WO2022198846A1 - 一种产泛解酸的微生物及其构建方法和应用 - Google Patents

一种产泛解酸的微生物及其构建方法和应用 Download PDF

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WO2022198846A1
WO2022198846A1 PCT/CN2021/105954 CN2021105954W WO2022198846A1 WO 2022198846 A1 WO2022198846 A1 WO 2022198846A1 CN 2021105954 W CN2021105954 W CN 2021105954W WO 2022198846 A1 WO2022198846 A1 WO 2022198846A1
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gene
promoter
sequence
escherichia coli
dna fragment
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PCT/CN2021/105954
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English (en)
French (fr)
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张学礼
刘萍萍
唐金磊
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中国科学院天津工业生物技术研究所
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Priority to US18/271,665 priority Critical patent/US20240011059A1/en
Priority to EP21932460.5A priority patent/EP4261272A1/en
Publication of WO2022198846A1 publication Critical patent/WO2022198846A1/zh

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Definitions

  • the invention relates to a pantoate-producing microorganism in the field of biotechnology and a construction method and application thereof.
  • Calcium pantothenate also known as vitamin B5
  • Vitamin B5 is one of the 13 essential vitamins for the human body.
  • Calcium pantothenate can only be synthesized in microorganisms and plants, and is an important precursor for the synthesis of coenzyme A and an essential vitamin for maintaining the normal physiological functions of organisms.
  • calcium pantothenate is also widely used as food additives and pharmaceutical raw materials.
  • calcium pantothenate is mainly obtained by splitting DL-pantolactone by biological or chemical methods to obtain high-purity D-pantolactone and then reacting with ⁇ -alanine.
  • Pantolactone is an important precursor for the synthesis of calcium pantothenate, and its synthesis is currently mainly accomplished by chemical means using petrochemical materials.
  • the materials used to synthesize DL-pantolactone are mainly petrochemical materials such as isobutyraldehyde and formaldehyde, which are realized through a series of chemical reactions, such as methylolation, acidification or aldol condensation. This process requires a large amount of highly toxic raw materials such as sodium cyanide or chemical reagents such as strong alkali and strong acid, and generates a large amount of waste water and waste gas, which is very destructive and irreparable to the environment.
  • the technical problem to be solved by the present invention is how to produce pantoic acid.
  • the present invention first provides a method for constructing a recombinant Escherichia coli, which comprises: carrying out the following A1-A25 transformations on the starting Escherichia coli to obtain a recombinant Escherichia coli:
  • A2 replace the promoter that drives the acetolactate synthase I large subunit gene ilvB gene with the M1-93 promoter, and the M1-93 promoter is any of the following DNA molecules:
  • nucleotide sequence of one chain is the DNA molecule of sequence 3 in the sequence listing;
  • a2) A DNA molecule that has more than 80% identity with the DNA molecule of a1) and has a promoter function;
  • A4 mutating the ilvH gene of the acetolactate synthase III regulatory subunit gene into an ilvH mutant gene, and the ilvH mutant gene encodes the protein shown in sequence 5 in the sequence listing;
  • A6 Introduce the ilvD gene encoding the dihydroxy acid dehydratase, and express the ilvD gene;
  • panB gene derived from Escherichia coli, denoted as E-panB gene, and express the E-panB gene;
  • panB gene derived from Corynebacterium glutamicum, which is denoted as C-panB gene, and express the C-panB gene;
  • the ilvE mutant gene encodes the protein shown in sequence 12 in the sequence listing;
  • A15 Introduce the phosphoserine/phosphohydroxythreonine aminotransferase gene serC gene and the phosphoserine phosphatase gene serB gene, and express the serC gene and the serB gene;
  • the alsS gene can be derived from Bacillus subtilis, such as Bacillus subtilis 168.
  • the ilvC gene can be derived from Escherichia coli, such as Escherichia coli ATCC8739.
  • the ilvD gene can be derived from Escherichia coli, such as Escherichia coli MG1655.
  • the panE gene can be derived from Escherichia coli, such as Escherichia coli MG1655.
  • the glyA gene can be derived from Escherichia coli, such as Escherichia coli ATCC 8739.
  • the serA gene can be derived from Corynebacterium glutamicum, such as Corynebacterium glutamicum ATCC13032.
  • the serC gene and the serB gene can be derived from Escherichia coli, such as Escherichia coli MG1655.
  • the E-panB gene can be derived from Escherichia coli MG1655.
  • the C-panB gene can be derived from Corynebacterium glutamicum ATCC13032.
  • the alsS gene can encode the alsS protein shown in SEQ ID NO: 2 in the sequence listing.
  • the C-panB gene can encode the C-panB protein shown in SEQ ID NO: 10 in the sequence listing.
  • the serA gene can encode the serA protein shown in sequence 14 in the sequence listing.
  • the serC gene can encode the serC protein shown in sequence 16 in the sequence listing.
  • the serB gene can encode the serB protein shown in sequence 17 in the sequence listing.
  • the sequence of the alsS gene may be sequence 1 in the sequence listing.
  • the sequence of the ilvH mutant gene can be sequence 4 in the sequence listing.
  • the sequence of the C-panB gene can be sequence 9 in the sequence listing.
  • the sequence of the ilvE mutant gene can be sequence 11 in the sequence listing.
  • the sequence of the serA gene can be sequence 13 in the sequence listing.
  • the sequence of the serC gene can be positions 89-1177 of sequence 15 in the sequence listing.
  • the sequence of the serB gene can be positions 1199-2167 of sequence 15 in the sequence listing.
  • A1 can be achieved by introducing an alsS gene expression cassette into the recipient E. coli, the alsS gene expression cassette containing a promoter and the alsS gene driven by the promoter.
  • A5 can be achieved by introducing into the recipient E. coli an ilvC gene expression cassette containing a promoter and the ilvC gene driven by the promoter.
  • A6 can be achieved by introducing into the recipient E. coli an ilvD gene expression cassette containing a promoter and the ilvD gene driven by the promoter.
  • A7 can be achieved by introducing into the recipient E. coli an E-panB gene expression cassette containing a promoter and the E-panB gene driven by the promoter.
  • A8 can be achieved by introducing into the recipient E. coli a panE gene expression cassette containing a promoter and the panE gene driven by the promoter.
  • A9 can be achieved by introducing into the recipient E. coli a glyA gene expression cassette containing a promoter and the glyA gene driven by the promoter.
  • A12 can be achieved by introducing into the recipient E. coli a C-panB gene expression cassette containing a promoter and the C-panB gene driven by the promoter.
  • A14 can be achieved by introducing into the recipient E. coli a serA gene expression cassette containing a promoter and the serA gene driven by the promoter.
  • A15 can be achieved by introducing a serCB gene expression cassette containing a promoter and the serC gene and the serB gene driven by the promoter into the recipient E. coli.
  • the promoter in A1, A7, A12, A14 or A15 may be the M1-93 promoter.
  • the promoter described in A5 or A9 can be the M1-46 promoter, and the M1-46 promoter is any of the following DNA molecules:
  • nucleotide sequence of one chain is the DNA molecule of sequence 6 in the sequence listing;
  • the promoter described in A6 can be the RBSL1 promoter, and the RBSL1 promoter is any of the following DNA molecules:
  • nucleotide sequence of one chain is the DNA molecule of sequence 7 in the sequence listing;
  • a2) A DNA molecule that is more than 80% identical to the DNA molecule of a1) and has a promoter function.
  • the promoter described in A8 can be the RBSL2 promoter, and the RBSL2 promoter is any of the following DNA molecules:
  • nucleotide sequence of one chain is the DNA molecule of sequence 8 in the sequence listing;
  • c2) A DNA molecule that is more than 80% identical to the DNA molecule of c1) and has a promoter function.
  • identity refers to sequence similarity to a native nucleic acid sequence. “Identity” includes nucleotide sequences that are 80% or more, or 85% or more, or 90% or more, or 95% or more identical to a nucleotide sequence of the invention. Identity can be assessed with the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
  • the above-mentioned 80% or more identity may be 85%, 90% or 95% or more identity.
  • the serCB gene expression cassette can be shown as sequence 15 in the sequence listing.
  • the starting Escherichia coli can be Escherichia coli ATCC 8739.
  • the recombinant Escherichia coli obtained by the method for constructing the recombinant Escherichia coli also belongs to the protection scope of the present invention.
  • the construction method of the recombinant Escherichia coli is used to obtain Escherichia coli Span050, Escherichia coli Span050 is deposited in the General Microbiology Center of China Microorganism Culture Collection Management Committee, and the registration number of the collection center is CGMCC No.21699.
  • the present invention also provides a method for producing pantoate, comprising: culturing the recombinant Escherichia coli to obtain a fermentation product; and obtaining pantoate from the fermentation product.
  • Cultivation of the recombinant E. coli can be carried out using a medium that contains glucose and can be used for culturing E. coli.
  • Described culture medium can be culture medium 1, culture medium 2 or culture medium 3, described culture medium 1 is made up of solvent and solute, solvent is water, solute and its concentration in described culture medium 1 are respectively: glucose 20g /L, (NH 4 ) 2 HPO 4 3.5g/L, KH 2 PO 4 3.91g/L, K 2 HPO 4 4.48g/L, MgSO 4 ⁇ 7H 2 O 0.18g/L, betaine-HCl 0.15g /L, FeCl 3 ⁇ 6H 2 O 1.5 ⁇ g/L, CoCl 2 ⁇ 6H 2 O 0.1 ⁇ g/L, CuCl 2 ⁇ 2H 2 O 0.1 ⁇ g/L, ZnCl 2 0.1 ⁇ g/L, Na 2 MoO 4 ⁇ 2H 2 O 0.1 ⁇ g/L, MnCl 2 ⁇ 4H 2 O 0.2 ⁇ g/L, H 3 BO 3 0.05 ⁇ g/L;
  • the medium 2 is composed of a solvent and a solute, the solvent is water, and the solute and its concentration in the medium 2 are respectively: glucose 50 g/L, (NH 4 ) 2 HPO 4 3.5 g/L, KH 2 PO 4 3.91g/L, K 2 HPO 4 4.48g/L, MgSO 4 7H 2 O 0.18g/L, Betaine-HCl 0.15g/L, 5g/L Serine, FeCl 3 6H 2 O 1.5 ⁇ g/L , CoCl 2 ⁇ 6H 2 O 0.1 ⁇ g/L, CuCl 2 ⁇ 2H 2 O 0.1 ⁇ g/L, ZnCl 2 0.1 ⁇ g/L, Na 2 MoO 4 ⁇ 2H 2 O 0.1 ⁇ g/L, MnCl 2 ⁇ 4H 2 O 0.2 ⁇ g/L, H 3 BO 3 0.05 ⁇ g/L;
  • Described culture medium 3 is made up of solvent and solute, and the solvent is water, and solute and its concentration in described culture medium 3 are respectively: glucose 30g/L, magnesium sulfate 5g/L, potassium dihydrogen phosphate 10.5g/L, Yeast powder 20g/L, diammonium hydrogen phosphate 6g/L, citric acid monohydrate 1.84g/L, FeCl 3 6H 2 O 1.5 ⁇ g/L, CoCl 2 6H 2 O 0.1 ⁇ g/L, CuCl 2 2H 2 O 0.1 ⁇ g/L, ZnCl 2 0.1 ⁇ g/L, Na 2 MoO 4 ⁇ 2H 2 O 0.1 ⁇ g/L, MnCl 2 ⁇ 4H 2 O 0.2 ⁇ g/L, H 3 BO 3 0.05 ⁇ g/L.
  • glucose can be added to the culturing system according to the culturing conditions.
  • the culturing can be performed at 37°C.
  • the present invention also provides any of the following applications:
  • Taxonomic name Escherichia coli
  • preservation unit General Microbiology Center of China Microorganism Culture Collection Management Committee
  • ATCC 8739, M1-93 and M1-46 are all E. coli.
  • two primers separated by "/" are used to form a corresponding primer pair for amplifying the target fragment.
