutylicum, C. beijerinckii, C. saccharoperbutylacetonicum or C. saccharobutylicum. 13) A method according to anyone of claims 1 to 14 wherein the culture is continuous and stable.
14) A method according to claim 13 comprising the following steps: a) Fermentation of the microorganism producing n-butanol b) Elimination of n-butanol during the fermentation by gas striping. c) Isolation of n-butanol from the condensate by distillation.
15) A microorganism as defined in any one of claims 1 to 12.
PCT/EP2006/067993 2006-10-31 2006-10-31 Process for the biological production of n-butanol with high yield WO2008052596A1 (en)

Priority Applications (19)

Application Number Priority Date Filing Date Title
PCT/EP2006/067993 WO2008052596A1 (en) 2006-10-31 2006-10-31 Process for the biological production of n-butanol with high yield
KR1020097011053A KR101444968B1 (en) 2006-10-31 2007-10-29 Process for the biological production of n-butanol with high yield
RU2009118372/10A RU2461627C2 (en) 2006-10-31 2007-10-29 METHOD FOR BIOLOGICAL PRODUCTION OF n-BUTANOL
BRPI0718142-6A2A BRPI0718142A2 (en) 2006-10-31 2007-10-29 PROCESS FOR THE HIGH-EFFICIENT N-BUTHANOL PRODUCTION
CN2007800391785A CN101528935B (en) 2006-10-31 2007-10-29 Process for the biological production of n-butanol with high yield
CA2665102A CA2665102C (en) 2006-10-31 2007-10-29 Process for the biological production of n-butanol with high yield
JP2009533879A JP5442441B2 (en) 2006-10-31 2007-10-29 Method for biologically producing n-butanol with high yield
EP07821988A EP2084287B1 (en) 2006-10-31 2007-10-29 Process for the biological production of n-butanol with high yield
AU2007316189A AU2007316189B2 (en) 2006-10-31 2007-10-29 Process for the biological production of n-Butanol with high yield
US12/447,726 US20100086982A1 (en) 2006-10-31 2007-10-29 PROCESS FOR THE BIOLOGICAL PRODUCTION OF n-BUTANOL WITH HIGH YIELD
PCT/EP2007/061634 WO2008052973A2 (en) 2006-10-31 2007-10-29 Process for the biological production of n-butanol with high yield
DK07821988.8T DK2084287T3 (en) 2006-10-31 2007-10-29 Process for biological production of high yield n-butanol
MX2009004660A MX2009004660A (en) 2006-10-31 2007-10-29 Process for the biological production of n-butanol with high yield.
TW096140669A TW200835792A (en) 2006-10-31 2007-10-30 Process for the biological production of n-butanol with high yield
ARP070104849A AR063762A1 (en) 2006-10-31 2007-10-31 PROCEDURE FOR THE BIOLOGICAL PRODUCTION OF N-BUTANOL WITH HIGH PERFORMANCE
ZA200902639A ZA200902639B (en) 2006-10-31 2009-04-16 Process for the biological production of n-butanol with high yield
IL198342A IL198342A (en) 2006-10-31 2009-04-23 Process for the biological production of n-butanol with high yield
JP2013172060A JP2014000087A (en) 2006-10-31 2013-08-22 Method for biological production of n-butanol with high yield
US14/321,173 US20140377825A1 (en) 2006-10-31 2014-07-01 PROCESS FOR THE BIOLOGICAL PRODUCTION OF n-BUTANOL WITH HIGH YIELD

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP2006/067993 WO2008052596A1 (en) 2006-10-31 2006-10-31 Process for the biological production of n-butanol with high yield

Publications (1)

Publication Number Publication Date
WO2008052596A1 true WO2008052596A1 (en) 2008-05-08

Family

ID=38229807

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/EP2006/067993 WO2008052596A1 (en) 2006-10-31 2006-10-31 Process for the biological production of n-butanol with high yield
PCT/EP2007/061634 WO2008052973A2 (en) 2006-10-31 2007-10-29 Process for the biological production of n-butanol with high yield

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/EP2007/061634 WO2008052973A2 (en) 2006-10-31 2007-10-29 Process for the biological production of n-butanol with high yield

Country Status (15)

