WO2009142541A1 - A microorganism producing 1-butanol and a method for producing 1-butanol - Google Patents

A microorganism producing 1-butanol and a method for producing 1-butanol Download PDF

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WO2009142541A1
WO2009142541A1 PCT/RU2009/000237 RU2009000237W WO2009142541A1 WO 2009142541 A1 WO2009142541 A1 WO 2009142541A1 RU 2009000237 W RU2009000237 W RU 2009000237W WO 2009142541 A1 WO2009142541 A1 WO 2009142541A1
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microorganism
seq
gene
coa
butanol
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PCT/RU2009/000237
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French (fr)
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Rustem Saidovich Shakulov
Aleksandra Yurievna Skorokhodova
Andrey Yurievich Gulevich
Aleksandr Sergeevich Mironov
Vladimir Georgievich Debabov
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State Research Institute Of Genetics And Selection Of Industrial Microorganisms (Gosniigenetika)
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention is related to microbiological industry, particularly to a method for producing 1-butanol using microorganism modified so that at least one of enzymes from pathway of fatty acids ⁇ -oxidation is involved into 1-butanol.
  • Butanol is four carbon alcohol widely used in the industry. Butanol is used as solvent in the paint and varnish industry, in resin and plasticizer production, in the synthesis of wide amount of organic compounds, particularly pesticides, perfumes, medicaments. At the time butanol is considered as an attractive compound which can be used as additive to motor fuel or even its complete substituent. Use of butanol as fuel both in mix with gasoline and in pure state does not require modification of existing combustion. Enegry value of butanol is 29 MJ/litre, octane rating is 96. The characterictics are rather close to corresponding characteristics of gasoline - 32 MJ/litre and 91-99, respectively (Durre P., 2007 Biobutanol: An attractive biofuel. Biotechnol.
  • Butanol is not hygroscopic and hydrophobic. It can be mixed with gasoline in any proportion. Low corrosive characteristics of butanol allow to use already existing infrastructure developed for oil products for transportation and storage of butanol.
  • butanol up to 5 million tons per year
  • Common methods of 1-butanol productins include hydrogenation of crotone aldehyde and oxosynthesis from propylene (Loktev et al, "High fatty " Moscow, Khikiya (Rus), 1970).
  • Disadvantages of above processes are high energy spending, aggressive conditions for the synthesis, requirement of oil products as raw materials.
  • Butanol can be obtained by biotransformation of wild spectrum of organic compounds using different microorganisms. Corresponding alcohls can be obtained using Pseudomonas strains (JP63017695). Aliphatic saturated hydrocarbons (from dibasic to twentybasic) can be oxydazied by monooxygenase of Rhodococcus ruber to yield corresponding aclohols (patent applicatins US2002028492, EPl 149918). Thus, in case of butane products can be 1-butanol (95%) and 2-butanol (5%).
  • Acetic acid, aceton and ethanol are by-products of the process (Final Report on the Production of Butyric Acid and Butanol from Biomass, which is based on work performed under: Contract No.: DE-F-G02-00ER86106, http://www.eere.energy.gov/afdc/pdfs/843183.pdf).
  • E. coli is one of them (WO2007041269; Atsumi S. et al., Metabolic engineering of Escherichia coli for 1- butanol production. Metab. Eng., 2007, doi:10.1016/j.ymben.2007.08.003; Inui M. et al., Expression of Clostridium acetobutylicum butanol synthetic genes in Esherichia coli. Appl. Microbiol. Biotechnol., 2008, 77(6): 1305-16).
  • B. subtilis is also one of them (WO2007041269).
  • the goal of the present invention is providing a microorganism producing 1 -butanol by fermentation and a method for producing 1 -butanol using such microorganism.
  • Such goal was achieved by constructing 1 -butanol producing microorganism modified so that at least one of enzymes from pathway of fatty acids ⁇ -oxidation is involved into 1 -butanol biosynthesis.
  • acyl-CoA dehydrogenase comprises amino acid sequence selected from any of SEQ ID NO: 1-17.
  • enoyl-CoA hydratase comprises amino acid sequence selected from any of SEQ ID NO: 18-38.
  • 3-hydroxyacetyl-CoA dehydrogenase comprises amino acid sequence selected from any of SEQ ID NO: 18, 19, 23-25, 27, 31-33, 39-44.
  • 3-ketoacyl-CoA thiolase comprises amino acid sequence selected from any of SEQ ID NO: 45-61.
  • microorganism belongs to the genus Clostridium, Escherichia, Salmonella, Shigella, Pseudomonas, Zymomonas, Bacillus, Lactobacillus, Enterococcus, Klebsiella, Corynebacterium, Brevibacterium, Streptomyces, Pichia, Candida or Sacchoromyces. It is a further object of the present invention to provide the microorganism as described above, wherein said microorganism is Clostridium acetobutylicum or Clostridium beijerinckii.
  • process of microorganism cultivation includes aerobic and anaerobic steps.
  • butanol The only native producers of butanol is bacterium belonging to the genus Clostridium, such as C. acetobutylicum, C. beijerinckii, C. saccharoperbutylacetonicum and others, in which biosynthesis of organic solvents (acetone, ethanol, butanol) is directly coupled with their vital functions.
  • Butanol biosynthetic pathway of such organisms as well as genes encoding enzymes of that pathway are known.
  • the pathway starts from formation of acetoacetyl-CoA from two molecules of acetyl-CoA and finishes after several steps with reduction of butyril-CoA to corresponding aldehyde and 1 -butanol.
  • Such pathway is accompanied with oxidation of three NADH molecules and one NADPH molecule (Jones, D. and Woods, R, Microbiol. Rev., 1986, 50 (4): 484-524).
  • microorganisms carrying definite regulatory mutations are able to utilize 1 -butanol even as a sole carbon source.
  • utilization of 1 -butanol includes formation of butyril-CoA by alcohol- and aldehyde dehydrogenases followed by its convertion into two acetyl-CoA molecules in the pathway of fatty acids ⁇ -oxidation (Neidhardt, F. C. et al. Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington D.C., chapter 21).
  • Fatty acids ⁇ -oxidation includes several reactions catalyzed by acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxyacetyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase, and accompanied with NAD + reduction.
  • Such activity is classified as EC 1.3.99.-, particularly EC 1.3.99.3.
  • Presence of acyl-CoA dehydrogenase activity can be determined by, for example, complementation of fadE62 mutation (inabilty to growth on the medium with fatty acids as a sole carbon source) using method described by Campbell, J. W.
  • FadE serine-CoA dehydrogenase activity
  • FadE serine-CoA dehydrogenase activity
  • FadE serine-CoA dehydrogenase activity
  • FadE serine-CoA dehydrogenase activity
  • FadE serine-CoA dehydrogenase activity
  • YdiO SEQ ID NO: 2
  • FadE SEQ ID NO: 3
  • YdiO SEQ ID NO: 4
  • STM0857 SEQ ID NO: 5
  • FadE SEQ ID NO: 6
  • PP 2437 SEQ ID NO: 7
  • PP 4948 SEQ ID NO: 8
  • Such activity is classified as EC 4.2.1.17.
  • Presence of enoyl-CoA hydratase activity can be determined, for example, using method described by Binstock, J. F. and Schulz, H. (Fatty acid oxidation complex from Escherichia coli, Methods Enzymol. 1981 , 71 Pt C; 403-11).
  • FadB semonims B3846, OldB
  • FadJ SEQ ID NO: 19
  • PaaG SEQ ID NO: 20
  • PaaF SEQ ID NO: 21
  • MaoC SEQ ID NO: 22
  • FadB SEQ ID NO: 23
  • FadJ SEQ ID NO: 24
  • Salmonella typhimurium FadB (SEQ ID NO: 25) and PhaL (SEQ ID NO: 26) from Pseudomonas putida, YusL (SEQ ID NO: 27), YngF (SEQ ID NO: 28), YsiB (SEQ ID NO: 29) and YhaR (SEQ ID NO: 30) from Bacillus subtilis, NCgl0882 (SEQ ID NO: 31) from Corynebacterium glutamicum, SCO6732 (SEQ ID NO: 30) from Bacillus subtilis, NCgl0882 (SEQ ID NO: 31) from Corynebacterium glutamicum, SCO6732 (
  • Such activity is classified as EC 1.1.1.35 or EC 1.1.1.157.
  • Presence of 3-hydroxyacetyl-CoA dehydrogenase activity can be determined, for example, using method described by Binstock, J. F. and Schulz, H. (Fatty acid oxidation complex from Escherichia coli, Methods Enzymol.
  • FadB from Escherichia coli (synonims B3846, OldB) (SEQ ID NO: 18), FadJ (SEQ ID NO: 19) and PaaH (SEQ ID NO: 39) from Escherichia coli, FadB (SEQ ID NO: 23) and FadJ (SEQ ID NO: 24) from Salmonella typhimurium, FadB (SEQ ID NO: 25), PP_0302 (SEQ ID NO: 40), PaaC (SEQ ID NO: 41) and PaaH (SEQ ID NO: 42) from Pseudomonas putida, YusL (SEQ ID NO: 27) and MmgB (SEQ ID NO: 43) from Bacillus subtilis, SCO6732 (SEQ ID NO: 32), SCO6789 (SEQ ID NO: 33) and SCO3834 (SEQ
  • Such activity is classified as E.C. 2.3.1.16 or E. C. 2.3.1.9.
  • Presence of 3-ketoacyl-CoA thiolase activity can be determined, for example, using method described by Binstock, J. F. and Schulz, H. (Fatty acid oxidation complex from Escherichia coli, Methods Enzymol.
  • FadA serine-CoA thiolase activity
  • Fadl SEQ ID NO: 46
  • AtoB SEQ ID NO: 47
  • YqeF SEQ ID NO: 48
  • FadA SEQ ID NO: 49
  • Fadl SEQ ID NO: 50
  • YqeF SEQ ID NO: 51
  • Salmonella typhimurium
  • FadA SEQ ID NO: 52
  • AtoB SEQ ID NO: 53
  • protein variant means proteins which have changes in the sequences, whether they are deletions, insertions, additions, or substitutions of amino acids which are not deteriorate activity of the protein.
  • the number of changes in the variant proteins depends on the position or the type of amino acid residues in the three dimensional structure of the protein. It may be 1 to 30, preferably 1 to 15, and more preferably 1 to 5 amino acid residues. These changes in the variants can occur in regions of the protein which are not critical for the three dimensional structure of the protein and its function.
  • protein variant includes proteins having homology no less then 80%, preferably no less then 90%, and more preferably no less then 95% regarding to whole amino acid sequence depicted in the Sequence Listing provided that activity of the protein is not deteriorated.
  • Homology between two amino acid sequences can be determined using the well-known methods, for example, the computer program BLAST 2.0, which calculates three parameters: score, identity and similarity.
  • substitution, deletion, insertion or addition of one or several amino acid residues should be conservative mutation(s) so that the activity is maintained.
  • the representative conservative mutation is a conservative substitution.
  • conservative substitutions include substitution of Ser or Thr for Ala, substitution of GIn, His or Lys for Arg, substitution of GIu, GIn, Lys, His or Asp for Asn, substitution of Asn, GIu or GIn for Asp, substitution of Ser or Ala for Cys, substitution of Asn, GIu, Lys, His, Asp or Arg for GIn, substitution of Asn, GIn, Lys or Asp for GIu, substitution of Pro for GIy, substitution of Asn, Lys, GIn, Arg or Tyr for His, substitution of Leu, Met, VaI or Phe for He, substitution of He, Met, VaI or Phe for Leu, substitution of Asn, GIu, GIn, His or Arg for Lys, substitution of He, Leu, VaI or Phe for Met, substitution of Trp, Tyr
  • NP 391598 acyl-CoA dehydrogenase [Bacillus subtilis subsp. 10 subtilis str. 168]
  • NP_389708 hypothetical protein BSUl 8260 [Bacillus subtilis 11 subsp. subtilis str.
  • NP_627009 acyl-CoA dehydrogenase [Streptomyces coelicolor 12 A3(2)]
  • NP 629821 acyl-CoA dehydrogenase
  • NP_630552 acyl-CoA dehydrogenase
  • NP_627272 acyl-CoA dehydrogenase
  • Streptomyces coelicolor 15 A3(2) NP 625964 acyl-CoA dehydrogenase [Streptomyces coelicolor 16 A3(2)]
  • NP 01 1310 Fatty-acyl coenzyme A oxidase, involved in the 17 fatty acid beta-oxidation pathway; localized to the peroxisomal matrix; Pox Ip [Saccharomyces cerevisiae]
  • NP_416843 fused enoyl-CoA hydratase and epimerase and 19 isomerase/3-hydroxyacyl-CoA dehydrogenase [Escherichia coli str. K- 12 substr. MG 1655]
  • NP_415912 acyl-CoA hydratase [Escherichia coli str. K- 12 20 substr. MG 1655]
  • NP_41591 1 enoyl-CoA hydratase-isomerase [Escherichia coli 21 str. K- 12 substr. MG 1655]
  • NP_462868 multifunctional fatty acid oxidation complex 23 subunit alpha [Salmonella typhimurium LT2] NP_461330 multifunctional fatty acid oxidation complex 24 subunit alpha [Salmonella typhimurium LT2] NP_744285 multifunctional fatty acid oxidation complex 25 subunit alpha [Pseudomonas putida KT2440] NP_745413 bifunctional aldehyde dehydrogenase/enoyl-CoA 26 hydratase [Pseudomonas putida KT2440] NP_391 163 hypothetical protein BSU32840 [Bacillus subtilis 27 subsp. subtilis str. 168] NP 389704 enoyl-CoA hydratase [Bacillus subtilis subsp. 28 subtilis str. 168]
  • Equilibrium of redox reaction (such as reactions of fatty acids ⁇ -oxidation catalyzed by alcohol- and aldehyde dehydrogenases) can be shifted to formation of desired product/substrate.
  • the equilibrium can be shifted, for example, by: a) changing the ratio of concentrations of substances involved into reaction; 6) changing the ratio of concentrations of oxidative and reducing equivalents required for passing reaction in direct or reversed, particularly in the case of biochemical reactions - NADH/NAD + , NADPH/NADP + , FADH 2 /FADH.
  • equilibrium can be shifted by chanching activity of catalyst, particularly in the case of enzymes as biologic catalysts due to mutations changing specific activity of the enzyme.
  • pathway of fatty acids ⁇ -can be used for butyril-CoA biosynthesis followed by formation of 1-butanol as a result of two consequtive reactions catalyzed by aldehyde dehydrogenase and alcohol dehydrogenase.
  • Such activity is generally classified as E. C. 1.2.1.3.
  • acetaldehyde dehydrogenase from E. coli acetaldehyde dehydrogenase (acetylating)
  • the enzyme can utilize acetaldehyde, butyraldehyde, propanaldehyde, glycolaldehyde and other compound as alternative substrates.
  • Presence of aldehyde dehydrogenase activity can be determined by, for example, method described by Rudolph, F. B. et al (Coenzyme A-linked aldehyde dehydrogenase from Escherichia coli. I. Partial purification, properties, and kinetic studies of the enzyme, J. Biol. Chem., 1968; 243(21); 5539-45).
  • Example of enzyme having activity of aldehyde dehydrogenase includes AdhE from Escherichia coli (synonims acetacldehyde:NAD + oxidoreductase (CoA-acetylated), ACDH, coenzyme A dependent acetaldehyde dehydrogenase) which is homopolimer with 3 Fe 2+ - dependent catalytic activities: alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate- formate lyase-deactivase (SEQ ID NO: 62).
  • Example of enzyme having activity of alcohol dehydrogenase includes AdhE (synonims - ADH, alcohol .NAD + oxidoreductase, aldehyde reductase described above (SEQ ID NO: 62 and YqhD (Sulzenbacher, G. Et al, J. MoI. Biol. 2004 10; 342(2):489-502) (SEQ ID NO: 63) from Escherichia coli
  • Term"protein variant is also for both acetaldehyde dehydrogenase and alcohol dehydrogenase.
  • Term"protein variant is understood as above.
  • Microorganism according to the present invention is a 1 -butanol producing microorganism modified so that at least one of enzymes from pathway of fatty acids ⁇ -oxidation is involved into 1 -butanol biosynthesis. Moreover, microorganism according to the present invention is a 1- butanol producing microorganism modified to have increased activity of one or several enzymes from pathway of fatty acids ⁇ -oxidation in the cell compare to non-modified.
  • 1 -butanol producing microorganism means microorganism having ability to produce and accumulate 1 -butanol in a medium when the microorganism in cultivated in the medium.
  • Ability to produce 1 -butanol can be imparted to the microorganism by selection, mutagenesis or genetic manipulations.
  • Phrase "1 -butanol producing microorganism” also means microorganism having ability to produce and accumulate 1 -butanol in a medium in higher amount then parent microorganism and preferably means ability to accumulate 1 -butanol in a medium in detectable amount.
  • Methods for detection of 1 -butanol include, for example, gas chromatography.
  • Such microorganism can be selected from group consisting of bacteria, yeast and fungi.
  • Microorganism according to the present invention is a microorganism containing at least one of enzymes from pathway of fatty acids ⁇ -oxidation. Such enzyme can be either native or heterogeneous for the microorganism.
  • Term “native enzyme” means natural enzyme inherent for the microorganism encoded by gene from the microorganism.
  • Term “heterogeneous enzyme” means enzyme encoded by gene introduced in the microorganism from another microorganism. Genes encoding heterogeneous enzyme can be introduced in the microorganism by genetic manipulations.
  • Microorganism according to the present invention is a microorganism having ability to utilize organic substrate efficiently or a microorganism to which such ability is imparted or increased by selection, mutagenesis or genetic manipulations.
  • Microorganism according to the present invention belongs to the genus Clostridium, Escherichia, Salmonella, Shigella, Pseudomonas, Zymomonas, Bacillus, Lactobacillus, Enter ococcus, Klebsiella, Corynebacterium, Brevibacterium, Streptomyces, Pichia, Candida or Sacchoromyces.
  • microorganism belongs to the genus means that the bacterium is classified as the bacterium belonging to the particular genus according to the classification known to a person skilled in the art of microbiology.
  • microorganisms belonging to the genera meantioned above include Escherichia coli, Salmonella enterica, Shigella flexneri, Pseudomonas aeruginosa, Pseudomonas putida, Zymomonas mobilis, Bacillus subtilis, Corynebacterium glutamicum, Streptomyces coelicolor, Saccharomyces cerevisiae, Clostridium acetobutylicum, Clostridium beijerinckii.
  • Microorganism according to the present invention is a microorganism modified to have increased activity of one or several said enzymes in comparison to non-modifieid microorganism compare to non-modified microorganism.
  • Phrase "activity of enzyme is increased” means that activity of enzyme in cell is higher compare to the non-modified microorganism, for example, wild-type strain.
  • Activity of one or several enzymes from pathway of fatty acids ⁇ -oxidation can be increased in the cell due to increasing the amount of corresponding mRNA, increasing amount of corresponding enzyme or increasing specific activity of enzyme.
  • Amount of mRNA can be increased by increasing copy number of corresponding gene or due to enhancing transcription of the gene by use of more potent promoter, releasing from repression or increasing mRNA stability.
  • Amount of enzyme can be increased by enhancing translation of corresponding mRNA due to modification of nucleotide sequence of ribosome binding site or by increasing the enzyme stability.
  • Specific activity of the enzyme can be increased by introduction of corresponding mutations into amino acid sequence of the enzyme.
  • Methods of enhancement of gene expression include increasing the gene copy number. Introducing a gene into a vector that is able to function in the microorganism according to the present invention increases the copy number of the gene.
  • Enhancement of gene expression may also be achieved by introduction of multiple copies of the gene into a bacterial chromosome by, for example, a method of homologous recombination, Mu integration, or the like.
  • Mu integration allows introduction of up to 3 copies of the gene into a bacterial chromosome.