  • the acetolactate synthase gene alsS from Bacillus subtilis 168 was inserted into the propionate kinase encoding gene tdcD and formate on the chromosome by two-step homologous recombination Acetyltransferase encodes the tdcE site of the gene, and the specific steps are as follows:
  • the primers tdcDE-incs-up/tdcDE-incs-down were used to amplify the 2719bp DNA fragment I, this fragment contains 50bp upstream homology arm of tdcDE, 2619bp cat-sacB fragment of chloramphenicol gene (cat) and fructan sucrose transferase gene (sacB) DNA, and 50bp downstream homology arm of tdcDE gene, using in the first step of homologous recombination.
  • the amplification system was: Phusion 5X buffer (NewEngland Biolabs) 10 ⁇ l, dNTP (10mM each dNTP) 1 ⁇ l, DNA template 20ng, primers (10 ⁇ M) 2 ⁇ l each, Phusion High-Fidelity DNA polymerase (2.5U/ ⁇ l) 0.5 ⁇ l, distilled water 33.5 ⁇ l, the total volume is 50 ⁇ l.
  • the amplification conditions were pre-denaturation at 98°C for 2 minutes (1 cycle); denaturation at 98°C for 10 seconds, annealing at 56°C for 10 seconds, extension at 72°C for 2 minutes (30 cycles); extension at 72°C for 10 minutes (1 cycle).
  • the above-mentioned DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid (CGSC Escherichia coli Collection, Yale University, CGSC#7739) was transformed into Escherichia coli ATCC 8739 by electroporation, and then the DNA fragment I was electroporated into E. coli ATCC 8739 with pKD46.
  • Electroporation conditions are: firstly prepare electrotransformed competent cells of Escherichia coli ATCC 8739 with pKD46 plasmid (preparation method is according to Dower et al., 1988, Nucleic Acids Res 16:6127-6145); 50 ⁇ l of competent cells are placed on ice Add 50ng of DNA Fragment I, place on ice for 2 minutes, and transfer to a 0.2cm Bio-Rad electroporation cup. A MicroPulser (Bio-Rad Company) electroporator was used, and the electric shock parameter was a voltage of 2.5 kV.
  • LB medium was transferred to the electric shock cup, pipetted 5 times and then transferred to a test tube, incubated at 75 rpm and 30 °C for 2 hours. Take 200 ⁇ l of bacterial solution and spread it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml).
  • XZ-tdcDE-up/XZ-tdcDE-down the correct colony amplification product is a fragment of 3615bp, including 845bp of tdcDE upstream homology arm, cat-sacB fragment of 2619bp and tdcDE downstream homology arm of 151bp. Pick a correct single colony and name it Span001.
  • DNA fragment II was amplified with primers tdcDE-alsSin-up/tdcDE-alsSin-down, including the 50bp upstream homology arm of tdcDE, alsS gene 1716bp and sacI restriction site and protection base total 10bp, and tdcDE downstream homology arm 50bp.
  • DNA fragment II was used for the second homologous recombination. Amplification conditions and systems were the same as described in the first step. DNA fragment II was electroporated into strain Span001.
  • the electroporation conditions are: firstly prepare the electrotransformed competent cells of Span001 with pKD46 plasmid; put 50 ⁇ l of competent cells on ice, add 50 ng DNA fragment II, place on ice for 2 minutes, transfer to 0.2 cm Bio-Rad for electroporation cup. A MicroPulser (Bio-Rad Company) electroporator was used, and the electric shock parameter was a voltage of 2.5 kV. Immediately after the electric shock, 1 ml of LB medium was transferred to the electric shock cup, pipetted 5 times and then transferred to a test tube, incubated at 75 rpm and 30 °C for 4 hours.
  • the bacterial liquid was transferred to LB liquid medium containing 10% sucrose without sodium chloride (50ml medium in a 250ml flask), and after culturing for 24 hours, it was streaked on the LB solid medium containing 6% sucrose without sodium chloride. line cultivation.
  • the primers used were XZ-tdcDE-up/XZ-tdcDE-down, and the correct colony amplification product was a fragment of 2722 bp, including the 845 bp upstream homology arm of tdcDE, alsS gene and sacI restriction sites totaling 1726 bp and tdcDE Downstream homology arm 151bp. Pick a single correct colony and name it Span002.
  • Span002 is a propionate kinase encoding gene tdcD and formate acetyltransferase that integrates the acetolactate synthase gene (alsS gene, the nucleotide sequence is sequence 1 in the sequence listing, the alsS protein shown in coding sequence 2) into Escherichia coli ATCC 8739
  • the first step using the pXZ-CS plasmid DNA as a template, use the primers tdcDE-incs-up/alsSPro-CS-down to amplify a 2719bp DNA fragment I, including the 50bp upstream homology arm of tdcDE, the cat-sacB fragment of 2619bp and alsS
  • the downstream homology arm of the gene is 50bp, which is used for the first step of homologous recombination.
  • the amplification system and amplification conditions were the same as described in Example 1. DNA Fragment I was electroporated into Span002.
  • DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span002, and then DNA fragment I was electroporated into E. coli Span002 with pKD46.
  • the electroporation conditions and procedures were the same as the first method described in Example 1 for the integration of alsS at the tdcDE site. Take 200 ⁇ l of bacterial solution and spread it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml), after overnight incubation at 30 °C, select a single colony for PCR verification, using primers For verification by XZ-tdcDE-up/tdcDE-YZ285-down, the correct PCR product should be 3749 bp, including 845 bp upstream homology arm of tdcDE, 2619 bp cat-sacB fragment and 285 bp downstream homology arm of alsS, pick a correct single colony, Named Span003.
  • DNA fragment II includes 50bp upstream homology arm of tdcDE, 88bp M1-93 promoter and 50bp downstream homology arm of alsS. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span003.
  • the electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site. Colony PCR was used to verify the clone. The primers used were XZ-tdcDE-up/tdcDE-YZ285-down. The correct colony amplification product was a fragment of 1218bp, including 8454bp upstream homology arm of tdcDE, 88bp M1-93 promoter sequence and alsS The downstream homology arm is 285bp, pick a correct single colony and name it Span004.
  • Span004 is a recombinant bacterium obtained by integrating the M1-93 promoter (nucleotide sequence is sequence 3 in the sequence listing) into the upstream of the alsS gene of E. coli Span002.
  • the M1-93 promoter in the recombinant bacterium drives the expression of the alsS gene.
  • DNA fragment I was amplified using the primers ilvB pro-catup/ilvB pro-catdown for the first step of homologous recombination.
  • DNA fragment I includes a 50 bp upstream homology arm of ilvB, a 2619 bp cat-sacB fragment and a 50 bp downstream homology arm of ilvB.
  • the amplification system and amplification conditions were the same as described in Example 1.
  • DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span004, and then DNA fragment I was electroporated into E. coli Span004 with pKD46.
  • the electroporation conditions and procedures were the same as the first method described in Example 1 for the integration of alsS at the tdcDE site. Take 200 ⁇ l of bacterial solution and spread it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml), after overnight incubation at 30 °C, select a single colony for PCR verification, using primers ilvB pro-YZup/ilvB pro-YZdown was verified, the correct PCR product should be 2996bp, including 123bp upstream homology arm of ilvB, 2619bp cat-sacB fragment and 254bp downstream homology arm of ilvB. Pick a correct single colony and name it Span005.
  • DNA fragment II includes 50 bp upstream homology arm of ilvB, 88 bp M1-93 promoter and 50 bp downstream homology arm of ilvB. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span005.
  • the electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site.
  • the clone was verified by colony PCR.
  • the primers used were ilvB pro-YZup/ilvB pro-YZdown.
  • the correct colony amplification product was a fragment of 465 bp, including the 123 bp upstream homology arm of ilvB, 88 bp of M1-93 promoter and ilvB downstream homology. Arm 254bp. Pick a single correct colony and name it Span006.
  • M1-93 promoter nucleotide sequence is sequence 3 in the sequence listing
  • acetolactate synthase II large subunit gene ilvG is regulated by the method of two-step homologous recombination using artificial regulatory element M1-93, and the specific steps are as follows:
  • DNA fragment I was amplified using the primers ilvG pro-catup/ilvG pro-catdown for the first step of homologous recombination.
  • DNA fragment I includes 50 bp upstream homology arm of ilvG, 2619 bp cat-sacB fragment and 50 bp downstream homology arm of ilvG.
  • the amplification system and amplification conditions were the same as described in Example 1.
  • DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span006, and then DNA fragment I was electroporated into E. coli Span006 with pKD46.
  • DNA fragment II includes 50 bp upstream homology arm of ilvG, 88 bp M1-93 promoter and 50 bp downstream homology arm of ilvG. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span007.
  • the electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site. Colony PCR was used to verify the clone. The primers used were ilvG pro-YZup/ilvG p-YZdown. The correct colony amplification product was a fragment of 462 bp, including the 179 bp upstream homology arm of ilvG, the 88 bp M1-93 fragment and the downstream homology arm of ilvG. 195bp. Pick a single correct colony and name it Span008.
  • a mutation was introduced into the ilvH gene of the acetolactate synthase III regulatory subunit gene by the method of two-step homologous recombination to relieve the feedback inhibition of L-valine.
  • the specific steps are as follows:
  • DNA fragment I includes a 50 bp upstream homology arm of ilvH, a 2619 bp cat-sacB fragment and a 50 bp downstream homology arm of ilvH.
  • the amplification system and amplification conditions were the same as described in Example 1.
  • DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span008, and then DNA fragment I was electroporated into E. coli Span008 with pKD46.
  • DNA fragment II was amplified with primers ilvH*-mut-up/ilvH*-mut-down.
  • DNA fragment II is the ilvH gene containing the mutation.
  • DNA fragment II was used for the second homologous recombination.
  • DNA fragment II was electroporated into strain Span009.
  • Span010 is a recombinant bacterium obtained by mutating the acetolactate synthase gene ilvH of Escherichia coli Span008 into the ilvH* gene (ie, the ilvH mutant gene).
  • the sequence of the ilvH* gene is the sequence 4 in the sequence table, and the coding sequence 5 ilvH* protein shown.
  • Example 6 Integration of the gene ilvC encoding acetohydroxy acid reductase isomerase at the adhE site of alcohol dehydrogenase and knockout of the adhE gene
  • the acetohydroxy acid reductoisomerase encoding gene ilvC from Escherichia coli was integrated into the alcohol dehydrogenase adhE site by a two-step homologous recombination method.
  • the specific steps include:
  • DNA fragment I includes a 50 bp upstream homology arm of adhE, a 2619 bp cat-sacB fragment and a 50 bp downstream homology arm of adhE.
  • the amplification system and amplification conditions were the same as described in Example 1.
  • DNA Fragment I was electroporated into Span010.
  • the DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid (Datsenko and Wanner 2000, Proc Natl Acad Sci USA 97:6640-6645; the plasmid was purchased from the Yale University CGSC Escherichia coli Collection, CGSC#7739) Transform into E. coli Span010 by electroporation, and then electroporate DNA fragment I into E. coli Span010 with pKD46.
  • the electroporation conditions and procedures were the same as the first method described in Example 1 for the integration of alsS at the tdcDE site. Take 200 ⁇ l of bacterial solution and spread it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml), after overnight incubation at 30 °C, select a single colony for PCR verification, using primers For verification by XZ-adhE-up/XZ-adhE-down, the correct PCR product should be 3167bp, including 221bp upstream homology arm of adhE, 2619bp cat-sacB fragment and 327bp downstream homology arm of adhE. Pick a correct single colony and name it Span011.
  • DNA fragment II was amplified with primers adhE-ilvC-up/adhE-ilvC-down.
  • DNA fragment II includes a 50 bp upstream homology arm of adhE, 1476 bp of ilvC gene and a 50 bp downstream homology arm of adhE.
  • DNA fragment II was used for the second homologous recombination.
  • DNA fragment II was electroporated into strain Span011.
  • the electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site.
  • the clone was verified by colony PCR.
  • the primers used were XZ-adhE-up/XZ-adhE-down.
  • the correct colony amplification product was a fragment of 2024bp, including the 221bp upstream homology arm of adhE, the ilvC gene 1476bp and the downstream homology arm of adhE. 327bp. Pick a correct single colony and name it Span012.
  • DNA fragment I used for the first step of homologous recombination.