Country Link
US (2) US20100086982A1 (en)
JP (2) JP5442441B2 (en)
KR (1) KR101444968B1 (en)
CN (1) CN101528935B (en)
AR (1) AR063762A1 (en)
AU (1) AU2007316189B2 (en)
BR (1) BRPI0718142A2 (en)
CA (1) CA2665102C (en)
DK (1) DK2084287T3 (en)
IL (1) IL198342A (en)
MX (1) MX2009004660A (en)
RU (1) RU2461627C2 (en)
TW (1) TW200835792A (en)
WO (2) WO2008052596A1 (en)
ZA (1) ZA200902639B (en)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2194120A1 (en) * 2008-12-02 2010-06-09 Total S.A. Bioprocessing ligno-cellulose into ethanol with recombinant clostridium
EP2204453A1 (en) 2008-12-30 2010-07-07 Sued-Chemie AG Process for cell-free production of chemicals
EP2267141A1 (en) * 2009-06-26 2010-12-29 Metabolic Explorer Process for the biological production of n-Butanol with high yield
EP2481793A2 (en) * 2009-09-22 2012-08-01 Korea Advanced Institute of Science and Technology Recombinant microorganism with increased butanol production ability, and preparation method of butanol using same
DE102011077705A1 (en) 2011-06-17 2012-12-20 Evonik Degussa Gmbh Microbial process for the preparation of low molecular weight organic compounds comprising the product absorption by isophorone
JP2013511283A (en) * 2009-11-23 2013-04-04 ビュータマックス・アドバンスド・バイオフューエルズ・エルエルシー Method for producing butanol using extractive fermentation with electrolyte addition
WO2013128230A1 (en) 2012-03-02 2013-09-06 Metabolic Explorer A process for butanol production
US8765446B2 (en) 2009-09-22 2014-07-01 Korea Advanced Institute Of Science And Technology Recombinant mutant microorganisms having increased ability to produce alcohols and method of producing alcohols using the same
GB2515366A (en) * 2013-02-05 2014-12-24 Green Biologics Ltd Production of butanol
CN107653208A (en) * 2017-11-15 2018-02-02 天津科技大学 One plant of hydrogenogen
US9902978B2 (en) 2013-03-26 2018-02-27 Nippon Shokubai Co., Ltd. Genetically modified Clostridium saccharoperbutylacetonicum
EP3348646A1 (en) 2017-01-17 2018-07-18 Evonik Degussa GmbH Microbial method for the preparation of acetone, isopropanol, butanol and/or ethanol comprising product absorption by water
US10961499B2 (en) 2016-07-08 2021-03-30 Metabolic Explorer Method for the fermentative production of molecules of interest by microorganisms comprising genes coding sugar phosphotransferase system (PTS)