  • Increasing the copy number of a gene can also be achieved by introducing multiple copies of the gene into the chromosomal DNA of the bacterium.
  • homologous recombination is carried out using a sequence whose multiple copies exist as targets in the chromosomal DNA.
  • Sequences having multiple copies in the chromosomal DNA include, but are not limited to repetitive DNA, or inverted repeats existing at the end of a transposable element. Also it is possible to incorporate the gene into a transposon, and allow it to be transferred to introduce multiple copies of the gene into the chromosomal DNA.
  • Enhancing gene expression may also be achieved by placing the DNA of the present invention under the control of a potent promoter.
  • a potent promoter for example, the lac promoter, the trp promoter, the trc promoter, the P R , or the P L promoters of lambda phage are all known to be potent promoters.
  • the use of a potent promoter can be combined with multiplication of gene copies.
  • the effect of a promoter can be enhanced by, for example, introducing a mutation into the promoter to increase the transcription level of a gene located downstream of the promoter.
  • substitution of several nucleotides in the spacer between ribosome binding site (RBS) and the start codon, especially the sequences immediately upstream of the start codon profoundly affect the mRNA translatability. For example, a 20-fold range in the expression levels was found, depending on the nature of the three nucleotides preceding the start codon (Gold et al, Annu. Rev. Microbiol., 35, 365-403, 1981 ; Hui et al, EMBO J., 3, 623-629, 1984).
  • a nucleotide substitution into a promoter region of a gene on the bacterial chromosome, which results in a stronger promoter function.
  • the alteration of the expression control sequence can be performed, for example, in the same manner as the gene substitution using a temperature-sensitive plasmid, as disclosed in WO 00/18935 and JP 1-215280 A.
  • Enhancement of translation can be achieved by introduction more effective Shine- Dalgarno sequence (SD-sequence) instead of native SD- ⁇ ocJie ⁇ Ba ⁇ ejibH ⁇ c ⁇ H provided that native SD-sequence is located upstream of start-codon of the mRNA contacting with 16S ribosomal RNA (Shine J. and Dalgarno L., Proc. Natl. Acad. Sci. U S A, 1974, 71, 4, 1342-6).
  • SD-sequence Shine- Dalgarno sequence
  • potent promoter can be combined with with use of more effective SD-sequence.
  • Microorganism according to the present invention is a microorganism additionally modified to have increased amount acetyl-CoA, the earlier precursor of 1-butanol.
  • Amount of acetyl-CoA can be increased by, for example, decreasing flux of acetyl-Coa to the pathways branching from direct pathway of butanol biosynthesis as well as by regulated decrease of phosphoenolpyruvate activity (see Example 7). So, one of the major pathways for acetyl-CoA utilization is tricarboxylic acid cycle. The cycle starts from formation of citrate from oxaloacetate and acetyl group of acetyl-CoA.
  • phosphoenolpyruvate carboxylase catalyzed reaction of formation oxaloacetate from phosphoenolpyruvate with CO 2 fixation.
  • Phosphoenolpyruvate is the intermediate of glycolysis and precursor of of acetyl-CoA. Thus, it is necessary to decrease activity of phosphoenolpyruvate carboxylase for effective butanol biosynthesis from acetyl-CoA.
  • ppc mutants for example E.coli
  • E.coli are auxotrophs for intermediates of tricarboxylic acid cycle and they can not grow in the medium containing most suitable for 1-butanol production carbon sources, such as sugars and glycerol [Sauer U. and Eikmanns B. 2005 The PEP-pyruvate-oxaloacetate node as the switch point for carbon flux distribution in bacteria. FEMS Microbiol. Rew., 29, 765-794].
  • complete inactivation of phosphoenolpyruvate carboxylase is not suitable.
  • Use of regulated decrease of the enzyme activity can solve the problem. Regulated decrease of the enzyme activity can be achieved by, for example, decrease of de novo synthesis of the enzyme by decrease of amount of corresponding mRNA as a result of counter transcription of target gene initiated from regulated promoter.
  • a method according to the present invention is a method for producing 1-butanol including cultivation of microorganism according to the present invention in culture medium and collecting produced 1-butanol from the medium.
  • collection and purification of 1 -butanol from culture medium and the like may be performed in a manner similar to conventional fermentation methods wherein 1-butanol is produced using a bacterium (distillation, rectification, liquid extraction, membrain filtration etc. Se, for example, Ezeji, T.C. et al, Butanol fermentation research: upstream and downstream manipulations. Chem Rec. 2004; 4(5): 305-14).
  • a medium used for culture may be either a synthetic or natural medium, so long as the medium includes a carbon source and a nitrogen source and minerals and, if necessary, appropriate amounts of nutrients which the bacterium requires for growth.
  • the carbon source may include various carbohydrates such as glucose and sucrose, and various organic acids and glycerol.
  • As the nitrogen source various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean-hydrolysate, and digested fermentative microorganism can be used.
  • As minerals potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like can be used.
  • vitamins, thiamine, yeast extract, and the like can be used.
  • the cultivation is preferably performed at the temperature from 30°C to 37°C, preferably from 35°C to 37°C. pH is adjusted from 5 to 9, preferably from 6.5 to 7.2.
  • the pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. Usually, a 1 to 5-day cultivation leads to accumulation of 1-butanol in the liquid medium.
  • Process of microorganism cultivation in the method of the present invention may be aerobic, anaerobic, microaerobic. Also the process of microorganism cultivation in the method of the present invention may include both aerobic and anaerobic steps.
  • solids such as cells can be removed from the liquid medium by centrifugation or membrane filtration.
  • Point mutation in the region "-35" of chromosomal promoter leading to formation of phenotype was introduced by the method developed by Datsenko, K.A. and Wanner, B. L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) also known as "Red-driven integration”.
  • DNA fragment containing Cm R marker encoded by cat gene was obtained by PCR using primers Pl (SEQ ID NO: 64) and P2 (SEQ ID NO: 65), and pMW1 18-attL-Cm-attR plasmid [Katashkina et al, 2005. MoI. Biol. (Rus), 39 (5), 823-831] as a template.
  • Primer Pl contains 36 nucleotides homologous to the DNA fragment on 3 '-end of mhpR gene and 28 nucleotides complementary to 3 '-end of attR region.
  • Primer P2 contains 36 nucleotides complementary except one point mutation to DNA fragment located immediately down-stream of stop-codon of mhpR gene and "-35" region of promoter, and 28 nucleotides homologous to the DNA fragment located in 5 '-end of attL region.
  • termerature profile was used: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 52°C, 1 minute at 72°C; and final polimerization: 7 minutes at 72°C.
  • Taq DNA polymerase corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
  • pKD46 plasmid contains DNA fragment from phage ⁇ (2154 base pairs; positions from 31088 to 33241 in nucleotide sequence with number J02459 in GenBank database) and genes of ⁇ Red-homologous recombination system ( ⁇ , ⁇ , exo genes) under control arabinose inducible promoter.
  • pKD46 plasmid is necessary for integration of PCR product into chromosome of strain MG1655.
  • Strain MG1655 is available in American Type Culture Collection. (P.O. Box 1549 Manassas, VA 20108, U.S.A.).
  • Electrocompetent cells were obtained as follows: night culture of E. coli strain MGl 655 containing pKD46 plasmid were grown at 30°C on LB medium containing ampicillin (100 mg/1), deluted 100 times with adding 10 ml of SOB medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) containing ampicillin and L-arabinose (10 mM). Obtained culture was grown with mixture at 30°C to reach O Then cells were made electrocompetent by concentrating in 100 times and washing three times with ice-chilled deionized water. Electroporation was performed with 70 ⁇ l of cells and of PCR-product.
  • Cm resistance gene (cat gene) was eliminated from chromosome of strain MGl 655 using int-xis system.
  • Strain MGl 655 was transformed with pMWts-Int/Xis plasmid (WO 2007013638; RU 2005123423). Selection of transformed clones was performed on LB agar medium containing ampicillin (100 mg/1). Plates were incubated during night at 30°C.
  • the cat gene was eliminated from transformed clones by culturing separate colonies at 37°C (at that termerature Cits repressor is partially inactivated while int/xis genes is activated) followed by selection of Cm s Ap R variants.
  • Cm s Ap R variants were healed from helper plasmid pMWts-Int/Xis containing temperature sensitive replicon by second spreading to get seperate colonies at 37°C followed by selection of Cm s Ap s clones.
  • Example 2 Construction of E. coli strain having alcohol dehydrogenase AdhE capable to exhibit enzymatic activity in aerobic conditions.
  • DNA fragment integrated into corresponding region of bacterial chromosome was performed in several stages.
  • DNA fragment containing BgIW restrictin site, promoter, SD sequence of ⁇ lO gene from phage T7 and 36 nucleotides complementary 5 '-end of coding region of adhE gene was obtained by PCR.
  • the DNA fragment was obtained in two stages. At first, DNA fragment containing restriction site, promoter and part of SD sequence of ⁇ lO gene from phage T7 was obtained by PCR using plasmid [Skorokhodova A. Yu. et al, 2006, Biotekhnologiya (Rus) 3, 6-16.].
  • PCR was performed using primers P5 (SEQ ID NO: 68) and P6 (SEQ ID NO: 69).
  • Primer P5 contains BgIW restrictin site and region homolous to 5'-end of promoter.
  • Primer P6 contains part of SD sequence of ⁇ 10 gene from phage T7 and region complementary to 3 '-end of promoter.
  • temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 55°C, 30 seconds at 72°C; and final polymerization: 7 minutes at 72°C.
  • Pfu DNA polymerase corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
  • PCR product purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to recommendations of manufacturer was used for the next round of PCR as template.
  • Primer P7 contains region complementary 3'-end of promoter, SD sequence of ⁇ lO gene from phage T7 and 36 nucleotides of opened reading frame of adhE gene. Following termerature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 55°C, 40 seconds at 72°C; and final polymerization: 7 minutes at 72°C.
  • Pfu DNA polymerase corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
  • DNA fragment containing Cm R marker encoded by cat gene was obtained by PCR using primers P8 (SEQ ID NO: 71) and P9 (SEQ ID NO: 72) and pMWl 18-attL-Cm-attR plasmid as template.
  • Primer P8 contains 36 hucleotides homologous to DNA fragment upstream of coding region of adhE gene and 28 nucleotides complementary to DNA fragment located at 3 '-end of attR region.
  • Primer P9 contains BgIW restriction site and 28 nucleotides homologous to the 5 '-end of attL region.
  • Product obtained after ligation was amplified by PCR using primers P7 (SEQ ID NO: 70) and P8 (SEQ ID NO: 71). Following temperature profile was used for PCR: denaturation at 98°C during 1 minute; 30 cycles: 15 seconds at 98°C, 15 seconds at 55°C, 1 minute at 72°C; and final polimerization: 7 minutes at 72°C. Phusion DNA polymease and corresponding buffer from Finnzymes; and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
  • Electrocompetent cells of E. coli strain MGl 655 lacf containing pKD46 plasmid were obtained as described in Example 1. Electroporation was performed with 70 ⁇ l of cells and of PCR product. After electroporation cells were incubated in 1 ml of SOC medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) at 37°C during 2.5 hours, then cells were spreaded on plates with L-agar containing 30 mg/1 chloramphenicol and grown at 37°C to select Cm R - recombinants. To eliminate pKD46 plassmid, cells were spreaded on LB agar to get separate colonies at 37°C and Cm Ap variants were selected.
  • Cm resistance gene ⁇ cat gene was eliminated from chromosome of strain MG1655 using int-xis system.
  • Strain MG1655 lacf attR-cat- was transformed with pMWts-Int/Xis plasmid (WO 2007013638; RU 2005123423). Selection of transformed clones was performed on LB agar medium containing ampicillin (100 mg/1). Plates were incubated during night at 30°C.
  • the cat gene was eliminated from transformed clones by culturing separate colonies at 37°C (at that termerature Cits repressor is partially inactivated while int/xis genes is activated) followed by selection of Cm Ap variants.
  • variants were healed from helper plasmid pMWts-Int/Xis containing temperature sensitive replicon by second spreading to get seperate colonies at 37°C followed by selection of clones.
  • strain MGl 655 lacf or abbreviated as MGl 655 l was obtained.
  • Primer Pl 2 contains 36 nucleotides homologous DNA of adhE gene in position 1657 to 1692 and region of homology to pUC4K plasmid.
  • Primer Pl 3 contains 36 nucleotides complementary to DNA of adhE gene in position 1749 to 1714 and region of homology to pUC4K plasmid.
  • temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 52°C, 1 minutes at 72°C; and final polymerization: 7 minutes at 72°C.
  • Taq DNA polymerase corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
  • Electrocompetent cells of E. coli strain MGl 655 l ⁇ cP containing pKD46 plasmid were obtained as described in Example 1. Electroporation was performed with 70 ⁇ l of cells and »300 ng of PCR product. After electroporation cells were incubated in 1 ml of SOC medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) at 37°C during 2.5 hours, then cells were spreaded on plates with L-agar containing 50 mg/1 kanamicin and grown at 37°C to select Km R - recombinants. To eliminate pKD46 plassmid, cells were spreaded on LB agar to get separate colonies at 37°C and Km R Ap s variants were selected.
  • Target double stranded oligonucleotide duplex was obtained by PCR using partialy overlapped primers Pl 6 (SEQ ID NO: 79) and Pl 7 (SEQ ID NO: 80).
  • Primers P16 and Pl 7 contain nucleotides in the overlapping region necessary for desired substitution in the protein product of adhE gene and 36 nucleotide non-overlapping regions necessary for integration of duplex into chromosome.
  • temperature profile was used for PCR: denaturation at 95°C during 30 seconds; 25 cycles: 15 seconds at 95°C, 15 seconds at 55°C, 30 seconds at 72°C; and final polymeerization: 3 minutes at 72°C.
  • Pfu DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
  • Electrocompetent cells of E. coli strain MGl 655 l ⁇ cP containing pKD46 plasmid were obtained as described in Example 1. Electroporation was performed with 70 ⁇ l of cells and «500 ng of PCR product. After electroporation cells were incubated in 1 ml of SOC medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) at 37°C during 2.5 hours, then cells were spreaded on plates with M9-agar medium containing 1 mM IPTG and 2% of ethanol as a sole carbon source and grown at 37°C to select + -recombinants. To eliminate pKD46 plassmid, cells were spreaded on LB agar to get separate colonies at 37°C and Km Ap variants were selected.
  • Example 3 Construction of E. coli strain with changed regulation of fadE gene expression. 1. Integration of hybrid regulatory region containing promoter and SD ⁇ io into chromosome of E. coli upstream of coding region of fadE gene. Construction of strain MG 1655 ⁇ ⁇
  • promoter hereinafter mentioned as jointed to SD sequence of ⁇ lO gene from phage T7 was integrated upstream of coding region of fadE gene in the chromosome of E. coli strain MG 1655 lacP using method developed by Datsenko, K.A. and Wanner, B. L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) also known as "Red-driven integration". Artificial DNA fragment integrated into chromosome also contained Cm R marker encoded by cat gene.
  • DNA fragment containing BgIU restriction site, promoter, SD sequence of ⁇ lO gene from phage T7 and 36 nucleotides complementary 5 '-end of coding region of fadE gene was obtained by PCR.
  • the DNA fragment was obtained in two stages. At first, DNA fragment containing restriction site, promoter and part of SD sequence of ⁇ lO gene from phage T7 was obtained by PCR using plasmid [Skorokhodova A. Yu. et al, 2006, Biotekhnologiya (Rus) 3, 6-16.].
  • PCR was performed using primers P5 (S ⁇ Q ID NO: 68) and P6 (S ⁇ Q ID NO: 69).
  • Primer P5 contains restrictin site and region homolous to 5 '-end of P promoter.
  • Primer P6 contains part of SD sequence of ⁇ lO gene from phage T7 and region complementary to 3 '-end of promoter.
  • temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 55°C, 30 seconds at 72°C; and final polymerization: 7 minutes at 72°C.
  • Pfu DNA polymerase corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
  • DNA fragment containing Cm marker encoded by cat gene was obtained by PCR using primers Pl 9 (SEQ ID NO: 82) and P9 (SEQ ID NO: 72) and pMWl 18-attL-Cm-attR plasmid as template.
  • Primer P19 contains 36 hucleotides homologous to DNA fragment upstream of coding region of fadE gene and 28 nucleotides complementary to DNA fragment located at 3 '-end o ⁇ attR region.
  • Primer P9 contains BgIW restriction site and 28 nucleotides homologous to the 5 '-end of attL region.
  • Product obtained after ligation was amplified by PCR using primers P19 (SEQ ID NO: 82) H Pl 8 (SEQ ID NO: 81). Following temperature profile was used for PCR: denaturation at 98°C during 1 minute; 30 cycles: 15 seconds at 98°C, 15 seconds at 55°C, 1 minute at 72°C; and final polimerization: 7 minutes at 72°C. Phusion DNA polymease and corresponding buffer from Finnzymes; and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
  • Electrocompetent cells of E. coli strain MG1655 containing pKD46 plasmid were obtained as described in Example 1. Electroporation was performed with 70 ⁇ l of cells and «300 ng of PCR product. After electroporation cells were incubated in 1 ml of SOC medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) at 37°C during 2.5 hours, then cells were spreaded on plates with L-agar containing 30 mg/1 chloramphenicol and grown at 37°C to select Cm R - recombinants. To eliminate pKD46 plassmid, cells were spreaded on LB agar to get separate colonies at 37°C and Cm R Ap s variants were selected.
  • Cm resistance gene ⁇ cat gene was eliminated from chromosome of strain MG 1655 using int-xis system.
  • Strain MG 1655 lacf R L was transformed with pMWts-Int/Xis plasmid (WO 2007013638; RU 2005123423). Selection of transformed clones was performed on LB agar medium containing ampicillin (100 mg/1). Plates were incubated during night at 30°C.
  • the cat gene was eliminated from transformed clones by culturing separate colonies at 37°C (at that termerature Cits repressor is partially inactivated while int/xis genes is activated) followed by selection of Cm Ap variants.
  • Cm Ap variants were healed from helper plasmid pMWts-Int/Xis containing temperature sensitive replicon by second spreading to get seperate colonies at 37°C followed by selcetion of Cm s Ap s clones.
  • strain MGl 655 lacf or abbreviated as MGl 655 l ⁇ cf was obtained.
  • Example 4 Construction of E. coli strain with changed regulation of fadB gene expression. 1. Integration of hybrid regulatory region containing promoter and into chromosome of E. coli upstream of coding region of fadB gene. Construction of strain MG 1655 ⁇ ⁇ /
  • promoter hereinafter mentioned as jointed to SD sequence of ⁇ lO gene from phage T7 was integrated upstream of coding region of fadB gene in the chromosome of E. coli strain MG 1655 using method developed by Datsenko, K. A. and Wanner, B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) also known as "Red- driven integration". Artificial DNA fragment integrated into chromosome also contained Cm R marker encoded by cat gene.
  • DNA fragment containing restriction site, promoter, SD sequence of ⁇ lO gene from phage T7 and 36 nucleotides complementary 5'-end of coding region of fadB gene was obtained by PCR.
  • the DNA fragment was obtained in two stages. At first, DNA fragment containing BgIU restriction site, promoter and part of SD sequence of ⁇ lO gene from phage T7 was obtained by PCR using plasmid [Skorokhodova A. Yu. et al, 2006, Biotekhnologiya (Rus) 3, 6-16.].
  • PCR was performed using primers P5 (S ⁇ Q ID NO: 68) and P6 (S ⁇ Q ID NO: 69).
  • Primer P5 contains Bgl ⁇ l restrictin site and region homolous to 5 '-end of promoter.
  • Primer P6 contains part of SD sequence of ⁇ lO gene from phage T7 and region complementary to 3 '-end of promoter.
  • temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 55°C, 30 seconds at 72°C; and final polymerization: 7 minutes at 72°C.
  • Pfu DNA polymerase corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
  • PCR product purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to the recommendations of manufacturer was used as template in the next round of PCR.