  • DNA fragment I includes a 50 bp upstream homology arm of adhE, a 2619 bp cat-sacB fragment and a 50 bp downstream homology arm of ilvC.
  • the amplification system and amplification conditions were the same as described in Example 1.
  • DNA Fragment I was electroporated into Span012.
  • the DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid (Datsenko and Wanner 2000, Proc Natl Acad Sci USA 97:6640-6645; the plasmid was purchased from the Yale University CGSC Escherichia coli Collection, CGSC#7739) Transform into E. coli Span012 by electroporation, and then electroporate DNA fragment I into E. coli Span012 with pKD46.
  • the electroporation conditions and procedures were the same as the first method described in Example 1 for the integration of alsS at the tdcDE site. Take 200 ⁇ l of bacterial solution and spread it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml), after overnight incubation at 30 °C, select a single colony for PCR verification, using primers For verification by XZ-adhE-up/ilvC-YZ347-down, the correct PCR product should be 3187bp, including the 221bp upstream homology arm of adhE, the 2619bp cat-sacB fragment and the 347bp downstream homology arm of ilvC. Pick a correct single colony and name it Span013.
  • DNA fragment II includes a 50 bp upstream homology arm of adhE, an 88 bp M1-46 promoter sequence and a 50 bp downstream homology arm of ilvC. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span013.
  • the electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site. Colony PCR was used to verify the clone. The primers used were XZ-adhE-up/ilvC-YZ347-down. The correct colony amplification product was a fragment of 656 bp, including 221 bp of the upstream homology arm of adhE, 88 bp of the M1-46 promoter and downstream of ilvC. Homology arm 347bp. Pick a single correct colony and name it Span014.
  • Span014 is a recombinant bacterium obtained by integrating the M1-46 promoter (nucleotide sequence is sequence 6 in the sequence listing) into the upstream of the ilvC gene of E. coli Span012.
  • the M1-46 promoter in the recombinant bacterium drives the expression of the ilvC gene.
  • Example 8 Integration of the gene ilvD encoding dihydroxy acid dehydratase at the pflB site of the gene encoding pyruvate formate lyase and knockout of the pflB gene
  • the dihydroxy acid dehydratase encoding gene ilvD from Escherichia coli was integrated into the pflB site of the pyruvate formate lyase encoding gene by two-step homologous recombination and the pflB gene was knocked out.
  • the specific steps are as follows:
  • DNA fragment I includes pflB upstream homology arm 50bp, cat-sacB fragment 2619bp and pflB downstream homology arm 50bp.
  • the amplification system and amplification conditions were the same as described in Example 1.
  • DNA Fragment I was electroporated into Span014.
  • DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span014, and then the DNA fragment I was electroporated into E. coli Span014 with pKD46.
  • the electroporation conditions and procedures were the same as the first method described in Example 1 for the integration of alsS at the tdcDE site. Take 200 ⁇ l of bacterial solution and spread it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml), after overnight incubation at 30 °C, select a single colony for PCR verification, using primers For verification by XZ-pflB-up600/XZ-pflB-down, the correct PCR product should be 3675bp, including pflB upstream homology arm 641bp, cat-sacB fragment 2619bp and pflB downstream homology arm 415bp. Pick a correct single colony and name it Span015.
  • DNA fragment I of 1951 bp was amplified from the gene of Escherichia coli MG1655 (from ATCC, No. 700926) with primers pflB-ilvD-up/pflB-ilvD-down.
  • DNA fragment II includes pflB upstream homology arm 50bp, ilvD gene 1851bp and pflB downstream homology arm 50bp.
  • DNA fragment II was used for the second homologous recombination.
  • DNA fragment II was electroporated into strain Span015.
  • the electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site.
  • the clone was verified by colony PCR.
  • the primers used were XZ-pflB-up600/XZ-pflB-down.
  • the correct colony amplification product was a fragment of 2996 bp, including pflB upstream homology arm 641 bp, ilvD gene 1851 bp and pflB downstream homology arm 415bp. Pick a single correct colony and name it Span016.
  • DNA fragment I includes pflB upstream homology arm 50bp, cat-sacB fragment 2619bp and ilvD downstream homology arm 50bp.
  • the amplification system and amplification conditions were the same as described in Example 1.
  • DNA Fragment I was electroporated into Span016.
  • DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span016, and then DNA fragment I was electroporated into E. coli Span016 with pKD46.
  • the electroporation conditions and procedures were the same as the first method described in Example 1 for the integration of alsS at the tdcDE site. Take 200 ⁇ l of bacterial solution and spread it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml), after overnight incubation at 30 °C, select a single colony for PCR verification, using primers XZ-pflB-up600/ilvD-YZ496-down was verified, the correct PCR product should be 3756bp, including pflB upstream homology arm 641bp, cat-sacB fragment 2619bp and ilvD downstream homology arm 496bp. Pick a correct single colony and name it Span017.
  • DNA fragment II includes a 50 bp upstream homology arm of pflB, an 89 bp artificial regulatory element RBSL1 sequence and a 50 bp downstream homology arm of ilvD. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span017.
  • the electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site.
  • the clone was verified by colony PCR.
  • the primers used were XZ-pflB-up600/ilvD-YZ496-down.
  • the correct colony amplification product was a fragment of 1226 bp, including the 641 bp upstream homology arm of pflB, the 89 bp RBSL1 sequence and the downstream homology arm of ilvD. 496bp. Pick a correct single colony and name it Span018.
  • Span018 is a recombinant bacterium obtained by integrating the RBSL1 promoter (nucleotide sequence is sequence 7 in the sequence listing) into the upstream of the ilvD gene of E. coli Span016.
  • the RBSL1 promoter in the recombinant bacterium drives the expression of the ilvD gene.
  • DNA fragment I includes frd upstream homology arm 50bp, cat-sacB fragment 2619bp and frd downstream homology arm 50bp.
  • the amplification system and amplification conditions were the same as described in Example 1.
  • DNA Fragment I was electroporated into Span018.
  • the DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span018, and then the DNA fragment I was electroporated into E. coli Span018 with pKD46.
  • the electroporation conditions and procedures were the same as the first method described in Example 1 for the integration of alsS at the tdcDE site. Take 200 ⁇ l of bacterial solution and spread it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml), after overnight incubation at 30 °C, select a single colony for PCR verification, using primers For verification by XZ-frd-up/XZ-frd-down, the correct PCR product should be 3440bp, including frd upstream homology arm 426bp, cat-sacB fragment 2619bp and frd downstream homology arm 395bp. Pick a correct single colony and name it Span019.
  • DNA fragment II of 895 bp was amplified with primers frd-panB-up/frd-panB-down.
  • DNA fragment II includes frd upstream homology arm 50bp, panB gene 795bp and frd downstream homology arm 50bp.
  • DNA fragment II was used for the second homologous recombination.
  • DNA fragment II was electroporated into strain Span019.
  • Example 2 The electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site. Colony PCR was used to verify the clone. The primers used were XZ-frd-up/XZ-frd-down. The correct colony amplification product was a fragment of 1661 bp, including frd upstream homology arm 426 bp, panB gene 795 bp and frd downstream homology arm 395bp. Pick a correct single colony and name it Span020.
  • DNA fragment I includes frd upstream homology arm 50bp, cat-sacB fragment 2619bp and panB downstream homology arm 50bp.
  • the amplification system and amplification conditions were the same as described in Example 1.
  • DNA Fragment I was electroporated into Span020.
  • DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span020, and then DNA fragment I was electroporated into E. coli Span020 with pKD46.
  • the electroporation conditions and procedures were the same as the first method described in Example 1 for the integration of alsS at the tdcDE site. Take 200 ⁇ l of bacterial solution and spread it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml), after overnight incubation at 30 °C, select a single colony for PCR verification, using primers For verification by XZ-frd-up/panB-YZ130-down, the correct PCR product should be 3175bp, including frd upstream homology arm 426bp, cat-sacB fragment 2619bp and panB downstream homology arm 130bp. Pick a correct single colony and name it Span021.
  • DNA fragment II includes frd upstream homology arm 50bp, M1-93 promoter sequence 88bp and panB downstream homology arm 50bp. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span021.
  • the electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site.
  • the clone was verified by colony PCR.
  • the primers used were XZ-frd-up/panB-YZ130-down.
  • the correct colony amplification product was a fragment of 644bp, including frd upstream homology arm 426bp, M1-93 promoter sequence 88bp and panB Downstream homology arm 130bp. Pick a single correct colony and name it Span022.
  • Span022 is a recombinant bacterium obtained by integrating the M1-93 promoter (nucleotide sequence is sequence 3 in the sequence listing) into the upstream of the panB gene of E. coli Span020.
  • the M1-93 promoter in the recombinant bacterium drives the expression of the panB gene.
  • DNA fragment I includes a 50 bp upstream homology arm of ldhA, a 2619 bp cat-sacB fragment and a 50 bp downstream homology arm of ldhA.
  • the amplification system and amplification conditions were the same as described in Example 1. DNA Fragment I was electroporated into Span022.
  • DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span022, and then the DNA fragment I was electroporated into E. coli Span022 with pKD46.
  • the electroporation conditions and procedures were the same as the first method described in Example 1 for the integration of alsS at the tdcDE site. Take 200 ⁇ l of bacterial solution and spread it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml), after overnight incubation at 30 °C, select a single colony for PCR verification, using primers For verification by XZ-ldhA-up/XZ-ldhA-down, the correct PCR product should be 3415bp, including ldhA upstream homology arm 380bp, cat-sacB fragment 2619bp and ldhA downstream homology arm 416bp. Pick a correct single colony and name it Span023.
  • DNA fragment II was amplified with primers ldhA-panE-up/ldhA-panE-down.
  • DNA fragment II includes a 50 bp upstream homology arm of ldhA, a 912 bp panE gene and a 50 bp downstream homology arm of ldhA.
  • DNA fragment II was used for the second homologous recombination.
  • DNA fragment II was electroporated into strain Span023.
  • Electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site.
  • Colony PCR was used to verify the clone.
  • the primers used were XZ-ldhA-up/XZ-ldhA-down.
  • the correct colony amplification product was a fragment of 1708 bp, including ldhA upstream homology arm of 380 bp, panE gene 912 bp and ldhA downstream homology arm. 416bp. Pick a single correct colony and name it Span024.
  • DNA fragment I includes 50 bp upstream homology arm of ldhA, 2619 bp cat-sacB fragment and 50 bp downstream homology arm of panE.
  • the amplification system and amplification conditions were the same as described in Example 1.
  • DNA Fragment I was electroporated into Span024.
  • DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span024, and then DNA fragment I was electroporated into E. coli Span024 with pKD46.
  • the electroporation conditions and procedures were the same as the first method described in Example 1 for the integration of alsS at the tdcDE site. Take 200 ⁇ l of bacterial solution and spread it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml), after overnight incubation at 30 °C, select a single colony for PCR verification, using primers For verification by XZ-ldhA-up/panE-YZ245-down, the correct PCR product should be 3244bp, including ldhA upstream homology arm 380bp, cat-sacB fragment 2619bp and panE downstream homology arm 245bp. Pick a correct single colony and name it Span025.
  • DNA fragment II includes 50bp upstream homology arm of ldhA, 89bp sequence of artificial promoter RBSL2 and 50bp downstream homology arm of panE. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span025.
  • the electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site.
  • the clone was verified by colony PCR.
  • the primers used were XZ-ldhA-up/panE-YZ245-down.
  • the correct colony amplification product was a fragment of 714 bp, including the 380 bp upstream homology arm of ldhA, the artificial promoter RBSL2 sequence of 89 bp and the downstream of panE. Homology arm 245bp. Pick a single correct colony and name it Span026.
  • Span026 is a recombinant bacterium obtained by integrating the RBSL2 promoter (nucleotide sequence is sequence 8 in the sequence listing) into the upstream of the panE gene of E. coli Span024.
  • the RBSL2 promoter in the recombinant bacterium drives the expression of the panE gene.