Families Citing this family (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090155869A1 (en) * 2006-12-01 2009-06-18 Gevo, Inc. Engineered microorganisms for producing n-butanol and related methods
CN101519673B (en) * 2008-02-27 2013-02-06 中国科学院上海生命科学研究院 Method for improving ratio of butanol produced from clostridium
US9249431B2 (en) 2008-02-28 2016-02-02 Green Biologics Limited Production process
WO2010022763A1 (en) * 2008-08-25 2010-03-04 Metabolic Explorer Method for the preparation of 2-hydroxy-isobutyrate
BRPI0923748B1 (en) 2008-12-31 2019-04-09 Metabolic Explorer METHOD FOR PREPARING DIOLS
WO2011003962A2 (en) 2009-07-08 2011-01-13 Metabolic Explorer Improved gas stripping process for the recovery of solvents from fermentation broths
JP2011177085A (en) * 2010-02-26 2011-09-15 Nippon Shokubai Co Ltd Method for producing 1-butanol by fermentation
US8911978B2 (en) 2010-07-02 2014-12-16 Metabolic Explorer Method for the preparation of hydroxy acids
TWI500768B (en) 2010-07-05 2015-09-21 Metabolic Explorer Sa Method for the preparation of 1,3-propanediol from sucrose
EP2614139A4 (en) * 2010-09-10 2014-03-26 Univ Delaware Recombinant clostridia that fix co2 and co and uses thereof
EP2678420A4 (en) * 2011-02-25 2014-10-29 Ohio State Innovation Foundation Autotrophic hydrogen bacteria and uses thereof
EP2540834A1 (en) 2011-06-29 2013-01-02 Metabolic Explorer Method for the preparation of 1,3-propanediol
EP2766492B1 (en) 2011-10-11 2018-06-06 Metabolic Explorer Fermentative production of prenol
ES2728279T3 (en) * 2011-11-03 2019-10-23 Easel Biotechnologies Llc Microbial production of n-butyraldehyde
EP2647718A3 (en) 2012-04-06 2014-12-24 Metabolic Explorer Process for producing 5-aminopentanoate empolying a recombinant microorganism
JP5638041B2 (en) 2012-07-25 2014-12-10 住友ゴム工業株式会社 Rubber composition for tire, tire member, and pneumatic tire
JP5536840B2 (en) 2012-09-07 2014-07-02 住友ゴム工業株式会社 Rubber composition for tire, tire member, and pneumatic tire
WO2014049382A2 (en) 2012-09-26 2014-04-03 Metabolic Explorer Ethylenediamine fermentative production by a recombinant microorganism
JP6012371B2 (en) * 2012-09-27 2016-10-25 株式会社日本触媒 Process for producing 4-hydroxy-2-butanone or butanol
WO2014062407A2 (en) 2012-10-19 2014-04-24 Dow Global Technologies Llc Anhydride-cured epoxy resin systems containing divinylarene dioxides
CN104781303B (en) 2012-11-13 2017-11-10 陶氏环球技术有限责任公司 Epoxy-resin systems for the tetramine containing polyethylene of resin transfer moulding method
US9321880B2 (en) 2012-11-13 2016-04-26 Dow Global Technologies Llc Epoxy resin system containing polyethylene tetramines and triethylene diamine catalyst for resin transfer molding processes
JP6236209B2 (en) * 2013-03-26 2017-11-22 株式会社日本触媒 Method for producing butanol
WO2017068385A1 (en) 2015-10-23 2017-04-27 Metabolic Explorer Microorganism modified for the assimilation of levulinic acid
CA3021033A1 (en) 2016-05-05 2017-11-09 Newpek S.A. De C.V. Enzymatic methods for butanol production
US11142751B2 (en) 2019-03-07 2021-10-12 Auburn University CRISPR-cas system for Clostridium genome engineering and recombinant strains produced thereof
JP2022179157A (en) 2021-05-21 2022-12-02 住友ゴム工業株式会社 Cap tread and passenger car tire
JP2022179159A (en) 2021-05-21 2022-12-02 住友ゴム工業株式会社 Cap tread and passenger car tire
JP2022179158A (en) 2021-05-21 2022-12-02 住友ゴム工業株式会社 Rubber composition for passenger car tire and passenger car tire

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050089979A1 (en) * 2003-09-18 2005-04-28 Ezeji Thaddeus C. Process for continuous solvent production