  • Primers P5 SEQ ID NO: 68
  • P22 SEQ ID NO: 85
  • Primer P22 contains region complementary to 3 '-end of promoter, SD sequence of ⁇ lO gene from phage T7 and 36 nucleotides from opened reading frame of fadB gene.
  • temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 55°C, 40 seconds at 72°C; and final polymerization: 7 minutes at 72°C.
  • Pfu DNA polymerase corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used. In para e , secon s age or construction o ragment was pe ⁇ orme .
  • NA fragment containing Cm R marker encoded by cat gene was obtained by PCR using primers P23 (SEQ ID NO: 86) and P9 (SEQ ID NO: 72) and pMW1 18-attL-Cm-attR plasmid as template.
  • Primer P23 contains 36 hucleotides homologous to DNA fragment upstream of coding region of fadB gene and 28 nucleotides complementary to DNA fragment located at 3 '-end o ⁇ region.
  • Primer P9 contains BgIW restriction site and 28 nucleotides homologous to the 5'-end of region. Following temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 52 °C, 1 minute at 72°C; and final polymerization: 7 minutes at 72°C. Taq DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
  • Product obtained after ligation was amplified by PCR using primers P23 (SEQ ID NO: 86) H P22 (SEQ ID NO: 85). Following temperature profile was used for PCR: denaturation at 98°C during 1 minute; 30 cycles: 15 seconds at 98°C, 15 seconds at 55°C, 1 minute at 72°C; and final polimerization: 7 minutes at 72°C. Phusion DNA polymease and corresponding buffer from Finnzymes; and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
  • Electrocompetent cells of E. coli strain MG 1655 lacP containing pKD46 plasmid were obtained as described in Example 1. Electroporation was performed with 70 ⁇ l of cells and »300 ng of PCR product. After electroporation cells were incubated in 1 ml of SOC medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) at 37°C during 2.5 hours, then cells were spreaded on plates with L-agar containing 30 mg/1 chloramphenicol and grown at 37°C to select Cm R -recombinants. To eliminate pKD46 plassmid, cells were spreaded on LB agar to get separate colonies at 37°C and Cm R Ap S variants were selected.
  • Cm resistance gene ⁇ cat gene was eliminated from chromosome of strain MG 1655 ⁇ ⁇ :/ ⁇ using int- xis system.
  • Strain MGl 655 lacf ⁇ ttR-c ⁇ t- ⁇ ttL- was transformed with pMWts-Int/Xis plasmid (WO 2007013638; RU 2005123423). Selection of transformed clones was performed on LB agar medium containing ampicillin (100 mg/1). Plates were incubated during night at 30°C.
  • the cat gene was eliminated from transformed clones by culturing separate colonies at 37°C (at that termerature Cits repressor is partially inactivated while int/xis genes is activated) followed by selection of Cm s Ap R variants. Elimination of cat gene from chromosome of the strain was confirmed by PCR using locus specific primers P24 (SEQ ID NO: 87) and P25 (SEQ ID NO: 88). PCR conditions for the test were as described above in the Example 1. Lenght of PCR product obtained using cells with eliminated cat gene was 323 base pairs.
  • Cm s Ap R variants were healed from helper plasmid pMWts-Int/Xis containing temperature sensitive replicon by second spreading to get seperate colonies at 37°C followed by selection of Cm Ap clones.
  • strain MGl 655 l ⁇ /c i d eai ⁇ io , c jd ea ⁇ 4 ⁇ io:M or abbreviated as MG 1655 lacf was obtained.
  • Example 5 Construction of E. coli strain with changed regulation o ⁇ fadA gene expression. 1. Integration of hybrid regulatory region containing promoter and SD ⁇ io into chromosome of E. coli upstream of coding region of f gene. Construction of strain MG 1655
  • promoter hereinafter mentioned as jointed to SD sequence of ⁇ lO gene from phage T7 was integrated upstream of coding region of fadA gene in the chromosome of E. coli strain MGl 655 lacf P, ⁇ C- using method developed by Datsenko, K.A. and Wanner, B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) also known as "Red-driven integration". Artificial DNA fragment integrated into chromosome also contained Cm R marker encoded by cat gene.
  • DNA fragment containing BgIW restriction site, promoter, SD sequence of ⁇ lO gene from phage T7 and 36 nucleotides complementary 5 '-end of coding region o ⁇ f gene was obtained by PCR.
  • the DNA fragment was obtained in two stages. At first, DNA fragment containing BgIW restriction site, Prrc-ideai-4 promoter and part of SD sequence of ⁇ lO gene from phage T7 was obtained by PCR using plasmid [Skorokhodova A. Yu. et al, 2006, Biotekhnologiya (Rus) 3, 6-16.].
  • PCR was performed using primers P5 (SEQ ID NO: 68) and P6 (SEQ ID NO: 69).
  • Primer P5 contains BgIW restrictin site and region homolous to 5 '-end of Prrc-jdeai-4 promoter.
  • Primer P6 contains part of SD sequence of ⁇ lO gene from phage T7 and region complementary to 3 '-end of P promoter.
  • temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 55°C, 30 seconds at 72°C; and final polymerization: 7 minutes at 72°C.
  • Pfu DNA polymerase corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
  • PCR product purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to the recommendations of manufacturer was used as template in the next round of PCR.
  • Primers P5 SEQ ID NO: 68
  • P26 SEQ ID NO: 89
  • Primer P26 contains region complementary to 3'-end of P promoter, SD sequence of ⁇ lO gene from phage T7 and 36 nucleotides from opened reading frame of fadA gene.
  • temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 55°C, 40 seconds at 72°C; and final polymerization: 7 minutes at 72°C.
  • DNA polymerase corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
  • second stage of construction of DNA fragment was performed.
  • DNA fragment containing Cm R marker encoded by cat gene was obtained by PCR using primers P27 (SEQ ID NO: 90) and P9 (SEQ ID NO: 72) and pMW1 18-attL-Cm-attR plasmid as template.
  • Primer P27 contains 36 hucleotides homologous to DNA fragment upstream of coding region of fadA gene and 28 nucleotides complementary to DNA fragment located at 3 '-end of attR region.
  • Primer P9 contains BgIW restriction site and 28 nucleotides homologous to the 5'-end of attL region. Following temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 52 °C, 1 minute at 72°C; and final polymerization: 7 minutes at 72°C. Taq DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
  • Product obtained after ligation was amplified by PCR using primers P27 (SEQ ID NO: 90) H P26 (SEQ ID NO: 89). Following temperature profile was used for PCR: denaturation at 98°C during 1 minute; 30 cycles: 15 seconds at 98°C, 15 seconds at 55°C, 1 minute at 72°C; and final polimerization: 7 minutes at 72°C. Phusion DNA polymease and corresponding buffer from Finnzymes; and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
  • Electrocompetent cells of £ coli strain MG1655 lacP P containing pKD46 plasmid were obtained as described in Example 1. Electroporation was performed with 70 ⁇ l of cells and »300 ng of PCR product. After electroporation cells were incubated in 1 ml of SOC medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) at 37°C during 2.5 hours, then cells were spreaded on plates with L-agar containing 30 mg/1 chloramphenicol and grown at 37°C to select Cm R -recombinants. To eliminate pKD46 plasmid, cells were spreaded on LB agar to get separate colonies at 37°C and Cm R Ap S variants were selected.
  • Cm resistance gene (cat gene) was eliminated from chromosome of strain MG 1655 ⁇ ⁇ ⁇ attR-Cat-attL- Pr rc- i deal - 4 - ofadA using int-xis system.
  • Strain MGl 655 fadE was transformed with pMWts- Int/Xis plasmid (WO 2007013638; RU 2005123423). Selection of transformed clones was performed on LB agar medium containing ampicillin (100 mg/1). Plates were incubated during night at 30°C.
  • the cat gene was eliminated from transformed clones by culturing separate colonies at 37°C (at that termerature Cits repressor is partially inactivated while int/xis genes is activated) followed by selection of Cm Ap variants. Elimination of cat gene from chromosome of the strain was confirmed by PCR using locus specific primers P28 (SEQ ID NO: 91) and P29 (SEQ ID NO: 92). PCR conditions for the test were as described above in the Example 1. Lenght of PCR product obtained using cells with eliminated cat gene was 338 base pairs.
  • Cm s Ap R variants were healed from helper plasmid pMWts-Int/Xis containing temperature sensitive replicon by second spreading to get seperate colonies at 37°C followed by selcetion of Cm s Ap s clones.
  • strain MGl 655 f or abbreviated as MG1655 was obtained.
  • Example 6 Construction of E. coli strain with changed regulation o ⁇ atoB gene expression.
  • promoter hereinafter mentioned as jointed to SD sequence of ⁇ lO gene from phage T7 was integrated upstream of coding region of atoB gene in the chromosome of E. coli strain MG 1655 lacP using method developed by Datsenko, K. A. and Wanner, B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) also known as "Red-driven integration". Artificial DNA fragment integrated into chromosome also contained Cm R marker encoded by cat gene.
  • DNA fragment containing BgM restriction site, promoter, SD sequence of ⁇ lO gene from phage T7 and 36 nucleotides complementary 5 '-end of coding region of atoB gene was obtained by PCR.
  • the DNA fragment was obtained in two stages. At first, DNA fragment containing BgIW restriction site, Pr rc -j dea i-4 promoter and part of SD sequence of ⁇ lO gene from phage T7 was obtained by PCR using plasmid [Skorokhodova A. Yu. et al, 2006, Biotekhnologiya (Rus) 3, 6-16.].
  • PCR was performed using primers P5 (SEQ ID NO: 68) and P6 (SEQ ID NO: 69).
  • Primer P5 contains restrictin site and region homolous to 5 '-end of promoter.
  • Primer P6 contains part of SD sequence of ⁇ lO gene from phage T7 and region complementary to 3 '-end of promoter.
  • temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 55°C, 30 seconds at 72°C; and final polymerization: 7 minutes at 72°C.
  • Pfu DNA polymerase corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
  • DNA fragment containing Cm R marker encoded by cat gene was obtained by PCR using primers P31 (SEQ ID NO: 94) and P9 (SEQ ID NO: 72) and pMW1 18-attL-Cm-attR plasmid as template.
  • Primer P31 contains 36 hucleotides homologous to DNA fragment upstream of coding region of atoB gene and 28 nucleotides complementary to DNA fragment located at 3 '-end of attR region.
  • Primer P9 contains BgIW restriction site and 28 nucleotides homologous to the 5 '-end of attL region.
  • Product obtained after ligation was amplified by PCR using primers P31 (SEQ ID NO: 94) H P30 (SEQ ID NO: 93). Following temperature profile was used for PCR: denaturation at 98°C during 1 minute; 30 cycles: 15 seconds at 98°C, 15 seconds at 55°C, 1 minute at 72°C; and final polimerization: 7 minutes at 72°C. Phusion DNA polymease and corresponding buffer from Finnzymes; and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
  • ⁇ lectrocompetent cells of E. coli strain MG1655 lac containing pKD46 plasmid were obtained as described in Example 1. Electroporation was performed with 70 ⁇ l of cells and «300 ng of PCR product. After electroporation cells were incubated in 1 ml of SOC medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) at 37°C during 2.5 hours, then cells were spreaded on plates with L-agar containing 30 mg/1 chloramphenicol and grown at 37°C to select Cm R -recombinants. To eliminate pKD46 plasmid, cells were spreaded on LB agar to get separate colonies at 37°C and Cm R Ap s variants were selected.
  • Cm resistance gene (cat gene) was eliminated from chromosome of strain MGl 655 ⁇ io using system.
  • the cat gene was eliminated from transformed clones by culturing separate colonies at 37°C (at that termerature Cits repressor is partially inactivated while int/xis genes is activated) followed by selection of Cm Ap variants. Elimination of cat gene from chromosome of the strain was confirmed by PCR using locus specific primers P32 (SEQ ID NO: 95) and P33 (SEQ ID NO: 96). PCR conditions for the test were as described above in the Example 1. Lenght of PCR product obtained using cells with eliminated cat gene was 341 base pairs.
  • Cm s Ap R variants were healed from helper plasmid pMWts-Int/Xis containing temperature sensitive replicon by second spreading to get seperate colonies at 37°C followed by selcetion of Cm s Ap s clones.
  • strain MGl 655 lacf attB or abbreviated as MG1655 was obtained.
  • Example 7 Construction of E. coli strain in which activity of phosphoenolpyruvate carboxylase can be decreased.
  • O promoter hereinafter promoter
  • chromosome of strain MG 1655 lacP downstream of coding region of ppc gene in orientation opposite to direction of native transcription of the gene under control its native promoter.
  • Artificail DNA fragment integrated into the chromosome also contained Cm R marker encoded by cat gene.
  • DNA fragment integrated into corresponding region of bacterial chromosome was performed in several steps. First, DNA fragment containing BgIW restriction site, Pr rc -i dea i-4 promoter, SD sequence of ⁇ lO gene from phage T7 and 36 nucleotides including 22 nucleotides homologous to 3-end of coding region of ppc gene and 14 nucleotides homologous to region downstream of stop-codon of the gene was obtained by PCR. The DNA fragment was obtained in two stages.
  • DNA fragment containing BgIW restriction site, Pr rc -i dea i-4 promoter and part of SD sequence of ⁇ lO gene from phage T7 was obtained by PCR using plasmid [Skorokhodova A. Yu. et al, 2006, Biotekhnologiya (Rus) 3, 6-16.].
  • PCR was performed using primers P5 (SEQ ID NO: 68) and P34 (SEQ ID NO: 97).
  • Primer P5 contains BgIW restrictin site and region homolous to 5'-end of P promoter.
  • Primer P34 contains 36 nucleotides necessary for integration into chromosome and region complementary to 3-end of promoter.
  • DNA fragment containing Cm R marker encoded by cat gene was obtained by PCR using primers P35 (SEQ ID NO: 98) and P9 (SEQ ID NO: 72) and pMW118-attL-Cm-attR plasmid as template.
  • Primer P35 contains 36 hucleotides homologous to DNA fragment downstream of coding region of ppc gene and 28 nucleotides complementary to DNA fragment located at 3 '-end of attR region.
  • Primer P9 contains BgIW restriction site and 28 nucleotides homologous to the 5'-end of attL region.
  • Product obtained after ligation was amplified by PCR using primers P34 (SEQ ID NO: 97) and P35 (SEQ ID NO: 98). Following temperature profile was used for PCR: denaturation at 98°C during 1 minute; 30 cycles: 15 seconds at 98°C, 15 seconds at 55°C, 1 minute at 72°C; and final polimerization: 7 minutes at 72°C. Phusion DNA polymease and corresponding buffer from Finnzymes; and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
  • Electrocompetent cells of E. coli strain MGl 655 l containing pKD46 plasmid were obtained as described in Example 1. Electroporation was performed with 70 ⁇ l of cells and »300 ng of PCR product. After electroporation cells were incubated in 1 ml of SOC medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) at 37°C during 2.5 hours, then cells were spreaded on plates wi -agar con ainin oramp enico an n a o se ec m - recombinants. To eliminate pKD46 plassmid, cells were spreaded on LB agar to get separate colonies at 37°C and Cm R Ap s variants were selected.
  • Cm resistance gene (cat gene) was eliminated from chromosome of strain MG 1655 E R using int-xis system.
  • Strain MG1655 P E was transformed with p plasmid (WO 2007013638; RU 2005123423). Selection of transformed clones was performed on LB agar medium containing ampicillin (100 mg/1). Plates were incubated during night at 30°C.
  • the cat gene was eliminated from transformed clones by culturing separate colonies at 37°C (at that termerature Cits repressor is partially inactivated while int/xis genes is activated) followed by selection of Cm s Ap R variants.
  • Cm s Ap R variants were healed from helper plasmid pMWts-Int/Xis containing temperature sensitive replicon by second spreading to get seperate colonies at 37°C followed by selection of Cm Ap clones.
  • strain MGl 655 attB or abbreviated as MG 1655 P was obtained.
  • Example 8 Production of 1-butanol by E. coli cells.
  • Incubation of resulted cell cultures is performed during 24 hours in 20 x 200 mm test tubes with ventilated plugs at 37°C on shaker (200 rpm) with or without presence of 1 mM IPTG.
  • Amount of 1-butanol in culture medium is determined by method of gas chromatography.
  • Amount of 1-butanol in culture medium is determined by method of gas chromatography as described above.

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Abstract

The present invention is related to microbiological industry particularly to the method for production of 1-using microorganism modified so that at least one of enzymes from pathway of fatty acids β-oxidation is involved into 1-butanol biosynthesis.

Description

A MICROORGANISM PRODUCING 1-BUTANOL AND A METHOD FOR PRODUCING 1-BUTANOL
Field of invention.
The present invention is related to microbiological industry, particularly to a method for producing 1-butanol using microorganism modified so that at least one of enzymes from pathway of fatty acids β-oxidation is involved into 1-butanol.
Description of the related art.
Butanol is four carbon alcohol widely used in the industry. Butanol is used as solvent in the paint and varnish industry, in resin and plasticizer production, in the synthesis of wide amount of organic compounds, particularly pesticides, perfumes, medicaments. At the time butanol is considered as an attractive compound which can be used as additive to motor fuel or even its complete substituent. Use of butanol as fuel both in mix with gasoline and in pure state does not require modification of existing combustion. Enegry value of butanol is 29 MJ/litre, octane rating is 96. The characterictics are rather close to corresponding characteristics of gasoline - 32 MJ/litre and 91-99, respectively (Durre P., 2007 Biobutanol: An attractive biofuel. Biotechnol. J., 2(12): 1525-34). Butanol is not hygroscopic and hydrophobic. It can be mixed with gasoline in any proportion. Low corrosive characteristics of butanol allow to use already existing infrastructure developed for oil products for transportation and storage of butanol.
At the present time major amount of butanol (up to 5 million tons per year) is produced by methods of oil chemistry. Common methods of 1-butanol productins include hydrogenation of crotone aldehyde and oxosynthesis from propylene (Loktev et al, "High fatty " Moscow, Khikiya (Rus), 1970). Disadvantages of above processes are high energy spending, aggressive conditions for the synthesis, requirement of oil products as raw materials.
Butanol can be obtained by biotransformation of wild spectrum of organic compounds using different microorganisms. Corresponding alcohls can be obtained using Pseudomonas strains (JP63017695). Aliphatic saturated hydrocarbons (from dibasic to twentybasic) can be oxydazied by monooxygenase of Rhodococcus ruber to yield corresponding aclohols (patent applicatins US2002028492, EPl 149918). Thus, in case of butane products can be 1-butanol (95%) and 2-butanol (5%).
Fermentation of different oligo- and monosaccharides containing pentose and hexose by different species of Clostridium, such as C. acetobutylicum, C. beijerinckii, C. saccharoperbutylacetonicum and others, leads to formation of acetone, butanol and ethanol mixture. Acetone-butanol-ethanol fermentation (ABE process) using different Clostridium strains have been initailly industrially used for acetone production and then for butanol production up to 1980th years (Zverlov V.V. et al., 2006 Bacterial acetone and butanol production by industrial fermentation in the Soviet Union: Use of hydrolyzed agricultural waste for biorefinery. Appl. Microbiol. Biotechnol., 71, 587-597). Production was stopped due to the economical reasons and complexity of the process. In the typical ABE process biomass growth is accompanied with acid accumulation, mainly acetic and butyric acid, and in low degree lactic and propionic acids. Acid accumulation leads to decrease of medium pH and to the metabolic shift inducing organic solvent production. Accumulated acids are re-utilized with formation of corresponding alcohols. The last process requiers utilization of additional molecule of sugar substrate yielding one molecule of aceton per each re-utilized molecule of the acid. At the end of the standard ABE process, aceton, butanol and ethanol is accumulated in the ratio 3:6: 1. Typical mass yield of alcohols per glucose usually does not exceed 32%. Concentration of butanol in the medium is usually no more then 1,5 - 2,0%.