  • Example 14 Integration of glycine hydroxymethyltransferase gene glyA at mgsA site of methylglyoxal synthase gene and knockout of mgsA site
  • the glycine hydroxymethyltransferase gene glyA is located at the mgsA site of the methylglyoxal synthase gene, and the specific steps are as follows:
  • the primers mgsA-cs-up/mgsA-cs-down were used to amplify a DNA fragment I of 2719 bp, which was used for the first step of homologous recombination.
  • the DNA fragment I includes a 50 bp upstream homology arm of mgsA, a 2619 bp cat-sacB fragment and a 50 bp downstream homology arm of mgsA.
  • the amplification system and amplification conditions were the same as described in Example 1.
  • DNA Fragment I was electroporated into Span026.
  • DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span026, and then DNA fragment I was electroporated into E. coli Span026 with pKD46.
  • the electroporation conditions and procedures were the same as the first method described in Example 1 for the integration of alsS at the tdcDE site. Take 200 ⁇ l of bacterial solution and spread it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml), after overnight incubation at 30 °C, select a single colony for PCR verification, using primers For verification by XZ-mgsA-up/XZ-mgsA-down, the correct PCR product should be 3646bp, including the 516bp upstream homology arm of mgsA, the 2619bp cat-sacB fragment and the 511bp downstream homology arm of mgsA. Pick a correct single colony and name it Span027.
  • DNA fragment II of 1354 bp was amplified with the primers mgsA-glyA-up/mgsA-glyA-down.
  • DNA fragment II includes a 50 bp upstream homology arm of mgsA, a 1254 bp glyA fragment and a 50 bp downstream homology arm of mgsA.
  • DNA fragment II was used for the second homologous recombination.
  • DNA fragment II was electroporated into strain Span027.
  • the electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site.
  • the clone was verified by colony PCR.
  • the primers used were XZ-mgsA-up/XZ-mgsA-down.
  • the correct colony amplification product was a fragment of 2281bp, including the 516bp upstream homology arm of mgsA, the 1254bp glyA fragment and the downstream homology arm of mgsA. 511bp. Pick a single correct colony and name it Span028.
  • DNA fragment I includes a 50 bp upstream homology arm of mgsA, a 2619 bp cat-sacB fragment and a 50 bp downstream homology arm of glyA.
  • the amplification system and amplification conditions were the same as described in Example 1. DNA Fragment I was electroporated into Span028.
  • DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span028, and then DNA fragment I was electroporated into E. coli Span028 with pKD46.
  • the electroporation conditions and procedures were the same as the first method described in Example 1 for the integration of alsS at the tdcDE site. Take 200 ⁇ l of bacterial solution and spread it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml), after overnight incubation at 30 °C, select a single colony for PCR verification, using primers For verification by XZ-mgsA-up/glyA-YZ364-down, the correct PCR product should be 3499bp, including the 516bp upstream homology arm of mgsA, the 2619bp cat-sacB fragment and the 364bp downstream homology arm of glyA. Pick a correct single colony and name it Span029.
  • DNA fragment II includes a 50 bp upstream homology arm of mgsA, an 88 bp M1-46 promoter and a 50 bp downstream homology arm of glyA. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span029.
  • the electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site. Colony PCR was used to verify the clone. The primers used were XZ-mgsA-up/glyA-YZ364-down. The correct colony amplification product was a 968bp fragment, including the 516bp upstream homology arm of mgsA, the 88bp M1-46 promoter and the downstream glyA. Homology arm 364bp. Pick a single correct colony and name it Span030.
  • Span030 is a recombinant bacterium obtained by integrating the M1-46 promoter (nucleotide sequence is sequence 6 in the sequence listing) into the upstream of the glyA gene of Span028.
  • the M1-46 promoter in the recombinant bacterium drives the expression of the glyA gene.
  • DNA fragment I includes a 50 bp upstream homology arm of gcvT gene, a 2619 bp cat-sacB fragment and a 50 bp downstream homology arm of gcvT.
  • the amplification system and amplification conditions were the same as described in Example 1.
  • DNA Fragment I was electroporated into Span030.
  • DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span030, and then DNA fragment I was electroporated into E. coli Span030 with pKD46.
  • the electroporation conditions and procedures were the same as the first method described in Example 1 for the integration of alsS at the tdcDE site. Take 200 ⁇ l of bacterial solution and spread it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml), after overnight incubation at 30 °C, select a single colony for PCR verification, using primers For verification by gcvT-up-500/gcvT-350-down, the correct PCR product should be 3197bp, including the 228bp upstream homology arm of gcvT gene, the 2619bp cat-sacB fragment and the 350bp downstream homology arm of gcvT. Pick a correct single colony and name it Span031.
  • DNA fragment II includes 50bp upstream homology arm of gcvT gene, 88bp M1-93 promoter and 50bp downstream homology arm of gcvT. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span031.
  • the electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site.
  • the clone was verified by colony PCR.
  • the primers used were gcvT-up-500/gcvT-350-down.
  • the correct colony amplification product was a fragment of 666 bp, including the 228 bp upstream homology arm of gcvT gene, 88 bp of M1-93 promoter and gcvT Downstream homology arm 350bp. Pick a single correct colony and name it Span032.
  • DNA fragment I includes a 50 bp upstream homology arm of gcvP gene, a 2619 bp cat-sacB fragment and a 50 bp downstream homology arm of gcvP.
  • the amplification system and amplification conditions were the same as described in Example 1. DNA Fragment I was electroporated into Span032.
  • DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span032, and then DNA fragment I was electroporated into E. coli Span032 with pKD46.
  • the electroporation conditions and procedures were the same as the first method described in Example 1 for the integration of alsS at the tdcDE site. Take 200 ⁇ l of bacterial solution and spread it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml), after overnight incubation at 30 °C, select a single colony for PCR verification, using primers For verification by gcvH-up/gcvP-390-down, the correct PCR product should be 3399bp, including 390bp upstream homology arm of gcvP gene, 2619bp cat-sacB fragment and 390bp downstream homology arm of gcvP. Pick a correct single colony and name it Span033.
  • DNA fragment II includes 50bp upstream homology arm of gcvP gene, 88bp M1-93 promoter and 50bp downstream homology arm of gcvP. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span033.
  • the electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site.
  • the clone was verified by colony PCR.
  • the primers used were gcvH-up/gcvP-390-down.
  • the correct colony amplification product was a fragment of 868 bp, including the 390 bp upstream homology arm of the gcvP gene, and the M1-93 promoter 88 bp and gcvP downstream homology.
  • Source arm 390bp. Pick a single correct colony and name it Span034.
  • M1-93 promoter nucleotide sequence is sequence 3 in the sequence listing
  • Example 18 Integration of the 3-methyl-2-oxobutyric acid hydroxymethyltransferase gene panB from Corynebacterium glutamicum at the phosphoacetyltransferase encoding gene pta and the acetate kinase encoding gene ackA site and ackA-pta site knockout
  • DNA fragment I includes 50 bp upstream homology arm of ackA-pta gene, 2619 bp cat-sacB fragment and 50 bp downstream homology arm of ackA-pta.
  • the amplification system and amplification conditions were the same as described in Example 1.
  • DNA Fragment I was electroporated into Span034.
  • DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span034, and then DNA fragment I was electroporated into E. coli Span034 with pKD46.
  • the electroporation conditions and procedures were the same as the first method described in Example 1 for the integration of alsS at the tdcDE site. Take 200 ⁇ l of bacterial solution and spread it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml), after overnight incubation at 30 °C, select a single colony for PCR verification, using primers For verification by XZ-ackA-up/XZ-pta-down, the correct PCR product should be 3350bp, including 320bp upstream homology arm of ackA-pta gene, 2619bp cat-sacB fragment and 411bp downstream homology arm of ackA-pta. Pick a correct single colony and name it Span035.
  • DNA fragment II was amplified with primers ackA-panBC-up/ackA-panBC-down.
  • DNA fragment II includes 50 bp upstream homology arm of ackA-pta gene, 816 bp and 50 bp downstream homology arm of ackA-pta gene from panB gene. for the second homologous recombination.
  • DNA fragment II was electroporated into strain Span035.
  • the electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site. Colony PCR was used to verify the clone. The primers used were XZ-ackA-up/XZ-pta-down. The correct colony amplification product was a fragment of 1547 bp, including the 320 bp upstream homology arm of the ackA-pta gene, and the panB gene from the corn stick. 816bp and 411bp downstream homology arm of ackA-pta. Pick a single correct colony and name it Span036.
  • panB gene the nucleotide sequence is sequence 9 in the sequence listing, and the
  • DNA fragment I includes 50 bp upstream homology arm of ackA-pta gene, 2619 bp cat-sacB fragment and 50 bp downstream homology arm of panB gene from bark.
  • the amplification system and amplification conditions were the same as described in Example 1.
  • DNA Fragment I was electroporated into Span036.
  • DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span036, and then DNA fragment I was electroporated into E. coli Span036 with pKD46.
  • the electroporation conditions and procedures were the same as the first method described in Example 1 for the integration of alsS at the tdcDE site. Take 200 ⁇ l of bacterial solution and spread it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml), after overnight incubation at 30 °C, select a single colony for PCR verification, using primers For verification by XZ-ackA-up/panBC-YZ425-down, the correct PCR product should be 3364bp, including 320bp upstream homology arm of ackA-pta gene, 2619bp cat-sacB fragment and 425bp downstream homology arm of panB. Pick a correct single colony and name it Span037.
  • DNA fragment II includes 50bp upstream homology arm of ackA-pta gene, 88bp M1-93 promoter and 50bp downstream homology arm of panB. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span037.
  • the electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site.
  • the clone was verified by colony PCR.
  • the primers used were XZ-ackA-up/panBC-YZ425-down.
  • the correct colony amplification product was a fragment of 833 bp, including the 320 bp upstream homology arm of the ackA-pta gene and the 88 bp M1-93 promoter.
  • the homology arm downstream of panB is 425bp. Pick a single correct colony and name it Span038.
  • Span038 is a recombinant bacterium obtained by integrating the M1-93 promoter (nucleotide sequence is sequence 3 in the sequence listing) into the upstream of the panB gene of E. coli Span036.
  • the M1-93 promoter in the recombinant bacterium drives the expression of the panB gene.
  • DNA fragment I includes 50 bp upstream homology arm of ilvE gene, 2619 bp cat-sacB fragment and 50 bp downstream homology arm of ilvE gene.
  • the amplification system and amplification conditions were the same as described in Example 1. DNA Fragment I was electroporated into Span038.
  • DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span038, and then DNA fragment I was electroporated into E. coli Span038 with pKD46.
  • the electroporation conditions and procedures were the same as the first method described in Example 1 for the integration of alsS at the tdcDE site. Take 200 ⁇ l of bacterial solution and spread it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml), after overnight incubation at 30 °C, select a single colony for PCR verification, using primers ilvM-up/ilvE-down for verification, the correct PCR product should be 3832bp, including ilvE gene upstream homology arm 283bp, cat-sacB fragment 2619bp and ilvE gene downstream homology arm 930bp. Pick a correct single colony and name it Span039.
  • DNA fragment II was amplified with primers ilvEGTG-up/ilvE-down.
  • DNA Fragment II includes the ilvE gene with the start codon ATG changed to GTG.
  • DNA fragment II was used for the second homologous recombination.
  • DNA fragment II was electroporated into strain Span039.
  • Example 2 The electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site.
  • Colony PCR was used to verify the clone.
  • the primers used were ilvM-up/ilvE-down.
  • the correct colony amplification product was a 1213bp fragment, including the 283bp upstream homology arm of the ilvE gene and the ilvE codon that replaced the start codon ATG with GTG. 930bp. Pick a single correct colony and name it Span040.
  • Span040 is a recombinant strain obtained by mutating the initiation codon of ilvE of Span038 from ATG to GTG.
  • the mutated gene is denoted as ilvE* gene (its sequence is sequence 11 in the sequence listing), and the ilvE* gene encodes ilvE* protein (which The sequence is sequence 12 in the sequence listing.
  • Example 21 Integration of the phosphoglycerate dehydrogenase gene serA at the ribokinase ara site and knockout of the ara site from Corynebacterium glutamicum
  • DNA fragment I includes a 50 bp homology arm upstream of the ara site, a 2619 bp cat-sacB fragment and a 50 bp downstream homology arm of the ara site.