Family Cites Families (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US1315585A (en) * 1919-09-09 Charles weizmann
JPS5831993A (en) * 1981-08-20 1983-02-24 Idemitsu Kosan Co Ltd Preparation of butanol
US4521516A (en) * 1982-11-18 1985-06-04 Cpc International Inc. Strain of Clostridium acetobutylicum and process for its preparation
US4539293A (en) * 1983-05-10 1985-09-03 The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration Production of butanol by fermentation in the presence of cocultures of clostridium
US4649112A (en) * 1984-10-11 1987-03-10 Cpc International Inc. Utilization of xylan and corn fiber for direct fermentation by clostridium acetobutylicum
US4777135A (en) * 1985-02-04 1988-10-11 The University Of Vermont And State Agricultural College Method for producing butanol by fermentation
US5254467A (en) * 1988-09-01 1993-10-19 Henkel Kommanditgesellschaft Auf Aktien Fermentive production of 1,3-propanediol
US5063156A (en) * 1990-04-30 1991-11-05 Glassner David A Process for the fermentative production of acetone, butanol and ethanol
RU2080382C1 (en) * 1995-03-13 1997-05-27 Государственный научно-исследовательский институт генетики и селекции промышленных микроорганизмов Strain of bacterium clostridium acetobutylicum - a producer of normal butyl alcohol and acetone
US6428767B1 (en) * 1995-05-12 2002-08-06 E. I. Du Pont De Nemours And Company Method for identifying the source of carbon in 1,3-propanediol
US5686276A (en) * 1995-05-12 1997-11-11 E. I. Du Pont De Nemours And Company Bioconversion of a fermentable carbon source to 1,3-propanediol by a single microorganism
US5633362A (en) * 1995-05-12 1997-05-27 E. I. Du Pont De Nemours And Company Production of 1,3-propanediol from glycerol by recombinant bacteria expressing recombinant diol dehydratase
US5599689A (en) * 1995-05-12 1997-02-04 E. I. Du Pont De Nemours And Company Process for making 1,3-propanediol from carbohydrates using mixed microbial cultures
US5753474A (en) * 1995-12-26 1998-05-19 Environmental Energy, Inc. Continuous two stage, dual path anaerobic fermentation of butanol and other organic solvents using two different strains of bacteria
IL129722A0 (en) * 1996-11-13 2000-02-29 Du Pont Method for the production of 1,3-propanediol by recombinant organisms
AU7384098A (en) * 1997-05-14 1998-12-08 Board Of Trustees Of The University Of Illinois, The A method of producing butanol using a mutant strain of (clostridium beijerinckii)
ATE305972T1 (en) * 1997-12-02 2005-10-15 Du Pont METHOD OF PRODUCTION OF GLYCERIN USING RECOMBINANT ORGANISMS
US7074608B1 (en) * 1998-05-12 2006-07-11 E. I. Du Pont De Nemours And Company Method for the production of 1,3-propanediol by recombinant organisms comprising genes for coenzyme B12 synthesis
US6432686B1 (en) * 1998-05-12 2002-08-13 E. I. Du Pont De Nemours And Company Method for the production of 1,3-propanediol by recombinant organisms comprising genes for vitamin B12 transport
US6468773B1 (en) * 1999-05-19 2002-10-22 Genencor International, Inc. Mutant 1,3-propandiol dehydrogenase
FR2796081B1 (en) * 1999-07-09 2003-09-26 Agronomique Inst Nat Rech PROCESS FOR THE PREPARATION OF 1,3-PROPANEDIOL BY A MICROORGANISM RECOMBINANT IN THE ABSENCE OF COENZYME B12 OR ONE OF ITS PRECURSORS
ATE335823T1 (en) * 1999-08-18 2006-09-15 Du Pont METHOD FOR THE BIOLOGICAL PRODUCTION OF 1,3-PROPANEDIOL
US6803218B1 (en) * 1999-09-24 2004-10-12 Genencor Intl., Inc. Enzymes which dehydrate glycerol
FR2800751B1 (en) * 1999-11-09 2003-08-29 Roquette Freres PROCESS FOR THE PRODUCTION OF 1.3 PROPANEDIOL BY FERMENTATION
BRPI0311517A2 (en) * 2002-05-30 2016-06-28 Cargill Dow Llc fermentation process employing specific oxygen uptake rates as a process control
DE60335574D1 (en) * 2002-10-04 2011-02-10 Du Pont PROCESS FOR THE BIOLOGICAL MANUFACTURE OF 1,3-PROPANDIOL WITH HIGH PROFIT
US20040152159A1 (en) * 2002-11-06 2004-08-05 Causey Thomas B. Materials and methods for the efficient production of acetate and other products
DK1647594T3 (en) * 2003-07-29 2010-05-25 Res Inst Innovative Tech Earth Coryneform bacterial transformant and method of producing dicarboxylic acid using it
JP2005102533A (en) * 2003-09-29 2005-04-21 Nippon Shokubai Co Ltd Method for producing 1,3-propanediol
US20070148749A1 (en) * 2004-03-26 2007-06-28 Shinzo Yasuda Process for producting 1,3-propanediol and or/3-hydroxypropionic acid
WO2006007530A2 (en) * 2004-07-01 2006-01-19 Rice University Blocking sporulation by inhibiting spoiie
BRPI0514734B1 (en) * 2004-08-27 2018-02-06 Rice University Modified E. Coli BACTERIAL VARIETY
BRPI0515273A (en) * 2004-09-17 2008-08-05 Rice University modified bacteria, genetically engineered bacterial cell, and method for producing carboxic acids in a bacterial culture
US7682811B2 (en) * 2006-01-27 2010-03-23 University Of Massachusetts Systems and methods for producing biofuels and related materials
US20070275447A1 (en) * 2006-05-25 2007-11-29 Lewis Randy S Indirect or direct fermentation of biomass to fuel alcohol
US20100330636A1 (en) * 2009-06-26 2010-12-30 Metabolic Explorer Process for the biological production of n-butanol with high yield

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050089979A1 (en) * 2003-09-18 2005-04-28 Ezeji Thaddeus C. Process for continuous solvent production