Attempts to improve standard ABE process were undertaken earlier and included several approaches. First one is the traditional selection of strains mostly producing butanol (for example US patent 6358717). Another approach is optimization or modification of existing two- step ABE process. For example, it was proposed to separate processes of acid formation and acid utilization (US patent 5753474). During the first step constructed Clostridium tyrobutyricum strain with inactivated pta gene produces butyric acid and in the second step non- modified acetobutylicum strain utilizes earlier obtained butyrate accompanied with carbohydrates yielding butanol accumulation. Acetic acid, aceton and ethanol are by-products of the process (Final Report on the Production of Butyric Acid and Butanol from Biomass, which is based on work performed under: Contract No.: DE-F-G02-00ER86106, http://www.eere.energy.gov/afdc/pdfs/843183.pdf).
Another approach was focused on the modification of metabolism of natural butanol producing Clostridium bacteria using rational design and genetic engineering methods (Nair R. et al., 1999, Regulation of the sol Locus Genes for Butanol and Acetone Formation in Clostridium acetobutylicum ATCC 824 by a Putative Transcriptional Repressor. J. Bacteriol. 181(1): 319-330; Desai R. and E. Papoutsakis, 1999, Antisense RNA Strategies for Metabolic Engineering of Clostridium acetobutylicum, Appl. Environ. Microbiol. 65(3): 936-945). However limited number of genetic methods and weak knowledge of metabolism of Clostridium did not allow to fulfill such approach.
The main disadvantage of traditional ABE process is low yield of butanol per utilized substrate caused by unavoidable formation of acetone. Maximum yeild could be achieved only by direct conversion of substrate to the target product avoiding step of formation and re- utilization of acids. Genes encoding for enzymes of butanol synthesis from Clostridium bacteria are known (see for example, Durre P. et al., 2002, Transcriptional Regulation of Solventogenesis in Clostridium acetobutylicum. J. MoI. Microbiol. Biotechnol., 4(3): 295-300). And several research group tried to realize one-step process of conversion of carbohydrates to 1- butanol by cloning corresponding genes from Clostridium bacteria into microorganisms more convenient for genetic manipulations with already investigated metabolism. E. coli is one of them (WO2007041269; Atsumi S. et al., Metabolic engineering of Escherichia coli for 1- butanol production. Metab. Eng., 2007, doi:10.1016/j.ymben.2007.08.003; Inui M. et al., Expression of Clostridium acetobutylicum butanol synthetic genes in Esherichia coli. Appl. Microbiol. Biotechnol., 2008, 77(6): 1305-16). B. subtilis is also one of them (WO2007041269).
New method for biosynthesis of high alcohols, particularly 1 -butanol, through decarboxylation 2-ketoacids was proposed and realized in E. coli by cloning heterologous kivD gene from Lactococcus lactis (Atsumi S. et al., 2008 Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature, doi:10.1038/nature06450).
However at the time there are no reports describing use of enzymes from pathway of fatty acids β-oxidation for production of 1 -butanol by fermentation.
Disclosure of the Invention
The goal of the present invention is providing a microorganism producing 1 -butanol by fermentation and a method for producing 1 -butanol using such microorganism.
Such goal was achieved by constructing 1 -butanol producing microorganism modified so that at least one of enzymes from pathway of fatty acids β-oxidation is involved into 1 -butanol biosynthesis.
It is an object of the present invention to provide a 1 -butanol producing microorganism modified so that at least one of enzymes from pathway of fatty acids β-oxidation is involved into 1 -butanol biosynthesis.
It is a further object of the present invention to provide the microorganism as described above, wherein the enzyme from pathway of fatty acids β-oxidation is native or heterogeneous for the microorganism.
It is a further object of the present invention to provide the microorganism as described above, wherein the enzyme from pathway of fatty acids β-oxidation is selected from group consisting of acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxyacetyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase. It is a further object of the present invention to provide the microorganism as described above, wherein acyl-CoA dehydrogenase comprises amino acid sequence selected from any of SEQ ID NO: 1-17.
It is a further object of the present invention to provide the microorganism as described above, wherein enoyl-CoA hydratase comprises amino acid sequence selected from any of SEQ ID NO: 18-38.
It is a further object of the present invention to provide the microorganism as described above, wherein 3-hydroxyacetyl-CoA dehydrogenase comprises amino acid sequence selected from any of SEQ ID NO: 18, 19, 23-25, 27, 31-33, 39-44.
It is a further object of the present invention to provide the microorganism as described above, wherein 3-ketoacyl-CoA thiolase comprises amino acid sequence selected from any of SEQ ID NO: 45-61.
It is a further object of the present invention to provide the microorganism as described above, wherein the microorganism is modified to have increased activity of one or several said enzymes in comparison to non-modifieid microorganism.
It is a further object of the present invention to provide the microorganism as described above, wherein activity of one or several enzymes is increased by increasing amount of corresponding mRNA, increasing amount of the enzyme, or increasing specific enzyme activity.
It is a further object of the present invention to provide the microorganism as described above, wherein amount of corresponding mRNA is increased by increasing copy number of gene or by enhancing gene expression due to use of potent promoter, removing repression or increasing mRNA stability.
It is a further object of the present invention to provide the microorganism as described above, wherein amount of enzyme is increased by enhancing translation of corresponding mRNA due to modification of ribosome binding site or increasing its stability.
It is a further object of the present invention to provide the microorganism as described above, wherein specific enzyme activity is increased by introducing mutatins into amino acid sequence of the enzyme.
It is a further object of the present invention to provide the microorganism as described above, wherein said microorganism is selected from group consisting of bacteria, yeast and fungi.
It is a further object of the present invention to provide the microorganism as described above, wherein said microorganism belongs to the genus Clostridium, Escherichia, Salmonella, Shigella, Pseudomonas, Zymomonas, Bacillus, Lactobacillus, Enterococcus, Klebsiella, Corynebacterium, Brevibacterium, Streptomyces, Pichia, Candida or Sacchoromyces. It is a further object of the present invention to provide the microorganism as described above, wherein said microorganism is Clostridium acetobutylicum or Clostridium beijerinckii.
It is a further object of the present invention to provide the microorganism as described above, wherein said microorganism is Escherichia coli.
It is a further object of the present invention to provide the microorganism as described above, wherein said microorganism is Salmonella enterica.
It is a further object of the present invention to provide the microorganism as described above, wherein said microorganism is Shigella flexneri.
It is a further object of the present invention to provide the microorganism as described above, wherein said microorganism is Pseudomonas aeruginosa or Pseudomonas putida.
It is a further object of the present invention to provide the microorganism as described above, wherein said microorganism is Zymomonas mobilis.
It is a further object of the present invention to provide the microorganism as described above, wherein said microorganism is Bacillus subtilis or Bacillus amyloliquefaciens .
It is a further object of the present invention to provide the microorganism as described above, wherein said microorganism is Lactobacillus plantarum.
It is a further object of the present invention to provide the microorganism as described above, wherein said microorganism is Enterococcus faecium.
It is a further object of the present invention to provide the microorganism as described above, wherein said microorganism is Corynebacterium glutamicum or Brevibacterium flavum.
It is a further object of the present invention to provide the microorganism as described above, wherein said microorganism is Streptomyces coelicolor.
It is a further object of the present invention to provide the microorganism as described above, wherein said microorganism is Pichiapastoris.
It is a further object of the present invention to provide the microorganism as described above, wherein said microorganism is Saccharomyces cerevisiae.
It is a further object of the present invention to provide the microorganism as described above, wherein said microorganism is additionally modified to increase intracellular amount of acetyl-CoA.
It is a further object of the present invention to provide the microorganism as described above, wherein intracellular amount of acetyl-CoA is increased by regulated decrease of phosphoenolpyruvate carboxylase activity.
It is a further object of the present invention to provide a method for producing 1-butanol including cultivation of microorganism according to claim 1 in culture medium and collecting produced an 1-butanol from the medium. It is a further object of the present invention to provide the method as described above, wherein process of microorganism cultivation is aerobic.
It is a further object of the present invention to provide the method as described above, wherein process of microorganism cultivation is anaerobic.
It is a further object of the present invention to provide the method as described above, wherein process of microorganism cultivation is microaerobic.
It is a further object of the present invention to provide the method as described above, wherein process of microorganism cultivation includes aerobic and anaerobic steps. The present invention is described in detail below.
Detailed Description of the Preferred Embodiments 1. 1-Butanol biosynthesis.
The only native producers of butanol is bacterium belonging to the genus Clostridium, such as C. acetobutylicum, C. beijerinckii, C. saccharoperbutylacetonicum and others, in which biosynthesis of organic solvents (acetone, ethanol, butanol) is directly coupled with their vital functions.
Butanol biosynthetic pathway of such organisms as well as genes encoding enzymes of that pathway are known. The pathway starts from formation of acetoacetyl-CoA from two molecules of acetyl-CoA and finishes after several steps with reduction of butyril-CoA to corresponding aldehyde and 1 -butanol. Such pathway is accompanied with oxidation of three NADH molecules and one NADPH molecule (Jones, D. and Woods, R, Microbiol. Rev., 1986, 50 (4): 484-524).
It is also known that microorganisms carrying definite regulatory mutations are able to utilize 1 -butanol even as a sole carbon source. Such utilization of 1 -butanol includes formation of butyril-CoA by alcohol- and aldehyde dehydrogenases followed by its convertion into two acetyl-CoA molecules in the pathway of fatty acids β-oxidation (Neidhardt, F. C. et al. Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington D.C., chapter 21).
Fatty acids β-oxidation includes several reactions catalyzed by acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxyacetyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase, and accompanied with NAD+ reduction.
Acyl-CoA dehydrogenase is the enzyme able to catalyze reversed reaction of conversion acyl-CoA (particularly, butyril-CoA) to 2-enoyl-CoA (particularly, crotonyl-CoA): acyl-CoA + FAD <=> FADH2 + 2,3-dehydroacyl-CoA (2-enoyl-CoA). Such activity is classified as EC 1.3.99.-, particularly EC 1.3.99.3. Presence of acyl-CoA dehydrogenase activity can be determined by, for example, complementation of fadE62 mutation (inabilty to growth on the medium with fatty acids as a sole carbon source) using method described by Campbell, J. W. and Cronan, J. E. Jr. (The enigmatic Escherichia coli fadE gene is yqfli, J. Bacteriol., 2002, 184(13):3759-64). Examples of enzyme having acyl-CoA dehydrogenase activity include FadE (synonims B0221, YafH, FadF) (SEQ ID NO: 1) and YdiO (SEQ ID NO: 2) from Escherichia coli, FadE (SEQ ID NO: 3), YdiO (SEQ ID NO: 4) and STM0857 (SEQ ID NO: 5) from Salmonella typhimurium, FadE (SEQ ID NO: 6), PP 2437 (SEQ ID NO: 7) and PP 4948 (SEQ ID NO: 8) from Pseudomonas putida, MmgC (SEQ ID NO: 9), AcdA (SEQ ID NO: 10) and YngJ (SEQ ID NO: 11) from Bacillus subtilis, SCO2779 (SEQ ID NO: 12), SCO5693 (SEQ ID NO: 13), SCO6469 (SEQ ID NO: 14), SCO3051 (SEQ ID NO: 15) and SCO1690 (SEQ ID NO: 16) from Streptomyces coelicolor, POXl(SEQ ID NO: 17) from Saccharomyces cerevisiae.
Enoyl-CoA hydratase is the enzyme able to catalyze reversed reaction of conversion 2- enoyl-CoA (particularly, crotonyl-CoA) to 3-hydroxyacyl-CoA (particularly, 3-hydroxybutyril- CoA): H2O + 2-enoyl-CoA <=> 3-hydroxyacyl-CoA. Such activity is classified as EC 4.2.1.17. Presence of enoyl-CoA hydratase activity can be determined, for example, using method described by Binstock, J. F. and Schulz, H. (Fatty acid oxidation complex from Escherichia coli, Methods Enzymol. 1981 , 71 Pt C; 403-11). Examples of enzyme having enoyl-CoA hydratase activity include FadB (synonims B3846, OldB) (SEQ ID NO: 18), FadJ (SEQ ID NO: 19), PaaG (SEQ ID NO: 20), PaaF (SEQ ID NO: 21) and MaoC (SEQ ID NO: 22) from Escherichia coli, FadB (SEQ ID NO: 23) and FadJ (SEQ ID NO: 24) from Salmonella typhimurium, FadB (SEQ ID NO: 25) and PhaL (SEQ ID NO: 26) from Pseudomonas putida, YusL (SEQ ID NO: 27), YngF (SEQ ID NO: 28), YsiB (SEQ ID NO: 29) and YhaR (SEQ ID NO: 30) from Bacillus subtilis, NCgl0882 (SEQ ID NO: 31) from Corynebacterium glutamicum, SCO6732 (SEQ ID NO: 32), SCO6789 (SEQ ID NO: 33), SCO5144 (SEQ ID NO: 34), SCO5979 (SEQ ID NO: 35), SCO0364 (SEQ ID NO: 36), SCO4384 (SEQ ID NO: 37) and SCO4930 (SEQ ID NO: 38) from Streptomyces coelicolor.
3-Hydroxyacetyl-CoA dehydrogenase is the enzyme able to catalyze reversed reaction of conversion 3-hydroxyacyl-CoA (particularly, 3-hydroxybutyril-CoA) to 3-ketoacyl-CoA (particularly, acetoacyl-CoA): NAD+ + 3-hydroxyacyl-CoA <=> NADH + 3-ketoacyl-CoA. Such activity is classified as EC 1.1.1.35 or EC 1.1.1.157. Presence of 3-hydroxyacetyl-CoA dehydrogenase activity can be determined, for example, using method described by Binstock, J. F. and Schulz, H. (Fatty acid oxidation complex from Escherichia coli, Methods Enzymol. 1981, 71 Pt C; 403-1 1). Examples of enzyme having 3-hydroxyacetyl-CoA dehydrogenase activity include FadB from Escherichia coli (synonims B3846, OldB) (SEQ ID NO: 18), FadJ (SEQ ID NO: 19) and PaaH (SEQ ID NO: 39) from Escherichia coli, FadB (SEQ ID NO: 23) and FadJ (SEQ ID NO: 24) from Salmonella typhimurium, FadB (SEQ ID NO: 25), PP_0302 (SEQ ID NO: 40), PaaC (SEQ ID NO: 41) and PaaH (SEQ ID NO: 42) from Pseudomonas putida, YusL (SEQ ID NO: 27) and MmgB (SEQ ID NO: 43) from Bacillus subtilis, SCO6732 (SEQ ID NO: 32), SCO6789 (SEQ ID NO: 33) and SCO3834 (SEQ ID NO: 44) from Streptomyces coelicolor.
3-Ketoacyl-CoA thiolase is the enzyme able to catalyze reversed reaction of conversion 3- ketoacyl-CoA (particularly, acetoacyl-CoA) to acyl-CoA (particularly, acetyl-CoA): acyl-CoA + acetyl-CoA <=> 3-ketoacyl-CoA + coenzyme A. Such activity is classified as E.C. 2.3.1.16 or E. C. 2.3.1.9. Presence of 3-ketoacyl-CoA thiolase activity can be determined, for example, using method described by Binstock, J. F. and Schulz, H. (Fatty acid oxidation complex from Escherichia coli, Methods Enzymol. 1981, 71 Pt C; 403-1 1). Examples of enzyme having 3- ketoacyl-CoA thiolase activity include FadA (synonims B3845, OldA) (SEQ ID NO: 45), Fadl (SEQ ID NO: 46), AtoB (SEQ ID NO: 47) and YqeF (SEQ ID NO: 48) from Escherichia coli, FadA (SEQ ID NO: 49), Fadl (SEQ ID NO: 50) and YqeF (SEQ ID NO: 51) from Salmonella typhimurium, FadA (SEQ ID NO: 52) and AtoB (SEQ ID NO: 53) from Pseudomonas putida, YusK (SEQ ID NO: 54) and MmgA (SEQ ID NO: 55) from Bacillus subtilis, NCgl2309 (SEQ ID NO: 56) from Coryne bacterium glutamicum, SCO6027 (SEQ ID NO: 57), SCO5399 (SEQ ID NO: 58) and SCO3079 (SEQ ID NO: 59) from Streptomyces coelicolor, POTl (SEQ ID NO: 60) and ERGlO (SEQ ID NO: 61) from Saccharomyces cerevisiae.
Since there may be some differences in amino acids sequences between the genera or strains of microorganism according to the present invention, the particular enzyme is not limited by protein sequence depicted in Sequence Listing but also includes variants of the proteins. The phrase "protein variant" as used in the present invention means proteins which have changes in the sequences, whether they are deletions, insertions, additions, or substitutions of amino acids which are not deteriorate activity of the protein. The number of changes in the variant proteins depends on the position or the type of amino acid residues in the three dimensional structure of the protein. It may be 1 to 30, preferably 1 to 15, and more preferably 1 to 5 amino acid residues. These changes in the variants can occur in regions of the protein which are not critical for the three dimensional structure of the protein and its function. This is because some amino acids have high homology to one another so the three dimensional structure is not affected by such a change. Therefore "protein variant" includes proteins having homology no less then 80%, preferably no less then 90%, and more preferably no less then 95% regarding to whole amino acid sequence depicted in the Sequence Listing provided that activity of the protein is not deteriorated. Homology between two amino acid sequences can be determined using the well-known methods, for example, the computer program BLAST 2.0, which calculates three parameters: score, identity and similarity.
The substitution, deletion, insertion or addition of one or several amino acid residues should be conservative mutation(s) so that the activity is maintained. The representative conservative mutation is a conservative substitution. Examples of conservative substitutions include substitution of Ser or Thr for Ala, substitution of GIn, His or Lys for Arg, substitution of GIu, GIn, Lys, His or Asp for Asn, substitution of Asn, GIu or GIn for Asp, substitution of Ser or Ala for Cys, substitution of Asn, GIu, Lys, His, Asp or Arg for GIn, substitution of Asn, GIn, Lys or Asp for GIu, substitution of Pro for GIy, substitution of Asn, Lys, GIn, Arg or Tyr for His, substitution of Leu, Met, VaI or Phe for He, substitution of He, Met, VaI or Phe for Leu, substitution of Asn, GIu, GIn, His or Arg for Lys, substitution of He, Leu, VaI or Phe for Met, substitution of Trp, Tyr, Met, He or Leu for Phe, substitution of Thr or Ala for Ser, substitution of Ser or Ala for Thr, substitution of Phe or Tyr for Tip, substitution of His, Phe or Tip for Tyr, and substitution of Met, He or Leu for VaI.