  • the amplification system and amplification conditions were the same as described in Example 1. DNA Fragment I was electroporated into Span041.
  • DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span041, and then DNA fragment I was electroporated into E. coli Span041 with pKD46.
  • the electroporation conditions and procedures were the same as the first method described in Example 1 for the integration of alsS at the tdcDE site. Take 200 ⁇ l of bacterial solution and spread it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml), after overnight incubation at 30 °C, select a single colony for PCR verification, using primers araBCD-YZ300-up/araBCD-YZ468-down was used for verification.
  • the correct PCR product should be 3378bp, including 291bp of homology arm upstream of ara site, 2619bp of cat-sacB fragment and 468bp of homology arm downstream of ara site. Pick a correct single colony and name it Span041.
  • DNA fragment II was amplified with primers araBCD-serA197-up/araBCD-serA197-down.
  • DNA fragment II includes 50bp of homology arm upstream of ara site, 1002bp of serA gene and 50bp of homology arm downstream of ara site.
  • DNA fragment II was used for the second homologous recombination.
  • DNA fragment II was electroporated into strain Span041.
  • the electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site.
  • Colony PCR was used to verify the clone.
  • the primers used were araBCD-YZ300-up/araBCD-YZ468-down.
  • the correct colony amplification product was a fragment of 1761bp, including the 291bp upstream homology arm of the ara site, 1002bp of the serA gene and the ara site. Point downstream homology arm 468bp. Pick a single correct colony and name it Span042.
  • DNA fragment I includes a 50 bp homology arm upstream of the ara site, a 2619 bp cat-sacB fragment and a 50 bp downstream homology arm of the serA site.
  • the amplification system and amplification conditions were the same as described in Example 1. DNA Fragment I was electroporated into Span042.
  • DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span042, and then DNA fragment I was electroporated into E. coli Span042 with pKD46.
  • DNA fragment II includes 50 bp upstream homology arm of ara site, 88 bp M1-93 promoter and 50 bp downstream homology arm of serA gene. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span043.
  • the electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site.
  • Colony PCR was used to verify the clone.
  • the primers used were araBCD-YZ300-up/SerA197-YZ358-down.
  • the correct colony amplification product was a fragment of 737 bp, including the 291 bp upstream homology arm of the ara site, the M1-93 promoter 88 bp and The homology arm downstream of the serA site is 358 bp. Pick a single correct colony and name it Span044.
  • Span044 is a recombinant bacterium obtained by integrating the M1-93 promoter (nucleotide sequence is sequence 3 in the sequence listing) into the upstream of the serA gene of E. coli Span042.
  • the M1-93 promoter in the recombinant bacterium drives the expression of the serA gene.
  • Example 23 Integration of phosphoserine/phosphohydroxythreonine aminotransferase gene serC and phosphoserine phosphatase gene serB from Escherichia coli in valine-pyruvate aminotransferase gene avtA site and knockout of avtA site
  • the phosphoserine/phosphohydroxythreonine aminotransferase gene serC and the phosphoserine phosphatase gene serB from Escherichia coli were integrated into the valine-pyruvate aminotransferase gene avtA site.
  • the specific steps are as follows:
  • DNA fragment I includes a homology arm of 50 bp upstream of the avtA site, a cat-sacB fragment of 2619 bp and a homology arm of 50 bp downstream of the avtA site.
  • the amplification system and amplification conditions were the same as described in Example 1.
  • DNA Fragment I was electroporated into Span044.
  • DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span044, and then DNA fragment I was electroporated into E. coli Span044 with pKD46.
  • fragment II of 1181 was amplified with primers avtA-serCB-up/serC-down.
  • a fragment III of 1062 bp was amplified using primers serB-up/avtA-serCB-down.
  • fragment IV is a DNA fragment of 2179 bp, which was used for the second homologous recombination. Fragment IV includes 50 bp upstream homology arm of avtA, 1089 bp of serC gene, 21 bp of RBS sequence for translation initiation of serB gene and 969 bp of serB gene, and 50 bp downstream homology arm of avtA. The DNA fragment IV was electroporated into strain Span045.
  • the electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site. Colony PCR was used to verify the clone. The primers used were avtA-YZ-up/avtA-YZ-down. The correct colony amplification product was a fragment of 2914bp, including 416bp of the upstream homology arm of the avtA site and 1089bp of the serC gene for serB. The RBS sequence of the gene translation initiation is 21bp, the serB gene is 969bp, and the downstream homology arm of the avtA site is 419bp. Pick a single correct colony and name it Span046.
  • the 1-88th position is the M1-93 promoter sequence
  • the 89-1177th position is the serC gene sequence
  • the 1178-1198th position is the RBS sequence used for the translation initiation of the serB gene
  • the 1199-2167th position is Sequence of the serB gene.
  • DNA fragment I includes a 50 bp upstream homology arm of the avtA site, a 2619 bp cat-sacB fragment and a 50 bp downstream homology arm of the serC gene.
  • the amplification system and amplification conditions were the same as described in Example 1.
  • DNA Fragment I was electroporated into Span046.
  • DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span046, and then DNA fragment I was electroporated into E. coli Span046 with pKD46.
  • DNA fragment II includes 50 bp upstream homology arm of avtA site, 88 bp M1-93 promoter sequence and 50 bp downstream homology arm of serC gene. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span047.
  • the electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site.
  • the clone was verified by colony PCR.
  • the primers used were avtA-YZ-up/serCB-YZ317-down.
  • the correct colony amplification product was a fragment of 925 bp, including the 416 bp upstream homology arm of the avtA site and the 88 bp M1-93 promoter sequence.
  • the homology arm downstream of serC gene is 421bp. Pick a single correct colony and name it Span048.
  • Span048 is a recombinant bacterium obtained by integrating the M1-93 promoter into the upstream of the serCB gene cluster of E. coli Span046, and contains the serCB gene cluster expression cassette shown in sequence 15.
  • the M1-93 promoter in the recombinant bacterium (the nucleotide sequence is Positions 1-88 of SEQ ID NO: 15 in the Sequence Listing) drive the expression of serC and serB genes in the serCB gene cluster.
  • DNA fragment I includes a 50 bp upstream homology arm of the sdaA site, a 2619 bp cat-sacB fragment and a 50 bp downstream homology arm of the sdaA gene.
  • the amplification system and amplification conditions were the same as described in Example 1. DNA Fragment I was electroporated into Span048.
  • DNA fragment I was used for the first homologous recombination: the pKD46 plasmid was first electroporated into E. coli Span048, and then DNA fragment I was electroporated into E. coli Span048 with pKD46.
  • the electroporation conditions and procedures were the same as the first method described in Example 1 for the integration of alsS at the tdcDE site. Take 200 ⁇ l of bacterial solution and spread it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml), after overnight incubation at 30 °C, select a single colony for PCR verification, using primers sdaA-YZ-up/sdaA-YZ-down for verification, the correct PCR product should be 3428bp, including the 383bp upstream homology arm of the sdaA site, the 2619bp cat-sacB fragment and the 426bp downstream homology arm of the sdaA gene. Pick a correct single colony and name it Span049.
  • DNA fragment II was amplified with primers sdaA-YZ-up/SdaAdel-down.
  • DNA fragment II includes 383 bp upstream homology arm and 50 bp downstream homology arm of sdaA.
  • DNA fragment II was used for the second homologous recombination.
  • DNA fragment II was electroporated into strain Span049.
  • the electroporation conditions and procedures were identical to the second-step method described in Example 1 for the integration of alsS at the tdcDE site.
  • the clone was verified by colony PCR.
  • the primers used were sdaA-YZ-up/sdaA-YZ-down.
  • the correct colony amplification product was a fragment of 809 bp, including the 383 bp upstream homology arm of the sdaA site and the 426 bp downstream homology arm of the sdaA gene. .
  • Span050 has been deposited in the General Microbiology Center of China Microorganism Culture Collection Management Committee on January 22, 2021, and the deposit number is CGMCC No.21699.
  • the seed medium consists of the following components (the solvent is water):
  • glucose 20g/L (NH 4 ) 2 HPO 4 3.5g/L, KH 2 PO 4 3.91g/L, K 2 HPO 4 4.48g/L, MgSO 4 7H 2 O 0.18g/L, sugar beet Alkali-HCl 0.15g/L;
  • Trace elements FeCl 3 ⁇ 6H 2 O 1.5 ⁇ g/L, CoCl 2 ⁇ 6H 2 O 0.1 ⁇ g/L, CuCl 2 ⁇ 2H 2 O 0.1 ⁇ g/L, ZnCl 2 0.1 ⁇ g/L, Na 2 MoO 4 ⁇ 2H 2 O 0.1 ⁇ g/L, MnCl 2 ⁇ 4H 2 O 0.2 ⁇ g/L, H 3 BO 3 0.05 ⁇ g/L.
  • the fermentation medium is the same as the seed medium, the difference is that the glucose concentration is 50g/L, and the fermentation medium also contains 5g/L serine.
  • Seed culture Inoculate fresh clones on the LB plate into a test tube containing 4 ml of seed medium, and cultivate overnight at 37°C with shaking at 250 rpm. Then, according to the inoculation amount of 2% (V/V), the culture was transferred to a 250ml Erlenmeyer flask containing 30ml of seed medium, and cultured with shaking at 37°C and 250rpm for 12 hours to obtain a seed broth for inoculation of fermentation medium.
  • composition and preparation of the seed medium, and the analytical methods were the same as described in Example 26.
  • Fermentation medium glucose 30g/L, magnesium sulfate 5g/L, potassium dihydrogen phosphate 10.5g/L, yeast powder 20g/L, diammonium hydrogen phosphate 6g/L, citric acid monohydrate 1.84g/L, the same trace elements
  • the solvent is water.
  • Feed medium 600 g/L glucose, and the solvent is water.
  • Fermentation was carried out in a 5L fermenter (Shanghai Baoxing, BIOTECH-5BG), including the following steps:
  • Seed culture 50 mL of seed medium in a 500 mL conical flask, sterilized at 115°C for 15 min. After cooling, the recombinant Escherichia coli Span050 was inoculated into the seed medium at an inoculation amount of 1% (V/V), and cultured at 37° C. and 250 rpm for 12 hours to obtain a seed liquid for inoculation of the fermentation medium.
  • Fermentation culture The volume of the fermentation medium in the 5L fermentor is 3L, and the volume is sterilized at 115°C for 25min.
  • Fermentation broth is all the substances in the fermenter.
  • the present invention successfully obtains a strain capable of producing pantoate by using the construction method of recombinant Escherichia coli, the pantoate synthesis pathway of the strain has been opened up and the accumulation of pantoate acid can be realized in the fermentation process.
  • the construction of the recombinant Escherichia coli of the present invention The method and the obtained recombinant Escherichia coli have good potential for industrial application.