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
DESAI RUCHIR P ET AL: "Antisense RNA strategies for metabolic engineering of Clostridium acetobutylicum", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 65, no. 3, March 1999 (1999-03-01), pages 936 - 945, XP002443073, ISSN: 0099-2240 *
GREEN EDWARD M ET AL: "GENETIC MANIPULATION OF ACID FORMATION PATHWAYS BY GENE ACTIVATION IN CLOSTRIDIUM ACETOBUTYLICUM ATCC 824", MICROBIOLOGY, SOCIETY FOR GENERAL MICROBIOLOGY, READING, GB, vol. 142, no. 8, 1996, pages 2079 - 2086, XP001248643, ISSN: 1350-0872 *
HARRIS LATONIA M ET AL: "Characterization of recombinant strains of the Clostridium acetobutylicum butyrate kinase inactivation mutant: Need for new phenomenological models for solventogenesis and butanol inhibition?", BIOTECHNOLOGY AND BIOENGINEERING, vol. 67, no. 1, 5 January 2000 (2000-01-05), pages 1 - 11, XP002443069, ISSN: 0006-3592 *
SONI B K ET AL: "CONTINUOUS ACETONE-BUTANOL FERMENTATION INFLUENCE OF VITAMINS ON THE METABOLIC ACTIVITY OF CLOSTRIDIUM-ACETOBUTYLICUM", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 27, no. 1, 1987, pages 1 - 5, XP008081386, ISSN: 0175-7598 *
TUMMALA SESHU B ET AL: "Design of antisense RNA constructs for downregulation of the acetone formation pathway of Clostridium acetobutylicum.", JOURNAL OF BACTERIOLOGY, vol. 185, no. 6, March 2003 (2003-03-01), pages 1923 - 1934, XP002443072, ISSN: 0021-9193 *
TUMMALA SESHU B ET AL: "Transcriptional analysis of product-concentration driven changes in cellular programs of recombinant Clostridium acetobutylicum strains.", BIOTECHNOLOGY AND BIOENGINEERING, vol. 84, no. 7, 30 December 2003 (2003-12-30), pages 842 - 854, XP002443071, ISSN: 0006-3592 *
WOODS D R: "The genetic engineering of microbial solvent production", TRENDS IN BIOTECHNOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 13, no. 7, July 1995 (1995-07-01), pages 259 - 264, XP004207180, ISSN: 0167-7799 *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010063766A1 (en) * 2008-12-02 2010-06-10 Total S.A. Bioprocessing ligno-cellulose into ethanol with recombinant clostridium
EP2194120A1 (en) * 2008-12-02 2010-06-09 Total S.A. Bioprocessing ligno-cellulose into ethanol with recombinant clostridium
EP2204453A1 (en) 2008-12-30 2010-07-07 Sued-Chemie AG Process for cell-free production of chemicals
WO2010076305A1 (en) 2008-12-30 2010-07-08 Süd-Chemie AG Process for cell-free production of chemicals
JP2012513759A (en) * 2008-12-30 2012-06-21 ズュード−ヒェミー・アクチェンゲゼルシャフト Cell-free chemical production method
EP2267141A1 (en) * 2009-06-26 2010-12-29 Metabolic Explorer Process for the biological production of n-Butanol with high yield
US8765446B2 (en) 2009-09-22 2014-07-01 Korea Advanced Institute Of Science And Technology Recombinant mutant microorganisms having increased ability to produce alcohols and method of producing alcohols using the same
EP2481793A2 (en) * 2009-09-22 2012-08-01 Korea Advanced Institute of Science and Technology Recombinant microorganism with increased butanol production ability, and preparation method of butanol using same
EP2481793A4 (en) * 2009-09-22 2013-07-10 Korea Advanced Inst Sci & Tech Recombinant microorganism with increased butanol production ability, and preparation method of butanol using same
JP2013511283A (en) * 2009-11-23 2013-04-04 ビュータマックス・アドバンスド・バイオフューエルズ・エルエルシー Method for producing butanol using extractive fermentation with electrolyte addition
DE102011077705A1 (en) 2011-06-17 2012-12-20 Evonik Degussa Gmbh Microbial process for the preparation of low molecular weight organic compounds comprising the product absorption by isophorone
WO2013128230A1 (en) 2012-03-02 2013-09-06 Metabolic Explorer A process for butanol production
US20150004664A1 (en) * 2012-03-02 2015-01-01 Assia Zigha Process for Butanol Production
US9434963B2 (en) 2012-03-02 2016-09-06 Metabolic Explorer Process for butanol production
GB2515366A (en) * 2013-02-05 2014-12-24 Green Biologics Ltd Production of butanol
US9499804B2 (en) 2013-02-05 2016-11-22 Green Biologics Ltd Cyclodextrin glucanotransferase
US9783831B2 (en) 2013-02-05 2017-10-10 Green Biologics Ltd Cyclodextrin glucanotransferase
US9902978B2 (en) 2013-03-26 2018-02-27 Nippon Shokubai Co., Ltd. Genetically modified Clostridium saccharoperbutylacetonicum
US10961499B2 (en) 2016-07-08 2021-03-30 Metabolic Explorer Method for the fermentative production of molecules of interest by microorganisms comprising genes coding sugar phosphotransferase system (PTS)
EP3348646A1 (en) 2017-01-17 2018-07-18 Evonik Degussa GmbH Microbial method for the preparation of acetone, isopropanol, butanol and/or ethanol comprising product absorption by water
CN107653208A (en) * 2017-11-15 2018-02-02 天津科技大学 One plant of hydrogenogen