Enzymes mentioned above as well as their variants can be chosed from proteins listed in
Figure imgf000010_0001
NP 391598 acyl-CoA dehydrogenase [Bacillus subtilis subsp. 10 subtilis str. 168] NP_389708 hypothetical protein BSUl 8260 [Bacillus subtilis 11 subsp. subtilis str. 168] NP_627009 acyl-CoA dehydrogenase [Streptomyces coelicolor 12 A3(2)] NP 629821 acyl-CoA dehydrogenase [Streptomyces coelicolor 13 A3(2)] NP_630552 acyl-CoA dehydrogenase {Streptomyces coelicolor 14 A3(2)] NP_627272 acyl-CoA dehydrogenase [Streptomyces coelicolor 15 A3(2)] NP 625964 acyl-CoA dehydrogenase [Streptomyces coelicolor 16 A3(2)] NP 01 1310 Fatty-acyl coenzyme A oxidase, involved in the 17 fatty acid beta-oxidation pathway; localized to the peroxisomal matrix; Pox Ip [Saccharomyces cerevisiae]
NP 418288 fused 3-hydroxybutyryl-CoA epimerase/delta(3)- cis-delta(2)-trans-enoyl-CoA isomerase/enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase [Escherichia coli str. K- 12 substr. MG 1655]
NP_416843 fused enoyl-CoA hydratase and epimerase and 19 isomerase/3-hydroxyacyl-CoA dehydrogenase [Escherichia coli str. K- 12 substr. MG 1655]
NP_415912 acyl-CoA hydratase [Escherichia coli str. K- 12 20 substr. MG 1655] NP_41591 1 enoyl-CoA hydratase-isomerase [Escherichia coli 21 str. K- 12 substr. MG 1655] NP_415905 fused aldehyde dehydrogenase/enoyl-CoA 22 hydratase [Escherichia coli str. K- 12 substr. MG1655]
NP_462868 multifunctional fatty acid oxidation complex 23 subunit alpha [Salmonella typhimurium LT2] NP_461330 multifunctional fatty acid oxidation complex 24 subunit alpha [Salmonella typhimurium LT2] NP_744285 multifunctional fatty acid oxidation complex 25 subunit alpha [Pseudomonas putida KT2440] NP_745413 bifunctional aldehyde dehydrogenase/enoyl-CoA 26 hydratase [Pseudomonas putida KT2440] NP_391 163 hypothetical protein BSU32840 [Bacillus subtilis 27 subsp. subtilis str. 168] NP 389704 enoyl-CoA hydratase [Bacillus subtilis subsp. 28 subtilis str. 168]
Figure imgf000012_0001
Figure imgf000013_0001
It is known that reactions catalyzed by the above enzymes are reversible as well as reactions catalyzed by alcohol- and aldehyde dehydrogenases.
Equilibrium of redox reaction (such as reactions of fatty acids β-oxidation catalyzed by alcohol- and aldehyde dehydrogenases) can be shifted to formation of desired product/substrate.
The equilibrium can be shifted, for example, by: a) changing the ratio of concentrations of substances involved into reaction; 6) changing the ratio of concentrations of oxidative and reducing equivalents required for passing reaction in direct or reversed, particularly in the case of biochemical reactions - NADH/NAD+, NADPH/NADP+, FADH2/FADH. In the case of catalyzed reactions equilibrium can be shifted by chanching activity of catalyst, particularly in the case of enzymes as biologic catalysts due to mutations changing specific activity of the enzyme.
In the case of several compiting catalytic reactions efficiency of each reaction can be changed by changing amount of corresponding catalyst (in the case of biochemical reactions by changing amount of enzyme).
Equilibrium also can be shifted by combination of several factors described above.
Thus, in definite conditions pathway of fatty acids β-can be used for butyril-CoA biosynthesis followed by formation of 1-butanol as a result of two consequtive reactions catalyzed by aldehyde dehydrogenase and alcohol dehydrogenase.
Aldehyde dehydrogenase is the enzyme able to catalyze reversed reaction of conversion acyl-CoA to corresponding aldehyde: NAD+ + coenzyme A + aldehyde <=> NADH + acyl- CoA. Such activity is generally classified as E. C. 1.2.1.3. However, in the particular case acetaldehyde dehydrogenase from E. coli (acetaldehyde dehydrogenase (acetylating)) is classified as 1.2.1.10. It is known that the enzyme can utilize acetaldehyde, butyraldehyde, propanaldehyde, glycolaldehyde and other compound as alternative substrates. Presence of aldehyde dehydrogenase activity can be determined by, for example, method described by Rudolph, F. B. et al (Coenzyme A-linked aldehyde dehydrogenase from Escherichia coli. I. Partial purification, properties, and kinetic studies of the enzyme, J. Biol. Chem., 1968; 243(21); 5539-45). Example of enzyme having activity of aldehyde dehydrogenase includes AdhE from Escherichia coli (synonims acetacldehyde:NAD+ oxidoreductase (CoA-acetylated), ACDH, coenzyme A dependent acetaldehyde dehydrogenase) which is homopolimer with 3 Fe2+- dependent catalytic activities: alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate- formate lyase-deactivase (SEQ ID NO: 62).
Alcohol dehydrogenase is the enzyme able to catalyze reversed reaction of conversion alcohol to aldehyde or ketone: alcohol + NAD+ <=> NADH + aldehyde or ketone. Such activity is classified as E. C. 1.1.1.1. It is known that the enzyme can utilize ethanol, n-butanol, n- propanol and other compounds as alternative substrates. Presence of alcohol dehydrogenase activity can be determined by, for example, method described by Clark, D. P. (The fermentation pathways of Escherichia coli. FEMS Microbiol Rev 1989 ;5(3); 223-34). Example of enzyme having activity of alcohol dehydrogenase includes AdhE (synonims - ADH, alcohol .NAD+ oxidoreductase, aldehyde reductase described above (SEQ ID NO: 62 and YqhD (Sulzenbacher, G. Et al, J. MoI. Biol. 2004 10; 342(2):489-502) (SEQ ID NO: 63) from Escherichia coli
In conditions with aeration, wild-type E. coli cells do not exhibit alcohol dehydrogenase activity. It is explained by fact that transcription of adhE gene is induced only in anaerobic conditions, and in the presence of oxygen protein encoded by the gene is not active. Consitutive mutation adhC leading to high level of active AdhE protein in both aerobic and anaerobic conditions is combination of mutation in promoter region of adhE gene and mutation Glu568Lys in coding region of corresponding protein. Absence of acidic amino acid in position 568 is assumed to be principal for function of the protein [Holland-Stalley C. et al., 2000 Aerobic activity of Escherichia coli Alcohol Dehydrogenase is determined by a single amino acid. J. Bacteriol. 128 (21) 6049-6054]. Thus, it is necessary to change transcriptional regulation ot the gene and introduce the mutation into the coding region to construct E. coli strain having alcohol dehydrogenase exhibiting ctivity in aerobic conditions (see Example 2).
Term"protein variant" is also for both acetaldehyde dehydrogenase and alcohol dehydrogenase. Term"protein variant" is understood as above.
2. Microorganism according to the present invention
Microorganism according to the present invention is a 1 -butanol producing microorganism modified so that at least one of enzymes from pathway of fatty acids β-oxidation is involved into 1 -butanol biosynthesis. Moreover, microorganism according to the present invention is a 1- butanol producing microorganism modified to have increased activity of one or several enzymes from pathway of fatty acids β-oxidation in the cell compare to non-modified.
According to the present invention phrase "1 -butanol producing microorganism" means microorganism having ability to produce and accumulate 1 -butanol in a medium when the microorganism in cultivated in the medium. Ability to produce 1 -butanol can be imparted to the microorganism by selection, mutagenesis or genetic manipulations.
Phrase "1 -butanol producing microorganism" according to the present invention also means microorganism having ability to produce and accumulate 1 -butanol in a medium in higher amount then parent microorganism and preferably means ability to accumulate 1 -butanol in a medium in detectable amount. Methods for detection of 1 -butanol include, for example, gas chromatography.
Such microorganism can be selected from group consisting of bacteria, yeast and fungi.
Microorganism according to the present invention is a microorganism containing at least one of enzymes from pathway of fatty acids β-oxidation. Such enzyme can be either native or heterogeneous for the microorganism. Term "native enzyme" means natural enzyme inherent for the microorganism encoded by gene from the microorganism. Term "heterogeneous enzyme" means enzyme encoded by gene introduced in the microorganism from another microorganism. Genes encoding heterogeneous enzyme can be introduced in the microorganism by genetic manipulations. icroorgan sm accor ng o e presen nven on s a m croorgan sm av ng natura resistance to 1 -butanol or a microorganism to which such resistance to 1 -butanol is imparted by selection, mutagenesis or genetic manipulations.
Microorganism according to the present invention is a microorganism having ability to utilize organic substrate efficiently or a microorganism to which such ability is imparted or increased by selection, mutagenesis or genetic manipulations.
Microorganism according to the present invention belongs to the genus Clostridium, Escherichia, Salmonella, Shigella, Pseudomonas, Zymomonas, Bacillus, Lactobacillus, Enter ococcus, Klebsiella, Corynebacterium, Brevibacterium, Streptomyces, Pichia, Candida or Sacchoromyces.
Phrase "microorganism belongs to the genus" means that the bacterium is classified as the bacterium belonging to the particular genus according to the classification known to a person skilled in the art of microbiology. Examples of microorganisms belonging to the genera meantioned above include Escherichia coli, Salmonella enterica, Shigella flexneri, Pseudomonas aeruginosa, Pseudomonas putida, Zymomonas mobilis, Bacillus subtilis, Corynebacterium glutamicum, Streptomyces coelicolor, Saccharomyces cerevisiae, Clostridium acetobutylicum, Clostridium beijerinckii.
3. Construction of 1 -butanol producing microorganism.
Microorganism according to the present invention is a microorganism modified to have increased activity of one or several said enzymes in comparison to non-modifieid microorganism compare to non-modified microorganism.
Phrase "activity of enzyme is increased" means that activity of enzyme in cell is higher compare to the non-modified microorganism, for example, wild-type strain.
Activity of one or several enzymes from pathway of fatty acids β-oxidation can be increased in the cell due to increasing the amount of corresponding mRNA, increasing amount of corresponding enzyme or increasing specific activity of enzyme. Amount of mRNA can be increased by increasing copy number of corresponding gene or due to enhancing transcription of the gene by use of more potent promoter, releasing from repression or increasing mRNA stability. Amount of enzyme can be increased by enhancing translation of corresponding mRNA due to modification of nucleotide sequence of ribosome binding site or by increasing the enzyme stability. Specific activity of the enzyme can be increased by introduction of corresponding mutations into amino acid sequence of the enzyme. Methods of enhancement of gene expression include increasing the gene copy number. Introducing a gene into a vector that is able to function in the microorganism according to the present invention increases the copy number of the gene.
Enhancement of gene expression may also be achieved by introduction of multiple copies of the gene into a bacterial chromosome by, for example, a method of homologous recombination, Mu integration, or the like. For example, one act of Mu integration allows introduction of up to 3 copies of the gene into a bacterial chromosome.
Increasing the copy number of a gene can also be achieved by introducing multiple copies of the gene into the chromosomal DNA of the bacterium. In order to introduce multiple copies of the gene into a bacterial chromosome, homologous recombination is carried out using a sequence whose multiple copies exist as targets in the chromosomal DNA. Sequences having multiple copies in the chromosomal DNA include, but are not limited to repetitive DNA, or inverted repeats existing at the end of a transposable element. Also it is possible to incorporate the gene into a transposon, and allow it to be transferred to introduce multiple copies of the gene into the chromosomal DNA.
Enhancing gene expression may also be achieved by placing the DNA of the present invention under the control of a potent promoter. For example, the lac promoter, the trp promoter, the trc promoter, the PR, or the PL promoters of lambda phage are all known to be potent promoters. The use of a potent promoter can be combined with multiplication of gene copies.
Alternatively, the effect of a promoter can be enhanced by, for example, introducing a mutation into the promoter to increase the transcription level of a gene located downstream of the promoter. Furthermore, it is known that substitution of several nucleotides in the spacer between ribosome binding site (RBS) and the start codon, especially the sequences immediately upstream of the start codon, profoundly affect the mRNA translatability. For example, a 20-fold range in the expression levels was found, depending on the nature of the three nucleotides preceding the start codon (Gold et al, Annu. Rev. Microbiol., 35, 365-403, 1981 ; Hui et al, EMBO J., 3, 623-629, 1984).
Moreover, it is also possible to introduce a nucleotide substitution into a promoter region of a gene on the bacterial chromosome, which results in a stronger promoter function. The alteration of the expression control sequence can be performed, for example, in the same manner as the gene substitution using a temperature-sensitive plasmid, as disclosed in WO 00/18935 and JP 1-215280 A.
Enhancement of translation can be achieved by introduction more effective Shine- Dalgarno sequence (SD-sequence) instead of native SD-πocJieΛθBaτejibHθcτH provided that native SD-sequence is located upstream of start-codon of the mRNA contacting with 16S ribosomal RNA (Shine J. and Dalgarno L., Proc. Natl. Acad. Sci. U S A, 1974, 71, 4, 1342-6).
Use of potent promoter can be combined with with use of more effective SD-sequence.
Microorganism according to the present invention is a microorganism additionally modified to have increased amount acetyl-CoA, the earlier precursor of 1-butanol. Amount of acetyl-CoA can be increased by, for example, decreasing flux of acetyl-Coa to the pathways branching from direct pathway of butanol biosynthesis as well as by regulated decrease of phosphoenolpyruvate activity (see Example 7). So, one of the major pathways for acetyl-CoA utilization is tricarboxylic acid cycle. The cycle starts from formation of citrate from oxaloacetate and acetyl group of acetyl-CoA. Major supplier of oxaloacetate in the bacterial cell is phosphoenolpyruvate carboxylase catalyzed reaction of formation oxaloacetate from phosphoenolpyruvate with CO2 fixation. Phosphoenolpyruvate is the intermediate of glycolysis and precursor of of acetyl-CoA. Thus, it is necessary to decrease activity of phosphoenolpyruvate carboxylase for effective butanol biosynthesis from acetyl-CoA. It is known that ppc mutants, for example E.coli, are auxotrophs for intermediates of tricarboxylic acid cycle and they can not grow in the medium containing most suitable for 1-butanol production carbon sources, such as sugars and glycerol [Sauer U. and Eikmanns B. 2005 The PEP-pyruvate-oxaloacetate node as the switch point for carbon flux distribution in bacteria. FEMS Microbiol. Rew., 29, 765-794]. Thus complete inactivation of phosphoenolpyruvate carboxylase is not suitable. Use of regulated decrease of the enzyme activity can solve the problem. Regulated decrease of the enzyme activity can be achieved by, for example, decrease of de novo synthesis of the enzyme by decrease of amount of corresponding mRNA as a result of counter transcription of target gene initiated from regulated promoter.
Methods for preparation of plasmid DNA, digestion and ligation of DNA, transformation, selection of an oligonucleotide as a primer and the like are well known to one skilled in the art. These methods are described, for instance, in Sambrook, J., Fritsch, E. F., and Maniatis. T., "Molecular Cloning A Laboratory Manual, Second Edition"; Cold Spring Harbor Laboratory Press (1989).
4. Method for production of 1-butanol.
A method according to the present invention is a method for producing 1-butanol including cultivation of microorganism according to the present invention in culture medium and collecting produced 1-butanol from the medium.
According to the present invention collection and purification of 1 -butanol from culture medium and the like may be performed in a manner similar to conventional fermentation methods wherein 1-butanol is produced using a bacterium (distillation, rectification, liquid extraction, membrain filtration etc. Se, for example, Ezeji, T.C. et al, Butanol fermentation research: upstream and downstream manipulations. Chem Rec. 2004; 4(5): 305-14).
A medium used for culture may be either a synthetic or natural medium, so long as the medium includes a carbon source and a nitrogen source and minerals and, if necessary, appropriate amounts of nutrients which the bacterium requires for growth. The carbon source may include various carbohydrates such as glucose and sucrose, and various organic acids and glycerol. As the nitrogen source, various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean-hydrolysate, and digested fermentative microorganism can be used. As minerals, potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like can be used. As vitamins, thiamine, yeast extract, and the like, can be used.
The cultivation is preferably performed at the temperature from 30°C to 37°C, preferably from 35°C to 37°C. pH is adjusted from 5 to 9, preferably from 6.5 to 7.2. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. Usually, a 1 to 5-day cultivation leads to accumulation of 1-butanol in the liquid medium.
Process of microorganism cultivation in the method of the present invention may be aerobic, anaerobic, microaerobic. Also the process of microorganism cultivation in the method of the present invention may include both aerobic and anaerobic steps.
After cultivation, solids such as cells can be removed from the liquid medium by centrifugation or membrane filtration.
Examples
The present invention will be more concretely explained below with reference to the following non-limiting examples.
Example 1. Construction of E. coli strain containing chromosomal mutation
Figure imgf000019_0003
1. Construction of strain
Figure imgf000019_0001
It is known that mutation
Figure imgf000019_0002
leading to 10-fold increasing of synthesis of lactose operon repressor is single point substitution in "-35" region of
Figure imgf000019_0004
promoter [Glascock CB. and Weickert M.J., 1998 Using chromosomal
Figure imgf000019_0006
to control expression of genes on high- copy-number plasamids in Escherichia coli. Gene, 223, 221-231].
Point mutation in the region "-35" of chromosomal
Figure imgf000019_0005
promoter leading to formation of phenotype was introduced by the method developed by Datsenko, K.A. and Wanner, B. L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) also known as "Red-driven integration". DNA fragment containing CmR marker encoded by cat gene was obtained by PCR using primers Pl (SEQ ID NO: 64) and P2 (SEQ ID NO: 65), and pMW1 18-attL-Cm-attR plasmid [Katashkina et al, 2005. MoI. Biol. (Rus), 39 (5), 823-831] as a template. Primer Pl contains 36 nucleotides homologous to the DNA fragment on 3 '-end of mhpR gene and 28 nucleotides complementary to 3 '-end of attR region. Primer P2 contains 36 nucleotides complementary except one point mutation to DNA fragment located immediately down-stream of stop-codon of mhpR gene and "-35" region of
Figure imgf000020_0001
promoter, and 28 nucleotides homologous to the DNA fragment located in 5 '-end of attL region. Following termerature profile was used: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 52°C, 1 minute at 72°C; and final polimerization: 7 minutes at 72°C. Taq DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
Obtained PCR product (1700 base pairs) purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to recommendations of manufacturerwas used for electroporation into E.coli strain MG1655 (ATCC 700926) containing pKD46 plasmid (Datsenko, K. A. and Wanner, B.L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645) with thermosensitive replicon. pKD46 plasmid contains DNA fragment from phage λ (2154 base pairs; positions from 31088 to 33241 in nucleotide sequence with number J02459 in GenBank database) and genes of λ Red-homologous recombination system (γ, β, exo genes) under control arabinose inducible
Figure imgf000020_0002
promoter. pKD46 plasmid is necessary for integration of PCR product into chromosome of strain MG1655. Strain MG1655 is available in American Type Culture Collection. (P.O. Box 1549 Manassas, VA 20108, U.S.A.).
Electrocompetent cells were obtained as follows: night culture of E. coli strain MGl 655 containing pKD46 plasmid were grown at 30°C on LB medium containing ampicillin (100 mg/1), deluted 100 times with adding 10 ml of SOB medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) containing ampicillin and L-arabinose (10 mM). Obtained culture was grown with mixture at 30°C to reach O Then cells were made electrocompetent by concentrating in 100 times
Figure imgf000020_0003
and washing three times with ice-chilled deionized water. Electroporation was performed with 70 μl of cells and
Figure imgf000020_0004
of PCR-product. After electroporation cells were incubated in 1 ml of SOC medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) at 37°C during 2.5 hours, then cells were spreaded on plates with L-agar containing 30 mg/1 chloramphenicol and grown at 37°C to select CmR- recombinants. To eliminate pKD4 plassmid, cells were spreaded on LB agar to get separate colonies at 37°C and CmRAps variants were selected.
Correct structure of chromosome of selected Cm Ap colonies was tested by PCR analysis using locus specific primers P3 (SEQ ID NO: 66) and P4 (SEQ ID NO: 67). Following temberature profile was used for PCR test: denaturatin at 95°C during 10 minutes; 30 cycles: 30 seconds at 95°C, 30 seconds at 52°C, 1 minute at 72°C; final step: 7minutes at 72°C. Lenght of PCR product obtained after reaction with cells of parent strain MGl 655 was 205 base pairs. Lenght of PCR product obtained after reaction with cells of mutant strain was 1833 base pairs. Mutant strain was temporarily named as MGl 655 a la (before determination
Figure imgf000021_0002
of nucleotide sequence of
Figure imgf000021_0003
region of
Figure imgf000021_0008
promoter).