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Abstract

提供了一种产泛解酸的微生物及其构建方法和应用。该产泛解酸的微生物是通过将大肠杆菌中的基因进行敲除并导入外源基因获得的，所得微生物在中国微生物菌种保藏管理委员会普通微生物中心保藏中心登记入册，是保藏编号为CGMCC No.21699的大肠埃希氏菌(Escherichia coli)，其泛解酸合成途径已打通并可以在发酵过程中实现泛解酸的积累。

Description

一种产泛解酸的微生物及其构建方法和应用 技术领域
本发明涉及生物技术领域中，一种产泛解酸的微生物及其构建方法和应用。
背景技术
泛酸钙又称维生素B5，是人体必需的13种维生素之一。泛酸钙只能在微生物和植物中合成，是合成辅酶A的重要前体和维持生物正常生理机能的必需维生素。同时，泛酸钙作为食品添加剂和医药原料药等也具有广泛的应用。目前，泛酸钙主要是通过生物或化学方法拆分DL-泛解酸内酯获得高纯度D-泛解酸内酯后与β-丙氨酸反应获得。
泛解酸内酯作为合成泛酸钙的重要前体物质，其合成目前主要是通过以石化材料通过化学方式完成。用于合成DL-泛解酸内酯的材料主要是异丁醛、甲醛等石化材料，经过一系列化学反应，如羟甲基化、酸化或者羟醛缩合等实现的。这个过程需要涉及大量氰化钠等剧毒原料或者强碱、强酸等化学试剂，并产生大量的废水、废气，对环境造成很大的破坏性和不可修复性。另一个值得注意的是，用于泛酸钙合成的DL-泛解酸内酯中L-泛解酸内酯需要去除的很干净才能获得高纯度的D-泛酸钙，这对用于DL-泛解酸内酯的拆分方法具有极高的要求。由于泛酸钙合成方法的局限，近年来泛酸钙合成企业经常受到环保督察，企业压力大，泛酸钙价格也从一吨几万到几十万元不等。
随着合成生物学和代谢工程的急速发展，通过设计和构建微生物细胞工厂以可再生原料通过发酵生产化学品成为替代以石化等不可再生资源为原料通过化学反应生产化学品的重要方式并已获得巨大的成功。大肠杆菌野生型细胞自身就有泛解酸合成路径。但是基于细胞自身复杂的代谢网络调控，野生型细胞难以积累可检测浓度的泛解酸。通过基因组规模系统代谢育种，设计并构建能够高效生产泛解酸的工程菌对于解决泛酸钙制造过程中污染重、原料贵等问题将具有极大的推动作用。另外，通过微生物发酵获得的泛解酸本身就是高纯度的D-泛解酸，无需后续拆分，这将极大的降低泛酸钙的生产成本和环保压力。
因此，研究并提供具有遗传稳定性、无需添加诱导剂和抗生素的能够以葡萄糖等为原料通过生物发酵生产高纯度泛解酸的微生物细胞对于推动泛酸钙生产、降低生产成本、减少环保压力将具有重要的意义。
发明公开
本发明所要解决的技术问题是如何生产泛解酸。
为解决上述技术问题，本发明首先提供了一种重组大肠杆菌的构建方法，所述包括：对出发大肠杆菌进行如下A1-A25的改造，得到重组大肠杆菌：
A1、导入乙酰乳酸合成酶基因alsS基因，并使所述alsS基因得到表达；
A2、将驱动乙酰乳酸合成酶I大亚基基因ilvB基因的启动子替换为M1-93启动子，所述M1-93启动子为下述任一种DNA分子：
a1)一条链的核苷酸序列为序列表中序列3的DNA分子；
a2)与a1)的DNA分子具有80％以上同一性且具有启动子功能的DNA分子；
A3、将驱动乙酰乳酸合成酶II大亚基基因ilvG基因的启动子替换为所述M1-93启动子；
A4、将乙酰乳酸合成酶III调控亚基基因ilvH基因突变为ilvH突变基因，所述ilvH突变基因编码序列表中序列5所示的蛋白质；
A5、导入乙酰羟基酸还原异构酶编码基因ilvC基因，并使所述ilvC基因得到表达；
A6、导入二羟酸脱水酶编码基因ilvD基因，并使所述ilvD基因得到表达；
A7、导入来源于大肠杆菌的3-甲基-2-氧代丁酸羟甲基转移酶基因panB基因，记为E-panB基因，并使所述E-panB基因得到表达；
A8、导入2-脱氢泛酸酯-2-还原酶基因panE基因，并使所述panE基因得到表达；
A9、导入甘氨酸羟甲基转移酶基因glyA基因，并使所述glyA基因得到表达；
A10、将驱动氨甲基转移酶基因gcvT基因的启动子替换为所述M1-93启动子；
A11、将驱动甘氨酸脱羧酶基因gcvP基因的启动子替换为所述M1-93启动子；
A12、导入来源于谷氨酸棒杆菌的3-甲基-2-氧代丁酸羟甲基转移酶基因panB基因，记为C-panB基因，并使所述C-panB基因得到表达；
A13、将支链氨基酸氨基转移酶基因ilvE基因突变为ilvE突变基因，所述ilvE突变基因编码序列表中序列12所示的蛋白质；
A14、导入磷酸甘油酸脱氢酶基因serA基因，并使所述serA基因得到表达；
A15、导入磷酸丝氨酸/磷酸羟基苏氨酸氨基转移酶基因serC基因和磷酸丝氨酸磷酸酶基因serB基因，并使所述serC基因和所述serB基因得到表达；
A16、敲除L-丝氨酸脱氨酶I基因sdaA基因；
A17、敲除丙酸激酶编码基因tdcD基因和甲酸乙酰转移酶编码基因tdcE基因；
A18、敲除醇脱氢酶基因adhE基因；
A19、敲除丙酮酸甲酸裂解酶编码基因pflB基因；
A20、敲除富马酸还原酶编码基因frd基因；
A21、敲除乳酸脱氢酶基因ldhA基因；
A22、敲除甲基乙二醛合酶基因mgsA基因；
A23、敲除乙酸激酶编码基因pta基因和ackA基因；
A24、敲除核糖激酶基因ara基因；
A25、敲除缬氨酸-丙酮酸转氨酶基因avtA基因。
上述方法中，所述alsS基因可来源于枯草芽孢杆菌(Bacillus subtilis)，如枯草芽孢杆菌(Bacillus subtilis)168。
所述ilvC基因可来源于大肠杆菌(Escherichia coli)，如大肠杆菌ATCC8739。
所述ilvD基因可来源于大肠杆菌，如大肠杆菌MG1655。
所述panE基因可来源于大肠杆菌，如大肠杆菌MG1655。
所述glyA基因可来源于大肠杆菌，如大肠杆菌ATCC 8739。
所述serA基因可来源于谷氨酸棒杆菌，如谷氨酸棒杆菌ATCC13032。
所述serC基因和所述serB基因可来源于大肠杆菌，如大肠杆菌MG1655。
所述E-panB基因可来源于大肠杆菌MG1655。
所述C-panB基因可来源于谷氨酸棒杆菌ATCC13032。
上述方法中，所述alsS基因可编码序列表中序列2所示的alsS蛋白质。
所述C-panB基因可编码序列表中序列10所示的C-panB蛋白质。
所述serA基因可编码序列表中序列14所示的serA蛋白质。
所述serC基因可编码序列表中序列16所示的serC蛋白质。
所述serB基因可编码序列表中序列17所示的serB蛋白质。
上述方法中，所述alsS基因的序列可为序列表中序列1。
所述ilvH突变基因的序列可为序列表中序列4。
所述C-panB基因的序列可为序列表中序列9。
所述ilvE突变基因的序列可为序列表中序列11。
所述serA基因的序列可为序列表中序列13。
所述serC基因的序列可为序列表中序列15的第89-1177位。
所述serB基因的序列可为序列表中序列15的第1199-2167位。
上述方法中，A1可通过向所述受体大肠杆菌中导入alsS基因表达盒实现，所述alsS基因表达盒含有启动子和由所述启动子驱动的所述alsS基因。
A5可通过向所述受体大肠杆菌中导入ilvC基因表达盒实现，所述ilvC基因表达盒含有启动子和由所述启动子驱动的所述ilvC基因。
A6可通过向所述受体大肠杆菌中导入ilvD基因表达盒实现，所述ilvD基因表达盒含有启动子和由所述启动子驱动的所述ilvD基因。
A7可通过向所述受体大肠杆菌中导入E-panB基因表达盒实现，所述E-panB基因表达盒含有启动子和由所述启动子驱动的所述E-panB基因。
A8可通过向所述受体大肠杆菌中导入panE基因表达盒实现，所述panE基因表达盒含有启动子和由所述启动子驱动的所述panE基因。
A9可通过向所述受体大肠杆菌中导入glyA基因表达盒实现，所述glyA基因表达盒含有启动子和由所述启动子驱动的所述glyA基因。
A12可通过向所述受体大肠杆菌中导入C-panB基因表达盒实现，所述C-panB基因表达盒含有启动子和由所述启动子驱动的所述C-panB基因。
A14可通过向所述受体大肠杆菌中导入serA基因表达盒实现，所述serA基因表达盒含有启动子和由所述启动子驱动的所述serA基因。
A15可通过向所述受体大肠杆菌中导入serCB基因表达盒实现，所述serCB基因表达盒含有启动子和由所述启动子驱动的所述serC基因和所述serB基因。
上述方法中，A1、A7、A12、A14或A15中所述启动子可为所述M1-93启动子。
A5或A9中所述启动子可为M1-46启动子，所述M1-46启动子为下述任一种DNA分子：
1)一条链的核苷酸序列为序列表中序列6的DNA分子；
2)与1)的DNA分子具有80％以上同一性且具有启动子功能的DNA分子。
A6中所述启动子可为RBSL1启动子，所述RBSL1启动子为下述任一种DNA分子：
a1)一条链的核苷酸序列为序列表中序列7的DNA分子；
a2)与a1)的DNA分子具有80％以上同一性且具有启动子功能的DNA分子。
A8中所述启动子可为RBSL2启动子，所述RBSL2启动子为下述任一种DNA分子：
c1)一条链的核苷酸序列为序列表中序列8的DNA分子；
c2)与c1)的DNA分子具有80％以上同一性且具有启动子功能的DNA分子。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的的核苷酸序列具有80％或更高，或85％或更高，或90％或更高，或95％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
上述80％以上同一性，可为85％、90％或95％以上的同一性。
所述serCB基因表达盒可如序列表中序列15所示。
上述方法中，所述出发大肠杆菌可为大肠杆菌ATCC 8739。
利用所述重组大肠杆菌的构建方法得到的重组大肠杆菌，也属于本发明的保护范围。
在本发明的一个实施例中，以大肠杆菌ATCC 8739为出发大肠杆菌，利用所述重组大肠杆菌的构建方法得到了大肠埃希氏菌(Escherichia coli)Span050，大肠埃希氏菌(Escherichia coli)Span050保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏中心登记入册编号：CGMCC No.21699。
本发明还提供了一种生产泛解酸的方法，所述方法包括：包括培养所述重组大肠杆菌，得到发酵产物；从所述发酵产物中得到泛解酸。
培养所述重组大肠杆菌可采用含有葡萄糖且能用于培养大肠杆菌的培养基进行。
所述培养基可为培养基1、培养基2或培养基3，所述培养基1由溶剂和溶 质组成，溶剂为水，溶质及其在所述培养基1中的浓度分别为：葡萄糖20g/L、(NH 4) 2HPO 4 3.5g/L、KH 2PO 4 3.91g/L、K 2HPO 4 4.48g/L、MgSO 4·7H 2O 0.18g/L、甜菜碱-HCl 0.15g/L，FeCl 3·6H 2O 1.5μg/L、CoCl 2·6H 2O 0.1μg/L、CuCl 2·2H 2O 0.1μg/L、ZnCl 2 0.1μg/L、Na 2MoO 4·2H 2O 0.1μg/L、MnCl 2·4H 2O 0.2μg/L,H 3BO 3 0.05μg/L；
所述培养基2由溶剂和溶质组成，溶剂为水，溶质及其在所述培养基2中的浓度分别为：葡萄糖50g/L、(NH 4) 2HPO 4 3.5g/L、KH 2PO 4 3.91g/L、K 2HPO 4 4.48g/L、MgSO 4·7H 2O 0.18g/L、甜菜碱-HCl 0.15g/L、5g/L丝氨酸，FeCl 3·6H 2O 1.5μg/L、CoCl 2·6H 2O 0.1μg/L、CuCl 2·2H 2O 0.1μg/L、ZnCl 2 0.1μg/L、Na 2MoO 4·2H 2O 0.1μg/L、MnCl 2·4H 2O 0.2μg/L,H 3BO 3 0.05μg/L；
所述培养基3由溶剂和溶质组成，溶剂为水，溶质及其在所述培养基3中的浓度分别为：葡萄糖30g/L、硫酸镁5g/L、磷酸二氢钾10.5g/L、酵母粉20g/L、磷酸氢二铵6g/L、一水柠檬酸1.84g/L，FeCl 3·6H 2O 1.5μg/L、CoCl 2·6H 2O 0.1μg/L、CuCl 2·2H 2O 0.1μg/L、ZnCl 2 0.1μg/L、Na 2MoO 4·2H 2O 0.1μg/L、MnCl 2·4H 2O 0.2μg/L,H 3BO 3 0.05μg/L。
在培养过程中，可根据培养情况向培养体系中添加葡萄糖。
所述培养可在37℃下进行。
本发明还提供了下述任一应用：
X1、所述重组大肠杆菌的构建方法在生产泛解酸中的应用；
X2、所述重组大肠杆菌的构建方法在生产泛酸钙中的应用；
X3、所述重组大肠杆菌在生产泛解酸中的应用；
X4、所述重组大肠杆菌在制备生产泛解酸产品中的应用；
X5、所述重组大肠杆菌在生产泛酸钙中的应用；
X6、所述重组大肠杆菌在制备生产泛酸钙产品中的应用；
X7、所述生产泛解酸的方法在生产泛酸钙中的应用。
生物材料保藏说明
分类命名：大肠埃希氏菌(Escherichia coli)
菌株编号：Span050
保藏单位名称：中国微生物菌种保藏管理委员会普通微生物中心
保藏单位简称：CGMCC
保藏单位地址：北京市朝阳区北辰西路1号院3号，邮政编码：100101
保藏日期：2021年01月22日
保藏中心登记入册编号：CGMCC No.21699
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限 制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂、仪器等，如无特殊说明，均可从商业途径得到。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。下述实施例中，如无特殊说明，序列表中各核苷酸序列的第1位均为相应DNA/RNA的5′末端核苷酸，末位均为相应DNA/RNA的3′末端核苷酸。
表1本发明所用的菌株和质粒
Figure PCTCN2021105954-appb-000001
Figure PCTCN2021105954-appb-000002
表1中，ATCC 8739、M1-93和M1-46均为大肠杆菌。
表2本发明所用的引物
Figure PCTCN2021105954-appb-000003
Figure PCTCN2021105954-appb-000004
Figure PCTCN2021105954-appb-000005
Figure PCTCN2021105954-appb-000006
Figure PCTCN2021105954-appb-000007
Figure PCTCN2021105954-appb-000008
Figure PCTCN2021105954-appb-000009
下文中，利用“/”隔开的两条引物组成相应的引物对用于扩增目标片段。
实施例1乙酰乳酸合成酶基因alsS在ATCC 8739菌株中丙酸激酶编码基因tdcD和甲酸乙酰转移酶编码基因tdcE位点的插入与tdcDE操纵子的敲除
从大肠杆菌ATCC 8739出发，采用两步同源重组的方法将来自枯草芽孢杆菌(Bacillus subtilis)168(来自ATCC，编号23857)的乙酰乳酸合成酶基因alsS插入染色体上丙酸激酶编码基因tdcD和甲酸乙酰转移酶编码基因tdcE位点，具体步骤如下：