Also Published As

Publication number Publication date
TW200835792A (en) 2008-09-01
JP2010508017A (en) 2010-03-18
KR101444968B1 (en) 2014-09-26
RU2461627C2 (en) 2012-09-20
IL198342A0 (en) 2011-08-01
CA2665102C (en) 2015-01-20
JP2014000087A (en) 2014-01-09
MX2009004660A (en) 2009-05-22
ZA200902639B (en) 2010-03-31
IL198342A (en) 2013-10-31
KR20090085650A (en) 2009-08-07
CN101528935B (en) 2013-08-07
AR063762A1 (en) 2009-02-18
US20100086982A1 (en) 2010-04-08
RU2009118372A (en) 2010-12-10
BRPI0718142A2 (en) 2013-11-05
AU2007316189B2 (en) 2014-07-03
CN101528935A (en) 2009-09-09
CA2665102A1 (en) 2008-05-08
WO2008052973A3 (en) 2008-07-31
US20140377825A1 (en) 2014-12-25
WO2008052973A2 (en) 2008-05-08
AU2007316189A1 (en) 2008-05-08
DK2084287T3 (en) 2012-07-23
JP5442441B2 (en) 2014-03-12

Similar Documents

Publication Publication Date Title
AU2007316189B2 (en) Process for the biological production of n-Butanol with high yield
US8236994B2 (en) Process for the biological production of 1,3-propanediol from glycerol with high yield
Cheng et al. Engineering Clostridium for improved solvent production: recent progress and perspective
Papoutsakis Engineering solventogenic clostridia
Schiel-Bengelsdorf et al. Butanol fermentation
Dürre Fermentative production of butanol—the academic perspective
US9284580B2 (en) Metabolic engineering of clostridium tyrobutyricum for butanol production
BRPI0813710B1 (en) A method of identifying a heterologous polypeptide having enzymatic activity to convert pyruvate, acetaldehyde or acetate to acetyl-coa in the yeast cell cytosol, vector for the expression of heterologous polypeptides in yeast, recombinant yeast cell and method of producing a fermentation product.
Dong et al. Biobutanol
Arslan et al. Engineering Acetobacterium woodii for the production of isopropanol and acetone from carbon dioxide and hydrogen
Amiri et al. Biobutanol production
Xue et al. 3.07-Biofuels and Bioenergy: Acetone and Butanol☆
US20100330636A1 (en) Process for the biological production of n-butanol with high yield
US9434963B2 (en) Process for butanol production
EP2267141A1 (en) Process for the biological production of n-Butanol with high yield
EP2084287B1 (en) Process for the biological production of n-butanol with high yield
Chang Acetone-butanol-ethanol fermentation by engineered Clostridium beijerinckii and Clostridium tyrobutyricum
US9957529B2 (en) Recombinant microorganism with improved butanol production ability and method for producing butanol by using the same
CN101935677A (en) Method for producing n-butanol by using high-yield organism
CN116478898A (en) Recombinant strain for transforming hrcA through CRISPR technology, construction method and application thereof
Gong et al. Hongjun Dong, Wenwen Tao, Zongjie Dai, Liejian Yang

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 06819207

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06819207

Country of ref document: EP

Kind code of ref document: A1