2. Construction of strain MG 1655 lacP.
Then, Cm resistance gene (cat gene) was eliminated from chromosome of strain MGl 655
Figure imgf000021_0004
using int-xis system. Strain MGl 655
Figure imgf000021_0005
was transformed with pMWts-Int/Xis plasmid (WO 2007013638; RU 2005123423). Selection of transformed clones was performed on LB agar medium containing ampicillin (100 mg/1). Plates were incubated during night at 30°C. The cat gene was eliminated from transformed clones by culturing separate colonies at 37°C (at that termerature Cits repressor is partially inactivated while int/xis genes is activated) followed by selection of CmsApR variants. Elimination of cat gene from chromosome of the strain was confirmed by PCR using locus specific primers P3 (SEQ ID NO: 66) and P4 (SEQ ID NO: 67). PCR conditions for the test were as described above. Lenght of PCR product obtained using cells with eliminated cat gene was 236 base pairs.
Then CmsApR variants were healed from helper plasmid pMWts-Int/Xis containing temperature sensitive replicon by second spreading to get seperate colonies at 37°C followed by selection of CmsAps clones.
Structure of "-35" region of promoter GTGCAA mutation) in obtained clones
Figure imgf000021_0009
Figure imgf000021_0010
was confirmed by sequence analysis. Thus, strain MG1655 attB-
Figure imgf000021_0006
or, shortly, MG1655 lacP was obtained.
Example 2. Construction of E. coli strain having alcohol dehydrogenase AdhE capable to exhibit enzymatic activity in aerobic conditions.
1. Integration of hybrid regulatory element containing P
Figure imgf000021_0007
promoter and SDφio upstream of coding region of chromosomal adhE gene. Construction of strain MG 1655 lacP attR-cat-
Figure imgf000021_0001
To optimize expression of adhE gene, promoter [Skorokhodova
Figure imgf000022_0005
A. Yu. et al, 2006, Biotekhnologiya (Rus) 3, 6-16.], hereinafter named as
Figure imgf000022_0006
promoter, jointed with Shine-Dalgarno sequence (SD sequence) of φlO gene from phage T7 was integrated upstream of adhE gene in the chromosome of E. coli strain MGl 655 lacP using method developed by Datsenko, K.A. and Wanner, B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) also known as "Red-driven integration". Artificial DNA fragment integrated into chromosome also contained CmR marker encoded by cat gene.
Construction of above mentioned DNA fragment integrated into corresponding region of bacterial chromosome was performed in several stages. First, DNA fragment containing BgIW restrictin site,
Figure imgf000022_0001
promoter, SD sequence of φlO gene from phage T7 and 36 nucleotides complementary 5 '-end of coding region of adhE gene was obtained by PCR. The DNA fragment was obtained in two stages. At first, DNA fragment containing
Figure imgf000022_0009
restriction site, promoter and part of SD sequence of φlO gene from phage T7 was obtained by PCR
Figure imgf000022_0008
using plasmid
Figure imgf000022_0004
[Skorokhodova A. Yu. et al, 2006, Biotekhnologiya (Rus) 3, 6-16.]. PCR was performed using primers P5 (SEQ ID NO: 68) and P6 (SEQ ID NO: 69). Primer P5 contains BgIW restrictin site and region homolous to 5'-end of
Figure imgf000022_0002
promoter. Primer P6 contains part of SD sequence of φ 10 gene from phage T7 and region complementary to 3 '-end of
Figure imgf000022_0003
promoter. Following temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 55°C, 30 seconds at 72°C; and final polymerization: 7 minutes at 72°C. Pfu DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
Obtained PCR product purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to recommendations of manufacturer was used for the next round of PCR as template. Primers P5 (SEQ ID NO: 68) and P7 (SEQ ID NO: 70) were used. Primer P7 contains region complementary 3'-end of
Figure imgf000022_0007
promoter, SD sequence of φlO gene from phage T7 and 36 nucleotides of opened reading frame of adhE gene. Following termerature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 55°C, 40 seconds at 72°C; and final polymerization: 7 minutes at 72°C. Pfu DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
In parallel, second stage of construction of DNA fragment was performed. DNA fragment containing CmR marker encoded by cat gene was obtained by PCR using primers P8 (SEQ ID NO: 71) and P9 (SEQ ID NO: 72) and pMWl 18-attL-Cm-attR plasmid as template. Primer P8 contains 36 hucleotides homologous to DNA fragment upstream of coding region of adhE gene and 28 nucleotides complementary to DNA fragment located at 3 '-end of attR region. Primer P9 contains BgIW restriction site and 28 nucleotides homologous to the 5 '-end of attL region. Following temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 52 °C, 1 minute at 72°C; and final polymerization: 7 minutes at 72°C. Taq DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
Obtained DNA fragments were purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to the recommendations of manufacturer followed by ethanol precipitation.
Two obtained DNA fragments were treated by BgIW restrictase followed by ligation using T4 DNA ligase (Maniatis T., Fritsch E.F., Sambrook, J.: Molecular Cloning:A Laboratory Manual. 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989).
Product obtained after ligation was amplified by PCR using primers P7 (SEQ ID NO: 70) and P8 (SEQ ID NO: 71). Following temperature profile was used for PCR: denaturation at 98°C during 1 minute; 30 cycles: 15 seconds at 98°C, 15 seconds at 55°C, 1 minute at 72°C; and final polimerization: 7 minutes at 72°C. Phusion DNA polymease and corresponding buffer from Finnzymes; and deoxynucleoside triphosphates from Fermentas (Lithuania) were used. Obtained PCR product (1823 base pairs) purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to the recommendations of manufacturer was used for electroporation of E.coli strain MG1655 lacf containing pKD46 plasmid with termosensitive replicon. pKD46 plasmid is necessary for integration of the DNA fragment into chromosome of strain MG 1655 lacf.
Electrocompetent cells of E. coli strain MGl 655 lacf containing pKD46 plasmid were obtained as described in Example 1. Electroporation was performed with 70 μl of cells and of PCR product. After electroporation cells were incubated in 1 ml of SOC medium
Figure imgf000023_0001
(Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) at 37°C during 2.5 hours, then cells were spreaded on plates with L-agar containing 30 mg/1 chloramphenicol and grown at 37°C to select CmR- recombinants. To eliminate pKD46 plassmid, cells were spreaded on LB agar to get separate colonies at 37°C and Cm Ap variants were selected.
Correctness of desired structure of chromosome of selected CmRAp colonies was confirmed by PCR analysis using locus specific primers PlO (SEQ ID NO: 73) and PI l (SEQ ID NO: 74). Temperature profile for PCR testing descirbed in the Example 1 was used. Length of PCR product obtained in the reaction with cells of parent strain MG1655 lacf was 316 base pairs. Length of PCR product obtained in the reaction with cells of mutant strain was 2067 base pairs. Mutant strain was temporarily named as
Figure imgf000024_0002
Figure imgf000024_0003
(before confirmation of sequence of promoter).
Figure imgf000024_0007
2. Construction of strain
Figure imgf000024_0004
φ
Then, Cm resistance gene {cat gene) was eliminated from chromosome of strain MG1655
Figure imgf000024_0008
using int-xis system. Strain MG1655 lacf attR-cat-
Figure imgf000024_0009
was transformed with pMWts-Int/Xis plasmid (WO 2007013638; RU 2005123423). Selection of transformed clones was performed on LB agar medium containing ampicillin (100 mg/1). Plates were incubated during night at 30°C. The cat gene was eliminated from transformed clones by culturing separate colonies at 37°C (at that termerature Cits repressor is partially inactivated while int/xis genes is activated) followed by selection of Cm Ap variants. Elimination of cat gene from chromosome of the strain was confirmed by PCR using locus specific primers PlO (SEQ ID NO: 73) and PI l (SEQ ID NO: 74). PCR conditions for the test were as described above in the Example 1. Lenght of PCR product obtained using cells with eliminated cat gene was 470 base pairs.
Then
Figure imgf000024_0010
variants were healed from helper plasmid pMWts-Int/Xis containing temperature sensitive replicon by second spreading to get seperate colonies at 37°C followed by selection of
Figure imgf000024_0011
clones.
Correctness of desired structure of new hybrid regulatory region introduced upstream of coding region of adhE gene was confirmed by sequence analysis. Thus, strain MGl 655 lacf
Figure imgf000024_0005
or abbreviated as MGl 655 l
Figure imgf000024_0006
was obtained.
3. Construction of strain MG1655
Figure imgf000024_0001
Introduction of point mutation into coding region of adhE gene was performed in two steps. First, region of 21 nucleotides including codon 568 of corresponding gene was replaced by DNA fragment containing KmR marker encoded by kan gene. Then the introduced marker was replaced by synthesized double stranded oligonucleotide duplex containing earlier deleted region of adhE gene, but including mutation leading to Glu568Lys substitution in the protein product of the gene.
Substitution of region of 21 nucleotides containing codon 568 of adhE gene was performed using method developed by Datsenko, K.A. and Wanner, B. L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) also known as "Red-driven integration". DNA fragment containing KmR marker encoded by kan gene was obtained by PCRusing primers Pl 2 (SEQ ID NO: 75) and Pl 3 (SEQ ID NO: 76), and pUC4K plasmid as template [accession number X06404 in the GenBak/EMBL]. Primer Pl 2 contains 36 nucleotides homologous DNA of adhE gene in position 1657 to 1692 and region of homology to pUC4K plasmid. Primer Pl 3 contains 36 nucleotides complementary to DNA of adhE gene in position 1749 to 1714 and region of homology to pUC4K plasmid. Following temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 52°C, 1 minutes at 72°C; and final polymerization: 7 minutes at 72°C. Taq DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
Obtained PCR product (1275 base pairs) purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to the recommendations of manufacturer was used for electroporation of E.coli strain
Figure imgf000025_0004
containing pKD46 plasmid with termosensitive replicon. pKD46 plasmid is necessary for integration of the DNA fragment into chromosome of strain MGl 655
Figure imgf000025_0001
Electrocompetent cells of E. coli strain MGl 655 lαcP containing
Figure imgf000025_0005
pKD46 plasmid were obtained as described in Example 1. Electroporation was performed with 70 μl of cells and »300 ng of PCR product. After electroporation cells were incubated in 1 ml of SOC medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) at 37°C during 2.5 hours, then cells were spreaded on plates with L-agar containing 50 mg/1 kanamicin and grown at 37°C to select KmR- recombinants. To eliminate pKD46 plassmid, cells were spreaded on LB agar to get separate colonies at 37°C and KmRAps variants were selected.
Correctness of desired structure of chromosome of selected Km Ap colonies was confirmed by PCR analysis using locus specific primers Pl 4 (SEQ ID NO: 77) and Pl 5 (SEQ ID NO: 78). Temperature profile for PCR testing descirbed in the Example 1 was used. Length of PCR product obtained in the reaction with cells of parent strain MG1655
Figure imgf000025_0008
Figure imgf000025_0006
was 200 base pairs. Length of PCR product obtained in the reaction with cells of mutant strain was 1382 base pairs. Mutant strain was temporarily named as MG1655
Figure imgf000025_0007
Figure imgf000025_0002
4. Construction of strain
Figure imgf000025_0003
Substitution of marker earlier introduced into coding region of αdhE gene by synthesized double stranded oligonucleotide duplex containing earlier deleted region of αdhE gene but containing mutation leading to Glu568Lys substitution in the protein product of the gene was performed by method developed by Datsenko, K.A. and Wanner, B. L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645).
Target double stranded oligonucleotide duplex was obtained by PCR using partialy overlapped primers Pl 6 (SEQ ID NO: 79) and Pl 7 (SEQ ID NO: 80). Primers P16 and Pl 7 contain nucleotides in the overlapping region necessary for desired substitution in the protein product of adhE gene and 36 nucleotide non-overlapping regions necessary for integration of duplex into chromosome. Following temperature profile was used for PCR: denaturation at 95°C during 30 seconds; 25 cycles: 15 seconds at 95°C, 15 seconds at 55°C, 30 seconds at 72°C; and final polymeerization: 3 minutes at 72°C. Pfu DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
Obtained PCR product (93 base pairs) purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to the recommendations of manufacturer was used for electroporation of E.coli strain MG1655 lacP
Figure imgf000026_0001
containing pKD46 plasmid with termosensitive replicon. pKD46 plasmid is necessary for integration of the DNA fragment into chromosome of strain MG1655
Figure imgf000026_0002
Electrocompetent cells of E. coli strain MGl 655 l
Figure imgf000026_0003
αcP containing pKD46 plasmid were obtained as described in Example 1. Electroporation was performed with 70 μl of cells and «500 ng of PCR product. After electroporation cells were incubated in 1 ml of SOC medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) at 37°C during 2.5 hours, then cells were spreaded on plates with M9-agar medium containing 1 mM IPTG and 2% of ethanol as a sole carbon source and grown at 37°C to select
Figure imgf000026_0004
+-recombinants. To eliminate pKD46 plassmid, cells were spreaded on LB agar to get separate colonies at 37°C and Km Ap variants were selected.
Ability of obtained recombinants to grow in aerobic conditions on the medium containing ethanol as a sole carbon source proved that protein product oiαdhE gene in the cells was active.
Correctness of desired structure of chromosome of selected αdhE? KmsAp colonies was confirmed by PCR analysis using locus specific primers P14 (SEQ ID NO: 77) and Pl 5 (SEQ ID NO: 78). Temperature profile for PCR testing descirbed in the Example 1 was used. Length of PCR product obtained in the reaction with cells of parent strain MG 1655 lαcf P,,-c-ideai-4- was 1382 base pairs. Length of PCR product obtained in the reaction with cells of mutant strain was 200 base pairs.
Correctness of desired nucleotide structure of obtained region around 568 codon of αdhE gene in the chromosome was confirmed by sequence analysis. Thus, the mutant strain having alcohol dehydrogenase AdhE capable to exhibit enzymatic activity in aerobic conditions was obtained. The strain was named as
Figure imgf000027_0001
Example 3. Construction of E. coli strain with changed regulation of fadE gene expression. 1. Integration of hybrid regulatory region containing
Figure imgf000027_0008
promoter and SDφio into chromosome of E. coli upstream of coding region of fadE gene. Construction of strain MG 1655
Figure imgf000027_0002
φ φ
For optimization of fadE gene expression,
Figure imgf000027_0003
promoter, hereinafter mentioned as
Figure imgf000027_0009
jointed to SD sequence of φlO gene from phage T7 was integrated upstream of coding region of fadE gene in the chromosome of E. coli strain MG 1655 lacP
Figure imgf000027_0015
Figure imgf000027_0004
using method developed by Datsenko, K.A. and Wanner, B. L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) also known as "Red-driven integration". Artificial DNA fragment integrated into chromosome also contained CmR marker encoded by cat gene.
Construction of artificial DNA fragment integrated into corresponding region of bacterial chromosome was performed in several steps. First, DNA fragment containing BgIU restriction site, promoter, SD sequence of φlO gene from phage T7 and 36 nucleotides
Figure imgf000027_0010
complementary 5 '-end of coding region of fadE gene was obtained by PCR. The DNA fragment was obtained in two stages. At first, DNA fragment containing
Figure imgf000027_0012
restriction site,
Figure imgf000027_0013
promoter and part of SD sequence of φlO gene from phage T7 was obtained by PCR using plasmid [Skorokhodova A. Yu. et al, 2006, Biotekhnologiya
Figure imgf000027_0005
(Rus) 3, 6-16.]. PCR was performed using primers P5 (SΕQ ID NO: 68) and P6 (SΕQ ID NO: 69). Primer P5 contains
Figure imgf000027_0011
restrictin site and region homolous to 5 '-end of P
Figure imgf000027_0014
promoter. Primer P6 contains part of SD sequence of φlO gene from phage T7 and region complementary to 3 '-end of
Figure imgf000027_0006
promoter. Following temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 55°C, 30 seconds at 72°C; and final polymerization: 7 minutes at 72°C. Pfu DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
Obtained PCR product purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to the recommendations of manufacturer was used as template in the next round of PCR. Primers P5 (SEQ ID NO: 68) and Pl 8 (SEQ ID NO: 81) were used. Primer Pl 8 contains region complementary to
Figure imgf000027_0007
promoter, SD sequence of φlO gene from phage T7 and 36 nucleotides from opened reading frame of fadE gene. Following temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 55°C, 40 seconds at 72°C; and final polymerization: 7 minutes at 72°C. Pfu DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
In parallel, second stage of construction of DNA fragment was performed. DNA fragment containing Cm marker encoded by cat gene was obtained by PCR using primers Pl 9 (SEQ ID NO: 82) and P9 (SEQ ID NO: 72) and pMWl 18-attL-Cm-attR plasmid as template. Primer P19 contains 36 hucleotides homologous to DNA fragment upstream of coding region of fadE gene and 28 nucleotides complementary to DNA fragment located at 3 '-end oϊattR region. Primer P9 contains BgIW restriction site and 28 nucleotides homologous to the 5 '-end of attL region. Following temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 52 °C, 1 minute at 72°C; and final polymerization: 7 minutes at 72°C. Taq DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
Obtained DNA fragments were purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to the recommendations of manufacturer followed by ethanol precipitation.
Two obtained DNA fragments were treated by
Figure imgf000028_0004
restrictase followed by ligation using T4 DNA ligase (Maniatis T., Fritsch E. F., Sambrook, J.: Molecular Cloning: A Laboratory Manual. 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989).
Product obtained after ligation was amplified by PCR using primers P19 (SEQ ID NO: 82) H Pl 8 (SEQ ID NO: 81). Following temperature profile was used for PCR: denaturation at 98°C during 1 minute; 30 cycles: 15 seconds at 98°C, 15 seconds at 55°C, 1 minute at 72°C; and final polimerization: 7 minutes at 72°C. Phusion DNA polymease and corresponding buffer from Finnzymes; and deoxynucleoside triphosphates from Fermentas (Lithuania) were used. Obtained PCR product (1823 base pairs) purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to the recommendations of manufacturer was used for electroporation of E.coli strain MG1655 l
Figure imgf000028_0003
containing pKD46 plasmid with termosensitive replicon. pKD46 plasmid is necessary for integration of the DNA fragment into chromosome of strain MGl 655
Figure imgf000028_0002
Electrocompetent cells of E. coli strain MG1655 containing
Figure imgf000028_0001
pKD46 plasmid were obtained as described in Example 1. Electroporation was performed with 70 μl of cells and «300 ng of PCR product. After electroporation cells were incubated in 1 ml of SOC medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) at 37°C during 2.5 hours, then cells were spreaded on plates with L-agar containing 30 mg/1 chloramphenicol and grown at 37°C to select CmR- recombinants. To eliminate pKD46 plassmid, cells were spreaded on LB agar to get separate colonies at 37°C and CmRAps variants were selected.
Correctness of desired structure of chromosome of selected CmRAps colonies was confirmed by PCR analysis using locus specific primers P20 (SEQ ID NO: 83) and P21 (SEQ ID NO: 84). Temperature profile for PCR testing descirbed in the Example 1 was used. Length of PCR product obtained in the reaction with cells of parent strain MG 1655
Figure imgf000029_0008
Figure imgf000029_0009
was 259 base pairs. Length of PCR product obtained in the reaction with cells of mutant strain was 2010 base pairs. Mutant strain was temporarily named as MG1655
Figure imgf000029_0010
Figure imgf000029_0003
(before confirmation of sequence of
Figure imgf000029_0011
promoter). 2. Construction of strain MGl 655
Figure imgf000029_0004
Then, Cm resistance gene {cat gene) was eliminated from chromosome of strain MG 1655
Figure imgf000029_0005
using int-xis system. Strain MG 1655 lacf
Figure imgf000029_0001
R L was transformed with pMWts-Int/Xis plasmid (WO 2007013638; RU 2005123423). Selection of transformed clones was performed on LB agar medium containing ampicillin (100 mg/1). Plates were incubated during night at 30°C. The cat gene was eliminated from transformed clones by culturing separate colonies at 37°C (at that termerature Cits repressor is partially inactivated while int/xis genes is activated) followed by selection of Cm Ap variants. Elimination of cat gene from chromosome of the strain was confirmed by PCR using locus specific primers P20 (SEQ ID NO: 83) and P21 (SEQ ID NO: 84). PCR conditions for the test were as described above in the Example 1. Lenght of PCR product obtained using cells with eliminated cat gene was 413 base pairs.