第一步，以pXZ-CS(Tan,et al.,Appl Environ Microbiol,2013,79:4838-4844)质粒DNA为模板，使用引物tdcDE-incs-up/tdcDE-incs-down扩增出2719bp的DNA片段I，该片段包含tdcDE上游同源臂50bp，氯霉素基因(cat)和果聚糖蔗糖转移酶基因(sacB)DNA的cat-sacB片段2619bp，和tdcDE基因下游同源臂50bp，用于第一步同源重组。
扩增体系为：Phusion 5X缓冲液(NewEngland Biolabs)10μl、dNTP(每种dNTP各10mM)1μl、DNA模板20ng、引物(10μM)各2μl、Phusion High-Fidelity DNA聚合酶(2.5U/μl)0.5μl、蒸馏水33.5μl，总体积为50μl。
扩增条件为98℃预变性2分钟(1个循环)；98℃变性10秒、56℃退火10秒、72℃延伸2分钟(30个循环)；72℃延伸10分钟(1个循环)。
将上述DNA片段I用于第一次同源重组：首先将pKD46质粒(美国耶鲁大学CGSC大肠杆菌保藏中心，CGSC#7739)通过电转化法转化至大肠杆菌ATCC 8739，然后将DNA片段I电转至带有pKD46的大肠杆菌ATCC 8739。
电转条件为：首先准备带有pKD46质粒的大肠杆菌ATCC 8739的电转化感受态细胞(准备方法按照Dower et al.,1988,Nucleic Acids Res 16:6127-6145)；将50μl感受态细胞置于冰上，加入50ng DNA片段I，冰上放置2分钟，转移至0.2cm的Bio-Rad电击杯。使用MicroPulser(Bio-Rad公司)电穿孔仪，电击参数为电压2.5kv。电击后迅速将1ml LB培养基转移至电击杯中，吹打5次后转移至试管中，75rpm，30℃孵育2小时。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，所用引物XZ-tdcDE-up/XZ-tdcDE-down，正确的菌落扩增产物为3615bp的片段，包含tdcDE上游同源臂845Bp，cat-sacB片段2619bp和tdcDE下游同源臂151bp。挑选一个正确的 单菌落，命名为Span001。
第二步，以野生型枯草芽孢杆菌(Bacillus subtilis)168基因组DNA为模板，用引物tdcDE-alsSin-up/tdcDE-alsSin-down扩增出1826bp的DNA片段II,包括tdcDE上游同源臂50bp，alsS基因1716bp和sacI的酶切位点及保护碱基共10bp，和tdcDE下游同源臂50bp。DNA片段II用于第二次同源重组。扩增条件和体系同第一步中所述。将DNA片段II电转至菌株Span001。
电转条件为：首先准备带有pKD46质粒的Span001的电转化感受态细胞；将50μl感受态细胞置于冰上，加入50ng DNA片段II，冰上放置2分钟，转移至0.2cm的Bio-Rad电击杯。使用MicroPulser(Bio-Rad公司)电穿孔仪，电击参数为电压2.5kv。电击后迅速将1ml LB培养基转移至电击杯中，吹打5次后转移至试管中，75rpm，30℃孵育4小时。将菌液转移至含有10％蔗糖的没有氯化钠的LB液体培养基(250ml烧瓶中装50ml培养基)，培养24小时后在含有6％蔗糖的没有氯化钠的LB固体培养基上划线培养。经过PCR验证，所用引物为XZ-tdcDE-up/XZ-tdcDE-down，正确的菌落扩增产物为2722bp的片段，包括tdcDE上游同源臂845bp，alsS基因和sacI酶切位点共1726bp和tdcDE下游同源臂151bp。挑选一个正确的单菌落，将其命名为Span002。
Span002是将乙酰乳酸合成酶基因(alsS基因，核苷酸序列是序列表中序列1，编码序列2所示的alsS蛋白质)整合到大肠杆菌ATCC 8739的丙酸激酶编码基因tdcD和甲酸乙酰转移酶编码基因tdcE位点得到的重组菌，该重组菌中丙酸激酶编码基因tdcD(编码的蛋白质序列为NCBI ACA76259.1，coded_by＝CP000946.1:626900..628108)和甲酸乙酰转移酶编码基因tdcE(编码的蛋白质序列为NCBI ACA76260.1，coded_by＝CP000946.1:628142..630436)同时被敲除。
实施例2乙酰乳酸合成酶基因alsS的调控
从Span002出发，使用人工调控元件调控整合在tdcDE位点的乙酰乳酸合成酶编码基因alsS的表达，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物tdcDE-incs-up/alsSPro-CS-down扩增出2719bp的DNA片段I，包括tdcDE上游同源臂50bp，cat-sacB片段2619bp和alsS基因下游同源臂50bp，用于第一步同源重组。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Span002。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span002，然后将DNA片段I电转至带有pKD46的大肠杆菌Span002。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物XZ-tdcDE-up/tdcDE-YZ285-down进行验证，正确的PCR产物应该3749 bp，包括tdcDE上游同源臂845bp，cat-sacB片段2619bp和alsS下游同源臂285bp，挑选一个正确的单菌落，命名为Span003。
第二步，以M1-93(Lu,et al.,Appl Microbiol Biotechnol,2012,93:2455-2462)的基因组DNA为模板，用引物alsS-Pro-up/alsS-Pro-down扩增出188bp的DNA片段II，包括tdcDE上游同源臂50bp，M1-93启动子88bp和alsS下游同源臂50bp。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span003。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为XZ-tdcDE-up/tdcDE-YZ285-down，正确的菌落扩增产物为1218bp的片段，包括tdcDE上游同源臂8454bp，M1-93启动子序列88bp和alsS下游同源臂285bp，挑选一个正确的单菌落，将其命名为Span004。
Span004是将M1-93启动子(核苷酸序列是序列表中序列3)整合到大肠杆菌Span002的alsS基因上游得到的重组菌，该重组菌中M1-93启动子驱动alsS基因的表达。
实施例3乙酰乳酸合成酶基因ilvB的调控
使用人工调控元件M1-93通过两步同源重组的方法调控乙酰乳酸合成酶I大亚基基因ilvB的表达，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物ilvB pro-catup/ilvB pro-catdown扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括ilvB上游同源臂50bp，cat-sacB片段2619bp和ilvB下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span004，然后将DNA片段I电转至带有pKD46的大肠杆菌Span004。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物ilvB pro-YZup/ilvB pro-YZdown进行验证，正确的PCR产物应该2996bp，包含ilvB上游同源臂123bp，cat-sacB片段2619bp和ilvB下游同源臂254bp。挑选一个正确的单菌落，命名为Span005。
第二步，以M1-93(Lu,et al.,Appl Microbiol Biotechnol,2012,93:2455-2462)的基因组DNA为模板，用引物ilvB pro-up/ilvB pro-down扩增出188bp的DNA片段II。DNA片段II包括ilvB上游同源臂50bp，M1-93启动子88bp和ilvB下游同源臂50bp。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span005。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为ilvB pro-YZup/ilvB  pro-YZdown，正确的菌落扩增产物为465bp的片段，包括ilvB上游同源臂123bp，M1-93启动子88bp和ilvB下游同源臂254bp。挑选一个正确的单菌落，将其命名为Span006。
Span006是将M1-93启动子(核苷酸序列是序列表中序列3)整合到大肠杆菌Span004的乙酰乳酸合成酶基因ilvB(编码的蛋白质序列为NCBI ACA75715.1，coded_by＝CP000946.1:28583..30271)的上游得到的重组菌，该重组菌中，M1-93启动子可以驱动ilvB基因的表达。
实施例4乙酰乳酸合成酶基因ilvG的调控
使用人工调控元件M1-93通过两步同源重组的方法调控乙酰乳酸合成酶II大亚基基因ilvG的表达，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物ilvG pro-catup/ilvG pro-catdown扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括ilvG上游同源臂50bp，cat-sacB片段2619bp和ilvG下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span006，然后将DNA片段I电转至带有pKD46的大肠杆菌Span006。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物ilvG pro-YZup/ilvG p-YZdown进行验证，正确的PCR产物应该2993bp，包括ilvG上游同源臂179bp，cat-sacB片段2619bp和ilvG下游同源臂195bp。挑选一个正确的单菌落，命名为Span007。
第二步，以M1-93(Lu,et al.,Appl Microbiol Biotechnol,2012,93:2455-2462)的基因组DNA质粒DNA为模板，用引物ilvG pro-up/ilvG pro-down扩增出188bp的DNA片段II。DNA片段II包括ilvG上游同源臂50bp，M1-93启动子88bp和ilvG下游同源臂50bp。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span007。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为ilvG pro-YZup/ilvG p-YZdown，正确的菌落扩增产物为462bp的片段，包括ilvG上游同源臂179bp，M1-93片段88bp和ilvG下游同源臂195bp。挑选一个正确的单菌落，将其命名为Span008。
Span008是将M1-93启动子(核苷酸序列是序列表中序列3)整合到大肠杆菌Span006的乙酰乳酸合成酶基因ilvG(编码的蛋白质序列为NCBI ACA79830.1，coded_by＝CP000946.1:4677780..4679426)的上游得到的重组菌，该重组菌中，M1-93启动子可以驱动ilvG基因的表达。
实施例5乙酰乳酸合成酶基因ilvH的突变
通过两步同源重组的方法在乙酰乳酸合成酶III调控亚基基因ilvH基因中引入突变解除L-缬氨酸的反馈抑制，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物ilvH*-cat-up/ilvH*-cat-down扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括ilvH上游同源臂50bp，cat-sacB片段2619bp和ilvH下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span008，然后将DNA片段I电转至带有pKD46的大肠杆菌Span008。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物ilvH*-mutYZ-up/ilvH*-mut-down进行验证，正确的PCR产物应该3165bp，包括ilvH上游同源臂202bp，cat-sacB片段2619bp和ilvH下游同源臂344bp。挑选一个正确的单菌落，命名为Span009。
第二步，以野生型大肠杆菌ATCC 8739的DNA为模板，用引物ilvH*-mut-up/ilvH*-mut-down扩增出467bp的DNA片段II。DNA片段II为含有突变的ilvH基因。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span009。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为ilvH*-mutYZ-up/ilvH*-mut-down，正确的菌落扩增产物为619bp的片段，包括ilvH基因上游163bp和ilvH基因456bp。挑选一个正确的单菌落，将其命名为Span010。
Span010是将大肠杆菌Span008的乙酰乳酸合成酶基因ilvH突变为ilvH*基因(即ilvH突变基因)得到的重组菌，该重组菌中，ilvH*基因的序列为序列表中序列4，编码序列5所示的ilvH*蛋白质。
实施例6乙酰羟基酸还原异构酶编码基因ilvC在醇脱氢酶adhE位点的整合和adhE基因的敲除
从Span010出发，通过两步同源重组的方法将来自大肠杆菌的乙酰羟基酸还原异构酶编码基因ilvC整合到醇脱氢酶adhE位点，具体步骤包括：
第一步，以pXZ-CS质粒DNA为模板，使用引物adhE-CS-up/adhE-CS-down扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括adhE上游同源臂50bp，cat-sacB片段2619bp和adhE下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Span010。
将DNA片段I用于第一次同源重组：首先将pKD46质粒(Datsenko and Wanner 2000,Proc Natl Acad Sci USA 97:6640-6645；质粒购买于美国耶鲁大学CGSC大肠杆菌保藏中心，CGSC#7739)通过电转化法转化至大肠杆菌Span010，然后将DNA片段I电转至带有pKD46的大肠杆菌Span010。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方 法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物XZ-adhE-up/XZ-adhE-down进行验证，正确的PCR产物应该3167bp，包括adhE上游同源臂221bp，cat-sacB片段2619bp和adhE下游同源臂327bp。挑选一个正确的单菌落，命名为Span011。