Then Cm Ap variants were healed from helper plasmid pMWts-Int/Xis containing temperature sensitive replicon by second spreading to get seperate colonies at 37°C followed by selcetion of CmsAps clones.
Correctness of desired structure of new hybrid regulatory region introduced upstream of coding region of
Figure imgf000029_0006
f gene was confirmed by sequence analysis. Thus, strain MGl 655 lacf
Figure imgf000029_0002
or abbreviated as MGl 655 lαcf
Figure imgf000029_0012
Figure imgf000029_0007
was obtained.
Example 4. Construction of E. coli strain with changed regulation of fadB gene expression. 1. Integration of hybrid regulatory region containing promoter and into
Figure imgf000030_0012
Figure imgf000030_0013
chromosome of E. coli upstream of coding region of fadB gene. Construction of strain MG 1655
Figure imgf000030_0001
φ φ /
For optimization of fadB gene expression,
Figure imgf000030_0005
promoter, hereinafter
Figure imgf000030_0004
mentioned as jointed to SD sequence of φlO gene from phage T7 was integrated
Figure imgf000030_0003
upstream of coding region of fadB gene in the chromosome of E. coli strain MG 1655
Figure imgf000030_0006
using method developed by Datsenko, K. A. and
Figure imgf000030_0002
Wanner, B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) also known as "Red- driven integration". Artificial DNA fragment integrated into chromosome also contained CmR marker encoded by cat gene.
Construction of artificial DNA fragment integrated into corresponding region of bacterial chromosome was performed in several steps. First, DNA fragment containing
Figure imgf000030_0007
restriction site, promoter, SD sequence of φlO gene from phage T7 and 36 nucleotides
Figure imgf000030_0014
complementary 5'-end of coding region of fadB gene was obtained by PCR. The DNA fragment was obtained in two stages. At first, DNA fragment containing BgIU restriction site,
Figure imgf000030_0008
promoter and part of SD sequence of φlO gene from phage T7 was obtained by PCR using plasmid
Figure imgf000030_0009
[Skorokhodova A. Yu. et al, 2006, Biotekhnologiya (Rus) 3, 6-16.]. PCR was performed using primers P5 (SΕQ ID NO: 68) and P6 (SΕQ ID NO: 69). Primer P5 contains Bglϊl restrictin site and region homolous to 5 '-end of
Figure imgf000030_0015
promoter. Primer P6 contains part of SD sequence of φlO gene from phage T7 and region complementary to 3 '-end of
Figure imgf000030_0010
promoter. Following temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 55°C, 30 seconds at 72°C; and final polymerization: 7 minutes at 72°C. Pfu DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
Obtained PCR product purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to the recommendations of manufacturer was used as template in the next round of PCR. Primers P5 (SEQ ID NO: 68) and P22 (SEQ ID NO: 85) were used. Primer P22 contains region complementary to 3 '-end of promoter, SD sequence of φlO gene
Figure imgf000030_0011
from phage T7 and 36 nucleotides from opened reading frame of fadB gene. Following temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 55°C, 40 seconds at 72°C; and final polymerization: 7 minutes at 72°C. Pfu DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used. In para e , secon s age or construction o ragment was peπorme . NA fragment containing CmR marker encoded by cat gene was obtained by PCR using primers P23 (SEQ ID NO: 86) and P9 (SEQ ID NO: 72) and pMW1 18-attL-Cm-attR plasmid as template. Primer P23 contains 36 hucleotides homologous to DNA fragment upstream of coding region of fadB gene and 28 nucleotides complementary to DNA fragment located at 3 '-end oϊ
Figure imgf000031_0008
region. Primer P9 contains BgIW restriction site and 28 nucleotides homologous to the 5'-end of
Figure imgf000031_0007
region. Following temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 52 °C, 1 minute at 72°C; and final polymerization: 7 minutes at 72°C. Taq DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
Obtained DNA fragments were purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to the recommendations of manufacturer followed by ethanol precipitation.
Two obtained DNA fragments were treated by B
Figure imgf000031_0005
restrictase followed by ligation using T4 DNA ligase (Maniatis T., Fritsch E.F., Sambrook, J.: Molecular Cloning:A Laboratory Manual. 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989).
Product obtained after ligation was amplified by PCR using primers P23 (SEQ ID NO: 86) H P22 (SEQ ID NO: 85). Following temperature profile was used for PCR: denaturation at 98°C during 1 minute; 30 cycles: 15 seconds at 98°C, 15 seconds at 55°C, 1 minute at 72°C; and final polimerization: 7 minutes at 72°C. Phusion DNA polymease and corresponding buffer from Finnzymes; and deoxynucleoside triphosphates from Fermentas (Lithuania) were used. Obtained PCR product (1823 base pairs) purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to the recommendations of manufacturer was used for electroporation of E.coli strain MG1655
Figure imgf000031_0001
containing pKD46 plasmid with termosensitive replicon. pKD46 plasmid is necessary for integration of the DNA fragment into chromosome of strain MG 1655
Figure imgf000031_0006
Figure imgf000031_0002
Electrocompetent cells of E. coli strain MG 1655 lacP
Figure imgf000031_0004
Figure imgf000031_0003
containing pKD46 plasmid were obtained as described in Example 1. Electroporation was performed with 70 μl of cells and »300 ng of PCR product. After electroporation cells were incubated in 1 ml of SOC medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) at 37°C during 2.5 hours, then cells were spreaded on plates with L-agar containing 30 mg/1 chloramphenicol and grown at 37°C to select CmR-recombinants. To eliminate pKD46 plassmid, cells were spreaded on LB agar to get separate colonies at 37°C and CmRApS variants were selected.
Correctness of desired structure of chromosome of selected CmRApS colonies was confirmed by PCR analysis using locus specific primers P24 (SEQ ID NO: 87) and P25 (SEQ ID NO: 88). Temperature profile for PCR testing descirbed in the Example 1 was used. Length of PCR product obtained in the reaction with cells of parent strain MGl 655 lacf P,rc-ideai-4-
Figure imgf000032_0001
P SD / /£ was 169 base pairs. Length of PCR product obtained in the reaction with cells of mutant strain was 1920 base pairs. Mutant strain was temporarily named as MGl 655
Figure imgf000032_0002
P φ R-cat-attL-
Figure imgf000032_0008
P SD fadB (before confirmation of sequence of
Figure imgf000032_0009
promoter).
2. Construction of strain MG1655 lacf
Figure imgf000032_0003
f/r ideai 4
Figure imgf000032_0010
φ
Then, Cm resistance gene {cat gene) was eliminated from chromosome of strain MG 1655
Figure imgf000032_0007
φ φ :/ φ using int- xis system. Strain MGl 655 lacf
Figure imgf000032_0004
αttR-cαt-αttL-
Figure imgf000032_0013
was transformed with pMWts-Int/Xis plasmid (WO 2007013638; RU
Figure imgf000032_0012
2005123423). Selection of transformed clones was performed on LB agar medium containing ampicillin (100 mg/1). Plates were incubated during night at 30°C. The cat gene was eliminated from transformed clones by culturing separate colonies at 37°C (at that termerature Cits repressor is partially inactivated while int/xis genes is activated) followed by selection of CmsApR variants. Elimination of cat gene from chromosome of the strain was confirmed by PCR using locus specific primers P24 (SEQ ID NO: 87) and P25 (SEQ ID NO: 88). PCR conditions for the test were as described above in the Example 1. Lenght of PCR product obtained using cells with eliminated cat gene was 323 base pairs.
Then CmsApR variants were healed from helper plasmid pMWts-Int/Xis containing temperature sensitive replicon by second spreading to get seperate colonies at 37°C followed by selection of Cm Ap clones.
Correctness of desired structure of new hybrid regulatory region introduced upstream of coding region of fadB gene was confirmed by sequence analysis. Thus, strain MGl 655 l
Figure imgf000032_0011
Figure imgf000032_0005
</c ideai φio , c jdeaι 4 φio:M or abbreviated as MG 1655 lacf
Figure imgf000032_0006
was obtained.
Example 5. Construction of E. coli strain with changed regulation oϊfadA gene expression. 1. Integration of hybrid regulatory region containing
Figure imgf000033_0009
promoter and SDφio into chromosome of E. coli upstream of coding region of
Figure imgf000033_0010
f gene. Construction of strain MG 1655
Figure imgf000033_0003
Figure imgf000033_0004
For optimization of
Figure imgf000033_0005
f gene expression,
Figure imgf000033_0001
promoter, hereinafter mentioned as
Figure imgf000033_0008
jointed to SD sequence of φlO gene from phage T7 was integrated upstream of coding region of fadA gene in the chromosome of E. coli strain MGl 655 lacf P,ΛC-
Figure imgf000033_0002
using method developed by Datsenko, K.A. and Wanner, B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) also known as "Red-driven integration". Artificial DNA fragment integrated into chromosome also contained CmR marker encoded by cat gene.
Construction of artificial DNA fragment integrated into corresponding region of bacterial chromosome was performed in several steps. First, DNA fragment containing BgIW restriction site,
Figure imgf000033_0013
promoter, SD sequence of φlO gene from phage T7 and 36 nucleotides complementary 5 '-end of coding region oϊf
Figure imgf000033_0011
gene was obtained by PCR. The DNA fragment was obtained in two stages. At first, DNA fragment containing BgIW restriction site, Prrc-ideai-4 promoter and part of SD sequence of φlO gene from phage T7 was obtained by PCR using plasmid
Figure imgf000033_0006
[Skorokhodova A. Yu. et al, 2006, Biotekhnologiya (Rus) 3, 6-16.]. PCR was performed using primers P5 (SEQ ID NO: 68) and P6 (SEQ ID NO: 69). Primer P5 contains BgIW restrictin site and region homolous to 5 '-end of Prrc-jdeai-4 promoter. Primer P6 contains part of SD sequence of φlO gene from phage T7 and region complementary to 3 '-end of P
Figure imgf000033_0007
promoter. Following temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 55°C, 30 seconds at 72°C; and final polymerization: 7 minutes at 72°C. Pfu DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
Obtained PCR product purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to the recommendations of manufacturer was used as template in the next round of PCR. Primers P5 (SEQ ID NO: 68) and P26 (SEQ ID NO: 89) were used. Primer P26 contains region complementary to 3'-end of P
Figure imgf000033_0012
promoter, SD sequence of φlO gene from phage T7 and 36 nucleotides from opened reading frame of fadA gene. Following temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 55°C, 40 seconds at 72°C; and final polymerization: 7 minutes at 72°C. Pfu DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used. In parallel, second stage of construction of DNA fragment was performed. DNA fragment containing CmR marker encoded by cat gene was obtained by PCR using primers P27 (SEQ ID NO: 90) and P9 (SEQ ID NO: 72) and pMW1 18-attL-Cm-attR plasmid as template. Primer P27 contains 36 hucleotides homologous to DNA fragment upstream of coding region of fadA gene and 28 nucleotides complementary to DNA fragment located at 3 '-end of attR region. Primer P9 contains BgIW restriction site and 28 nucleotides homologous to the 5'-end of attL region. Following temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 52 °C, 1 minute at 72°C; and final polymerization: 7 minutes at 72°C. Taq DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
Obtained DNA fragments were purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to the recommendations of manufacturer followed by ethanol precipitation.
Two obtained DNA fragments were treated by
Figure imgf000034_0005
restrictase followed by ligation using T4 DNA ligase (Maniatis T., Fritsch E.F., Sambrook, J.: Molecular Cloning: A Laboratory Manual. 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989).
Product obtained after ligation was amplified by PCR using primers P27 (SEQ ID NO: 90) H P26 (SEQ ID NO: 89). Following temperature profile was used for PCR: denaturation at 98°C during 1 minute; 30 cycles: 15 seconds at 98°C, 15 seconds at 55°C, 1 minute at 72°C; and final polimerization: 7 minutes at 72°C. Phusion DNA polymease and corresponding buffer from Finnzymes; and deoxynucleoside triphosphates from Fermentas (Lithuania) were used. Obtained PCR product (1823 base pairs) purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to the recommendations of manufacturer was used for electroporation of E.coli strain MGl 655 lacP
Figure imgf000034_0001
ideai-4-SDφi Q-fadB containing pKD46 plasmid with termosensitive replicon. pKD46 plasmid is necessary for integration of the DNA fragment into chromosome of strain MGl 655 lacf
Figure imgf000034_0006
φ
Figure imgf000034_0003
Electrocompetent cells of £ coli strain MG1655 lacP
Figure imgf000034_0002
P
Figure imgf000034_0004
containing pKD46 plasmid were obtained as described in Example 1. Electroporation was performed with 70 μl of cells and »300 ng of PCR product. After electroporation cells were incubated in 1 ml of SOC medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) at 37°C during 2.5 hours, then cells were spreaded on plates with L-agar containing 30 mg/1 chloramphenicol and grown at 37°C to select CmR-recombinants. To eliminate pKD46 plasmid, cells were spreaded on LB agar to get separate colonies at 37°C and CmRApS variants were selected.
Correctness of desired structure of chromosome of selected CmRAps colonies was confirmed by PCR analysis using locus specific primers P28 (SEQ ID NO: 91) and P29 (SEQ ID NO: 92). Temperature profile for PCR testing descirbed in the Example 1 was used. Length of PCR product obtained in the reaction with cells of parent strain MG 1655 lacf Pfrc-ideai-4-
Figure imgf000035_0001
was 184 base pairs. Length of PCR product obtained in the reaction with cells of mutant strain was 1935 base pairs. Mutant strain was temporarily named as MGl 655 lacf
Figure imgf000035_0009
(before confirmation of sequence of P<rc-ideai-4
Figure imgf000035_0010
promoter).
2. Construction of strain MG1655 lacf
Figure imgf000035_0002
φ
Figure imgf000035_0004
Then, Cm resistance gene (cat gene) was eliminated from chromosome of strain MG 1655
Figure imgf000035_0005
φ φ φ attR-Cat-attL- Prrc-ideal-4-
Figure imgf000035_0013
ofadA using int-xis system. Strain MGl 655
Figure imgf000035_0012
fadE
Figure imgf000035_0011
was transformed with pMWts- Int/Xis plasmid (WO 2007013638; RU 2005123423). Selection of transformed clones was performed on LB agar medium containing ampicillin (100 mg/1). Plates were incubated during night at 30°C. The cat gene was eliminated from transformed clones by culturing separate colonies at 37°C (at that termerature Cits repressor is partially inactivated while int/xis genes is activated) followed by selection of Cm Ap variants. Elimination of cat gene from chromosome of the strain was confirmed by PCR using locus specific primers P28 (SEQ ID NO: 91) and P29 (SEQ ID NO: 92). PCR conditions for the test were as described above in the Example 1. Lenght of PCR product obtained using cells with eliminated cat gene was 338 base pairs.
Then CmsApR variants were healed from helper plasmid pMWts-Int/Xis containing temperature sensitive replicon by second spreading to get seperate colonies at 37°C followed by selcetion of CmsAps clones.
Correctness of desired structure of new hybrid regulatory region introduced upstream of coding region of fadA gene was confirmed by sequence analysis. Thus, strain MGl 655
Figure imgf000035_0006
f
Figure imgf000035_0003
or abbreviated as MG1655
Figure imgf000035_0008
Figure imgf000035_0007
was obtained. Example 6. Construction of E. coli strain with changed regulation oϊatoB gene expression.
1. Integration of hybrid regulatory region containing promoter and SDφi0 into
Figure imgf000036_0004
chromosome of E. coli upstream of coding region of atoB gene. Construction of strain MG1655
Figure imgf000036_0001
φ
For optimization of atoB gene expression,
Figure imgf000036_0010
promoter, hereinafter mentioned as jointed to SD sequence of φlO gene from phage T7 was integrated
Figure imgf000036_0008
upstream of coding region of atoB gene in the chromosome of E. coli strain MG 1655 lacP
Figure imgf000036_0009
Figure imgf000036_0002
using method developed by Datsenko, K. A. and Wanner, B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) also known as "Red-driven integration". Artificial DNA fragment integrated into chromosome also contained CmR marker encoded by cat gene.
Construction of artificial DNA fragment integrated into corresponding region of bacterial chromosome was performed in several steps. First, DNA fragment containing BgM restriction site,
Figure imgf000036_0007
promoter, SD sequence of φlO gene from phage T7 and 36 nucleotides complementary 5 '-end of coding region of atoB gene was obtained by PCR. The DNA fragment was obtained in two stages. At first, DNA fragment containing BgIW restriction site, Prrc-jdeai-4 promoter and part of SD sequence of φlO gene from phage T7 was obtained by PCR using plasmid
Figure imgf000036_0005
[Skorokhodova A. Yu. et al, 2006, Biotekhnologiya (Rus) 3, 6-16.]. PCR was performed using primers P5 (SEQ ID NO: 68) and P6 (SEQ ID NO: 69). Primer P5 contains
Figure imgf000036_0011
restrictin site and region homolous to 5 '-end of
Figure imgf000036_0012
promoter. Primer P6 contains part of SD sequence of φlO gene from phage T7 and region complementary to 3 '-end of
Figure imgf000036_0003
promoter. Following temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 55°C, 30 seconds at 72°C; and final polymerization: 7 minutes at 72°C. Pfu DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
Obtained PCR product purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to the recommendations of manufacturer was used as template in the next round of PCR. Primers P5 (SEQ ID NO: 68) and P30 (SEQ ID NO: 93) were used. Primer P30 contains region complementary to 3'-end of
Figure imgf000036_0006
promoter, SD sequence of φlO gene from phage T7 and 36 nucleotides from opened reading frame of atoB gene. Following temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 55°C, 40 seconds at 72°C; and final polymerization: 7 minutes at 72°C. Pfu DNA po ymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
In parallel, second stage of construction of DNA fragment was performed. DNA fragment containing CmR marker encoded by cat gene was obtained by PCR using primers P31 (SEQ ID NO: 94) and P9 (SEQ ID NO: 72) and pMW1 18-attL-Cm-attR plasmid as template. Primer P31 contains 36 hucleotides homologous to DNA fragment upstream of coding region of atoB gene and 28 nucleotides complementary to DNA fragment located at 3 '-end of attR region. Primer P9 contains BgIW restriction site and 28 nucleotides homologous to the 5 '-end of attL region. Following temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 52 °C, 1 minute at 72°C; and final polymerization: 7 minutes at 72°C. Taq DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
Obtained DNA fragments were purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to the recommendations of manufacturer followed by ethanol precipitation.
Two obtained DNA fragments were treated by BgIW restrictase followed by ligation using T4 DNA ligase (Maniatis T., Fritsch E. F., Sambrook, J.: Molecular Cloning: A Laboratory Manual. 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989).
Product obtained after ligation was amplified by PCR using primers P31 (SEQ ID NO: 94) H P30 (SEQ ID NO: 93). Following temperature profile was used for PCR: denaturation at 98°C during 1 minute; 30 cycles: 15 seconds at 98°C, 15 seconds at 55°C, 1 minute at 72°C; and final polimerization: 7 minutes at 72°C. Phusion DNA polymease and corresponding buffer from Finnzymes; and deoxynucleoside triphosphates from Fermentas (Lithuania) were used. Obtained PCR product (1823 base pairs) purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to the recommendations of manufacturer was used for electroporation of E.coli strain MGl 655 lacP
Figure imgf000037_0004
containing pKD46 plasmid with termosensitive replicon. pKD46 plasmid is
Figure imgf000037_0005
necessary for integration of the DNA fragment into chromosome of strain MG 1655
Figure imgf000037_0006
Figure imgf000037_0001
Εlectrocompetent cells of E. coli strain MG1655
Figure imgf000037_0002
lac
Figure imgf000037_0003
containing pKD46 plasmid were obtained as described in Example 1. Electroporation was performed with 70 μl of cells and «300 ng of PCR product. After electroporation cells were incubated in 1 ml of SOC medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) at 37°C during 2.5 hours, then cells were spreaded on plates with L-agar containing 30 mg/1 chloramphenicol and grown at 37°C to select CmR-recombinants. To eliminate pKD46 plasmid, cells were spreaded on LB agar to get separate colonies at 37°C and CmRAps variants were selected.
Correctness of desired structure of chromosome of selected CmRAps colonies was confirmed by PCR analysis using locus specific primers P32 (SEQ ID NO: 95) and P33 (SEQ ID NO: 96). Temperature profile for PCR testing descirbed in the Example 1 was used. Length of PCR product obtained in the reaction with cells of parent strain MG 1655 lacP Pπ-c-ideai-4-
Figure imgf000038_0001
was 187 base pairs. Length of PCR product obtained in the reaction with cells of mutant strain was 1938 base pairs. Mutant strain was temporarily named as MG1655 lαcP
Figure imgf000038_0002
φ P<rc-jdeai-4-
Figure imgf000038_0003
promoter).
2. Construction of strain MG1655 lαcf
Figure imgf000038_0007
P SD f/ E* P SD dr
Figure imgf000038_0004
P SDφ β
Then, Cm resistance gene (cat gene) was eliminated from chromosome of strain MGl 655
Figure imgf000038_0005
Figure imgf000038_0011
φio using
Figure imgf000038_0012
system. Strain MGl 655
Figure imgf000038_0013
P fadE φ /αd# a
Figure imgf000038_0009
Figure imgf000038_0008
ttR-cat-attL-
Figure imgf000038_0010
was transformed with pMWts- Int/Xis plasmid (WO 2007013638; RU 2005123423). Selection of transformed clones was performed on LB agar medium containing ampicillin (100 mg/1). Plates were incubated during night at 30°C. The cat gene was eliminated from transformed clones by culturing separate colonies at 37°C (at that termerature Cits repressor is partially inactivated while int/xis genes is activated) followed by selection of Cm Ap variants. Elimination of cat gene from chromosome of the strain was confirmed by PCR using locus specific primers P32 (SEQ ID NO: 95) and P33 (SEQ ID NO: 96). PCR conditions for the test were as described above in the Example 1. Lenght of PCR product obtained using cells with eliminated cat gene was 341 base pairs.
Then CmsApR variants were healed from helper plasmid pMWts-Int/Xis containing temperature sensitive replicon by second spreading to get seperate colonies at 37°C followed by selcetion of CmsAps clones.
Correctness of desired structure of new hybrid regulatory region introduced upstream of coding region of atoB gene was confirmed by sequence analysis. Thus, strain MGl 655 lacf
Figure imgf000038_0006
attB or abbreviated as MG1655
Figure imgf000039_0001
Figure imgf000039_0012
was obtained.
Example 7. Construction of E. coli strain in which activity of phosphoenolpyruvate carboxylase can be decreased.
1. Integration of promoter into chromosome of E. coli downstream of coding region of
Figure imgf000039_0003
ppc gene in orientation opposite to direction of native transcription of the gene under control its native promoter. Construction of strain MG1655
Figure imgf000039_0002
φ φ
To realize counter transcription of
Figure imgf000039_0010
gene, O promoter, hereinafter
Figure imgf000039_0005
Figure imgf000039_0006
promoter, was integrated into chromosome of strain MG 1655 lacP
Figure imgf000039_0011
downstream of coding
Figure imgf000039_0004
region of ppc gene in orientation opposite to direction of native transcription of the gene under control its native promoter. For integration method developed by Datsenko, K.A. and Wanner, B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) also known as "Red-driven integration" was used. Artificail DNA fragment integrated into the chromosome also contained CmR marker encoded by cat gene.
Construction of artificial DNA fragment integrated into corresponding region of bacterial chromosome was performed in several steps. First, DNA fragment containing BgIW restriction site, Prrc-ideai-4 promoter, SD sequence of φlO gene from phage T7 and 36 nucleotides including 22 nucleotides homologous to 3-end of coding region of ppc gene and 14 nucleotides homologous to region downstream of stop-codon of the gene was obtained by PCR. The DNA fragment was obtained in two stages. At first, DNA fragment containing BgIW restriction site, Prrc-ideai-4 promoter and part of SD sequence of φlO gene from phage T7 was obtained by PCR using plasmid
Figure imgf000039_0007
[Skorokhodova A. Yu. et al, 2006, Biotekhnologiya (Rus) 3, 6-16.]. PCR was performed using primers P5 (SEQ ID NO: 68) and P34 (SEQ ID NO: 97). Primer P5 contains BgIW restrictin site and region homolous to 5'-end of P
Figure imgf000039_0009
promoter. Primer P34 contains 36 nucleotides necessary for integration into chromosome and region complementary to 3-end of
Figure imgf000039_0008
promoter. Following temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 55°C, 30 seconds at 72°C; and final polymerization: 7 minutes at 72°C. Pfu DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used. In parallel, second stage of construction of DNA fragment was performed. DNA fragment containing CmR marker encoded by cat gene was obtained by PCR using primers P35 (SEQ ID NO: 98) and P9 (SEQ ID NO: 72) and pMW118-attL-Cm-attR plasmid as template. Primer P35 contains 36 hucleotides homologous to DNA fragment downstream of coding region of ppc gene and 28 nucleotides complementary to DNA fragment located at 3 '-end of attR region. Primer P9 contains BgIW restriction site and 28 nucleotides homologous to the 5'-end of attL region. Following temperature profile was used for PCR: denaturation at 95°C during 5 minutes; 25 cycles: 30 seconds at 95°C, 30 seconds at 52°C, 1 minute at 72°C; and final polymerization: 7 minutes at 72°C. Taq DNA polymerase, corresponding buffer and deoxynucleoside triphosphates from Fermentas (Lithuania) were used.
Obtained DNA fragments were purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to the recommendations of manufacturer followed by ethanol precipitation.
Two obtained DNA fragments were treated by BgIW restrictase followed by ligation using T4 DNA ligase (Maniatis T., Fritsch E. F., Sambrook, J.: Molecular Cloning: A Laboratory Manual. 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989).
Product obtained after ligation was amplified by PCR using primers P34 (SEQ ID NO: 97) and P35 (SEQ ID NO: 98). Following temperature profile was used for PCR: denaturation at 98°C during 1 minute; 30 cycles: 15 seconds at 98°C, 15 seconds at 55°C, 1 minute at 72°C; and final polimerization: 7 minutes at 72°C. Phusion DNA polymease and corresponding buffer from Finnzymes; and deoxynucleoside triphosphates from Fermentas (Lithuania) were used. Obtained PCR product (1808 base pairs) purified in agarose gel and isolated using Qiagen QIAquick Gel Extraction Kit according to the recommendations of manufacturer was used for electroporation of E.coli strain E.coli MG1655
Figure imgf000040_0004
fadE
Figure imgf000040_0005
containing pKD46 plasmid with termosensitive replicon. pKD46 plasmid is necessary for integration of the DNA fragment into chromosome of strain MG1655 l P
Figure imgf000040_0001
Figure imgf000040_0006
Electrocompetent cells of E. coli strain MGl 655 l
Figure imgf000040_0002
Figure imgf000040_0003
containing pKD46 plasmid were obtained as described in Example 1. Electroporation was performed with 70 μl of cells and »300 ng of PCR product. After electroporation cells were incubated in 1 ml of SOC medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) at 37°C during 2.5 hours, then cells were spreaded on plates wi -agar con ainin oramp enico an n a o se ec m - recombinants. To eliminate pKD46 plassmid, cells were spreaded on LB agar to get separate colonies at 37°C and CmRAps variants were selected.
Correctness of desired structure of chromosome of selected CmRAps colonies was confirmed by PCR analysis using locus specific primers P36 (SEQ ID NO: 99) and P37 (SEQ ID NO: 100). Temperature profile for PCR testing descirbed in the Example 1 was used. Length of PCR product obtained in the reaction with cells of parent strain MG 1655 lacF
Figure imgf000041_0006
was 293 base
Figure imgf000041_0001
pairs. Length of PCR product obtained in the reaction with cells of mutant strain was 2029 base pairs. Mutant strain was temporarily named as MG1655 lacF
Figure imgf000041_0005
φ
Figure imgf000041_0007
(before confirmation of sequence of
Figure imgf000041_0008
promoter).
2. Construction of Strain M
Figure imgf000041_0012
Figure imgf000041_0013
Then, Cm resistance gene (cat gene) was eliminated from chromosome of strain MG 1655 E
Figure imgf000041_0002
R using int-xis system. Strain MG1655
Figure imgf000041_0003
Figure imgf000041_0015
Figure imgf000041_0004
P E was transformed with p plasmid (WO 2007013638; RU 2005123423). Selection
Figure imgf000041_0014
of transformed clones was performed on LB agar medium containing ampicillin (100 mg/1). Plates were incubated during night at 30°C. The cat gene was eliminated from transformed clones by culturing separate colonies at 37°C (at that termerature Cits repressor is partially inactivated while int/xis genes is activated) followed by selection of CmsApR variants. Elimination of cat gene from chromosome of the strain was confirmed by PCR using locus specific primers P36 (SEQ ID NO: 99) and P37 (SEQ ID NO: 100). PCR conditions for the test were as described above in the Example 1. Lenght of PCR product obtained using cells with eliminated cat gene was 432 base pairs.
Then CmsApR variants were healed from helper plasmid pMWts-Int/Xis containing temperature sensitive replicon by second spreading to get seperate colonies at 37°C followed by selection of Cm Ap clones.
Correctness of desired structure of promoter introduced downstream of coding
Figure imgf000041_0011
region of ppc gene was confirmed by sequence analysis. Thus, strain MGl 655
Figure imgf000041_0010
Figure imgf000041_0009
attB or abbreviated as MG 1655
Figure imgf000042_0001
P was obtained.
Figure imgf000042_0007
Example 8. Production of 1-butanol by E. coli cells.
1. Production of 1-butanol using strain MG1655 l
Figure imgf000042_0008
Figure imgf000042_0002
f , c dea 4 φio in aerobic conditions.
Cells of the strain MGl 655
Figure imgf000042_0003
P and control strains MGl 655 and
Figure imgf000042_0010
Figure imgf000042_0004
MG1655 are grown overnight in M9 medium containing 2 g/1 of glucose at 37°C. One ml of obtained night cultures are deluted in 50 times with 49 ml of M9 medium containing 2 g/1 of glucose. Obtained cultures are grown in 250 ml flasks with ventilated plugs at 37°C on the shaker (250 rpm) during 6 hours. Obtained cell suspensions are centrifuged during 15 minutes at 4000 rpm and 4°C. Pellets are resuspended in 3 ml of M9 medium containing 30 g/1 of glucose and 10 g/1 of glycerol. Incubation of resulted cell cultures is performed during 24 hours in 20 x 200 mm test tubes with ventilated plugs at 37°C on shaker (200 rpm) with or without presence of 1 mM IPTG. Amount of 1-butanol in culture medium is determined by method of gas chromatography.
2. Production of 1-butanol using strain MG1655
Figure imgf000042_0011
f
Figure imgf000042_0009
in semiaerobic conditions.
Cells of strain MG 1655
Figure imgf000042_0005
Figure imgf000042_0006
and MG1655 were grown overnight in M9 medium containing 2 g/1 of glucose at 37°C. One ml of obtained night cultures were deluted in 50 times with 49 ml of M9 medium containing 2 g/1 of glucose. Obtained cultures were grown in 250 ml flasks with ventilated plugs at 37°C on the shaker (250 rpm) during 6 hours. Obtained cell suspensions were centrifuged during 15 minutes at 4000 rpm and 4°C. Pellets were resuspended in 7 ml of M9 medium containing 30 g/1 of glucose and 10 g/1 of glycerol. Incubation of resulted cell cultures was performed during 24 hours in 20 x 200 mm test tubes with ventilated plugs at 37°C on shaker (150 rpm) with or without presence of 1 mM IPTG. Amount of 1-butanol in culture medium was determined by method of gas chromatography using flame ionization detector on quartz capillary 30 meters column 30 M with internal diameter 0,30 mm and stationary phase FFAP (film thickness 0,30 micrometer). Preasure of gas (helium) on the entrence to the column was 1 ,6 atmosphere. Injector disposal was 1 :30. Thermostate temperature was 3 minutes at 70°C followed by increase of temperature up to 235°C with velocity 10°C per minute, holding 15 minute at the high temperature and decreasing to starting temperature. Temperature of injector and detector was 240°C. Results of measurement are presented in Table 2.
Also cells fo strain MGl 655
Figure imgf000043_0001
φ P
Figure imgf000043_0002
5 and control strains MGl 655 lacP
Figure imgf000043_0003
and MGl 655 were grown overnight in LB medium at 37°C. 0,5 ml of obtained night cultures were deluted 100 times with addition of 49 ml of LB medium. Obtained cultures were grown in 250 ml flasks with ventilated plugs at 37°C on the shaker (250 rpm) during 3 hours. Obtained cell suspensions were centrifuged during 15 minutes at 4000 rpm and 4°C. Pellets were resuspended in 7 ml of LB medium containing 20 g/1 of glucose. Incubation of resulted cell cultures was performed during 24 hours in 20 x 200 mm test tubes with ventilated plugs at 37°C on shaker (150 rpm) in presence of 1 mM IPTG. Amount of 1-butanol in culture medium was determined by method of gas chromatography as described above. Results of measurement are presented in Table 3.
3. Production of 1-butanol using strain MGl 655 lacf
Figure imgf000043_0004
Prrc-ideai-4-SDφl0-
Figure imgf000043_0008
in anaerobic conditions.
Cells of strain MGl 655
Figure imgf000043_0005
P
Figure imgf000043_0006
,re ideai 4 φio and control strains MGl 655 lαcP
Figure imgf000043_0007
and MG 1655 were grown overnight in M9 medium containing 2 g/1 of glucose at 37°C. One ml of obtained night cultures were deluted in 50 times with 49 ml of M9 medium containing 2 g/1 of glucose. Obtained cultures were grown in 250 ml flasks with ventilated plugs at 37°C on the shaker (250 rpm) during 6 hours. Obtained cell suspensions were centrifuged during 15 minutes at 4000 rpm and 4°C. Pellets were resuspended in 15 ml of M9 medium containing 30 g/1 of glucose. Incubation of resulted cell cultures was performed during 24 hours in 15 ml test tubes with non-ventilated plugs at 37°C on shaker (150 rpm) with or without presence of 1 mM IPTG. Amount of 1-butanol in culture medium was determined by method of gas chromatography as described above. Results of measurement are presented in Table 4.
4. Production of 1-butanol using strain MG1655
Figure imgf000043_0010
Figure imgf000043_0009
Cultivation of the strain
Figure imgf000043_0012
in aerobic, semiaerobic and anaerobic
Figure imgf000043_0011
conditions is performed as described above. Amount of 1-butanol in culture medium is determined by method of gas chromatography as described above.
While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. Each of the aforementioned documents is incorporated by reference herein in its entirety.
Figure imgf000045_0001

Claims

What we claim is:
1. A l -butanol producing microorganism modified so that at least one of enzymes from pathway of fatty acids β-oxidation is involved into 1 -butanol biosynthesis.
2. The microorganism according to claim 1, wherein the enzyme from pathway of fatty acids β-oxidation is native or heterogeneous for the microorganism.
3. The microorganism according to claim 1 , wherein the enzyme from pathway of fatty acids β-oxidation is selected from group consisting of acyl-CoA dehydrogenase, enoyl- CoA hydratase, 3-hydroxyacetyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase.
4. The microorganism according to claim 3, wherein acyl-CoA dehydrogenase comprises amino acid sequence selected from any of SEQ ID NO: 1-17.
5. The microorganism according to claim 3, wherein enoyl-CoA hydratase comprises amino acid sequence selected from any of SEQ ID NO: 18-38.
6. The microorganism according to claim 3, wherein 3-hydroxyacetyl-CoA dehydrogenase comprises amino acid sequence selected from any of SEQ ID NO: 18, 19, 23-25, 27, 31- 33, 39-44.
7. The microorganism according to claim 3, wherein 3-ketoacyl-CoA thiolase comprises amino acid sequence selected from any of SEQ ID NO: 45-61.
8. The microorganism according to claim 3, wherein the microorganism is modified to have increased activity of one or several said enzymes in comparison to non-modifieid microorganism.
9. The microorganism according to claim 8, wherein activity of one or several enzymes is increased by increasing amount of corresponding mRNA, increasing amount of the enzyme, or increasing specific enzyme activity.
10. The microorganism according to claim 9, wherein amount of corresponding mRNA is increased by increasing copy number of gene or by enhancing gene expression due to use of potent promoter, removing repression or increasing mRNA stability.
11. The microorganism according to claim 9, wherein amount of enzyme is increased by enhancing translation of corresponding mRNA due to modification of ribosome binding site or increasing its stability.
12. The microorganism according to claim 9, wherein specific enzyme activity is increased by introducing mutatins into amino acid sequence of the enzyme.
13. The microorganism according to claim 1, wherein said microorganism is selected from group consisting of bacteria, yeast and fungi.
14. The microorganism according to claim 13, wherein said microorganism belongs to the genus Clostridium, Escherichia, Salmonella, Shigella, Pseudomonas, Zymomonas, , , , , , Streptomyces, Pichia, Candida or Sacchoromyces.
15. The microorganism according to claim 14, wherein said microorganism is Clostridium acetobutylicum or Clostridium beijerinckii.
16. The microorganism according to claim 14, wherein said microorganism is Escherichia coli.
17. The microorganism according to claim 14, wherein said microorganism is Salmonella ent erica.
18. The microorganism according to claim 14, wherein said microorganism is Shigella βexneri.
19. The microorganism according to claim 14, wherein said microorganism is Pseudomonas aeruginosa or Pseudomonas putida
20. The microorganism according to claim 14, wherein said microorganism is Zymomonas mobilis.
21. The microorganism according to claim 14, wherein said microorganism is Bacillus subtilis or Bacillus amyloliquefaciens.
22. The microorganism according to claim 14, wherein said microorganism is Lactobacillus plantarum.
23. The microorganism according to claim 14, wherein said microorganism is Enterococcus faecium.
24. The microorganism according to claim 14, wherein said microorganism is Corynebacterium glutamicum or Brevibacterium flavum.
25. The microorganism according to claim 14, wherein said microorganism is Streptomyces coelicolor.
26. The microorganism according to claim 14, wherein said microorganism is Pichia pastoris.
27. The microorganism according to claim 14, wherein said microorganism is Saccharomyces cerevisiae.
28. The microorganism according to claim 1, wherein said microorganism is additionally modified to increase intracellular amount of acetyl-CoA.
29. The microorganism according to claim 28, wherein intracellular amount of acetyl-CoA is increased by regulated decrease of phosphoenolpyruvate carboxylase activity.
30. A method for producing 1-butanol including cultivation of microorganism according to claim 1 in culture medium and collecting produced 1-butanol from the medium.
31. The method according to claim 30, wherein process of microorganism cultivation is aerobic.
32. The method according to claim 30, wherein process of microorganism cultivation is anaerobic.
33. The method according to claim 30, wherein process of microorganism cultivation is microaerobic.
34. The method according to claim 30, wherein process of microorganism cultivation includes aerobic and anaerobic steps.
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CN113652440A (en) * 2021-08-05 2021-11-16 昆明理工大学 3-ketoacyl-coenzyme A thiolase gene RKACAA1-2 and application thereof
CN113621630B (en) * 2021-08-05 2023-03-24 昆明理工大学 3-ketoacyl-CoA thiolase gene RkACAA1-1 and application thereof
CN113652440B (en) * 2021-08-05 2023-04-21 昆明理工大学 3-ketoacyl coenzyme A thiolase gene RKACAA1-2 and application thereof

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