第二步，以野生型大肠杆菌ATCC 8739的基因组DNA为模板，用引物adhE-ilvC-up/adhE-ilvC-down扩增出1576bp的DNA片段II。DNA片段II包括adhE上游同源臂50bp，ilvC基因1476bp和adhE下游同源臂50bp。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span011。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为XZ-adhE-up/XZ-adhE-down，正确的菌落扩增产物为2024bp的片段，包括adhE上游同源臂221bp，ilvC基因1476bp和adhE下游同源臂327bp。挑选一个正确的单菌落，将其命名为Span012。
Span012是将乙酰羟基酸还原异构酶编码基因ilvC(编码的蛋白质序列为NCBI ACA79824.1，coded_by＝CP000946.1:4670539..4672014)整合到大肠杆菌Span010的adhE位点得到的重组菌，该重组菌中醇脱氢酶基因adhE(编码的蛋白质序列为NCBI ACA78022.1，coded_by＝CP000946.1:2627307..2629982)同时被敲除。
实施例7乙酰羟基酸还原异构酶编码基因ilvC的调控
从Span012出发，使用人工调控元件调控整合在醇脱氢酶基因adhE位点的乙酰羟基酸还原异构酶编码基因ilvC的表达，具体步骤如下：
第一步，以pXZ-CS质粒(Tan,et al.,Appl Environ Microbiol,2013,79:4838-4844)DNA为模板，使用引物adhE-cs-up/ilvC-ProCS-down扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括adhE上游同源臂50bp，cat-sacB片段2619bp和ilvC下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Span012。
将DNA片段I用于第一次同源重组：首先将pKD46质粒(Datsenko and Wanner 2000,Proc Natl Acad Sci USA 97:6640-6645；质粒购买于美国耶鲁大学CGSC大肠杆菌保藏中心，CGSC#7739)通过电转化法转化至大肠杆菌Span012，然后将DNA片段I电转至带有pKD46的大肠杆菌Span012。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物XZ-adhE-up/ilvC-YZ347-down进行验证，正确的PCR产物应该3187bp，包括adhE上游同源臂221bp，cat-sacB片段2619bp和ilvC下游同源臂347bp。挑选一个正确的单菌落，命名为Span013。
第二步，以M1-46(Lu,et al.,Appl Microbiol Biotechnol,2012,93:2455-2462)的基因组DNA为模板，用引物ilvC-Pro-up/ilvC-Pro-down 扩增出188bp的DNA片段II。DNA片段II包括adhE上游同源臂50bp，M1-46启动子序列88bp和ilvC下游同源臂50bp。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span013。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为XZ-adhE-up/ilvC-YZ347-down，正确的菌落扩增产物为656bp的片段，包括adhE上游同源臂221bp，M1-46启动子88bp和ilvC下游同源臂347bp。挑选一个正确的单菌落，将其命名为Span014。
Span014是将M1-46启动子(核苷酸序列是序列表中序列6)整合到大肠杆菌Span012的ilvC基因上游得到的重组菌，该重组菌中M1-46启动子驱动ilvC基因的表达。
实施例8二羟酸脱水酶编码基因ilvD在丙酮酸甲酸裂解酶编码基因pflB位点的整合和pflB基因的敲除
从Span014出发，通过两步同源重组的方法将来自大肠杆菌的二羟酸脱水酶编码基因ilvD整合到丙酮酸甲酸裂解酶编码基因pflB位点并敲除pflB基因，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物pflB-CS-up/pflB-CS-down扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括pflB上游同源臂50bp，cat-sacB片段2619bp和pflB下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Span014。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span014，然后将DNA片段I电转至带有pKD46的大肠杆菌Span014。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物XZ-pflB-up600/XZ-pflB-down进行验证，正确的PCR产物应该3675bp，包括pflB上游同源臂641bp，cat-sacB片段2619bp和pflB下游同源臂415bp。挑选一个正确的单菌落，命名为Span015。
第二步，以大肠杆菌MG1655(来自ATCC，编号700926)的基因，用引物pflB-ilvD-up/pflB-ilvD-down扩增出1951bp的DNA片段I。DNA片段II包括pflB上游同源臂50bp，ilvD基因1851bp和pflB下游同源臂50bp。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span015。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为XZ-pflB-up600/XZ-pflB-down，正确的菌落扩增产物为2996bp的片段，包括pflB上游同源臂641bp，ilvD基因1851bp和pflB下游同源臂415bp。挑选一个正确的单菌落，将其命名为Span016。
Span016是将二羟酸脱水酶编码基因ilvD(编码的蛋白质序列为NCBI QPA17447.1，coded_by＝CP032679.1:3943375..3945225)整合到大肠杆菌Span014的pflB位点得到的重组菌，该重组菌中丙酮酸甲酸裂解酶编码基因pflB(编码的蛋白质序列为NCBI ACA78322.1，coded_by＝CP000946.1:2956804..2959086)同时被敲除。
实施例9二羟酸脱水酶编码基因ilvD的表达调控
从Span016出发，使用人工调控元件调控整合在丙酮酸甲酸裂解酶编码基因pflB位点的二羟酸脱水酶编码基因ilvD的表达，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物pflB-CS-up/pflB-Pcs-down扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括pflB上游同源臂50bp，cat-sacB片段2619bp和ilvD下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Span016。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span016，然后将DNA片段I电转至带有pKD46的大肠杆菌Span016。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物XZ-pflB-up600/ilvD-YZ496-down进行验证，正确的PCR产物应该3756bp，包括pflB上游同源臂641bp，cat-sacB片段2619bp和ilvD下游同源臂496bp。挑选一个正确的单菌落，命名为Span017。
第二步，以M1-93(Lu,et al.,Appl Microbiol Biotechnol,2012,93:2455-2462)的基因组DNA为模板，用引物pflB-Pro-up/ilvD-Pro-down扩增出189bp的DNA片段II。DNA片段II包括pflB上游同源臂50bp，人工调控元件RBSL1序列89bp和ilvD下游同源臂50bp。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span017。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为XZ-pflB-up600/ilvD-YZ496-down，正确的菌落扩增产物为1226bp的片段，包括pflB上游同源臂641bp，RBSL1序列89bp和ilvD下游同源臂496bp。挑选一个正确的单菌落，将其命名为Span018。
Span018是将RBSL1启动子(核苷酸序列是序列表中序列7)整合到大肠杆菌Span016的ilvD基因上游得到的重组菌，该重组菌中RBSL1启动子驱动ilvD基因的表达。
实施例10 3-甲基-2-氧代丁酸羟甲基转移酶基因panB在富马酸还原酶编码基因frd位点的整合和frd位点的敲除
从Span018出发，将3-甲基-2-氧代丁酸羟甲基转移酶编码基因panB整合到富马酸还原酶编码基因frd位点，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物frd-cs-up/frd-cs-down扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括frd上游同源臂50bp，cat-sacB片段2619bp和frd下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Span018。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span018，然后将DNA片段I电转至带有pKD46的大肠杆菌Span018。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物XZ-frd-up/XZ-frd-down进行验证，正确的PCR产物应该3440bp，包括frd上游同源臂426bp，cat-sacB片段2619bp和frd下游同源臂395bp。挑选一个正确的单菌落，命名为Span019。
第二步，以大肠杆菌MG1655(来自ATCC，编号700926)的基因组DNA为模板，用引物frd-panB-up/frd-panB-down扩增出895bp的DNA片段II。DNA片段II包括frd上游同源臂50bp，panB基因795bp和frd下游同源臂50bp。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span019。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为XZ-frd-up/XZ-frd-down，正确的菌落扩增产物为1661bp的片段，包括frd上游同源臂426bp，panB基因795bp和frd下游同源臂395bp。挑选一个正确的单菌落，将其命名为Span020。
Span020是将3-甲基-2-氧代丁酸羟甲基转移酶基因panB(编码的蛋白质序列为NCBI QPA14045.1，coded_by＝CP032679.1:148806..149600)整合到大肠杆菌Span018的frd位点得到的重组菌，该重组菌中富马酸还原酶编码基因frd(编码的蛋白质序列为NCBI ACA79462.1，coded_by＝CP000946.1:4217304..4217699)同时被敲除。
实施例11 3-甲基-2-氧代丁酸羟甲基转移酶基因panB的表达调控
从Span020出发，使用人工调控元件调控整合在富马酸还原酶编码基因frd位点的3-甲基-2-氧代丁酸羟甲基转移酶基因panB的表达，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物frd-cs-up/panB-Pcs-down扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括frd上游同源臂50bp，cat-sacB片段2619bp和panB下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Span020。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span020，然后将DNA片段I电转至带有pKD46的大肠杆菌Span020。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证， 使用引物XZ-frd-up/panB-YZ130-down进行验证，正确的PCR产物应该3175bp，包括frd上游同源臂426bp，cat-sacB片段2619bp和panB下游同源臂130bp。挑选一个正确的单菌落，命名为Span021。
第二步，以M1-93(Lu,et al.,Appl Microbiol Biotechnol,2012,93:2455-2462)的基因组DNA为模板，用引物panB-Pro-up/panB-Pro-down扩增出188bp的DNA片段II。DNA片段II包括frd上游同源臂50bp，M1-93启动子序列88bp和panB下游同源臂50bp。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span021。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为XZ-frd-up/panB-YZ130-down，正确的菌落扩增产物为644bp的片段，包括frd上游同源臂426bp，M1-93启动子序列88bp和panB下游同源臂130bp。挑选一个正确的单菌落，将其命名为Span022。
Span022是将M1-93启动子(核苷酸序列是序列表中序列3)整合到大肠杆菌Span020的panB基因上游得到的重组菌，该重组菌中M1-93启动子驱动panB基因的表达。
实施例12 2-脱氢泛酸酯-2-还原酶基因panE在乳酸脱氢酶ldhA位点的整合和ldhA位点的敲除
从Span022出发，将2-脱氢泛酸酯-2-还原酶基因panE整合到乳酸脱氢酶基因ldhA位点，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物ldhA-csin-up/ldhA-csin-down扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括ldhA上游同源臂50bp，cat-sacB片段2619bp和ldhA下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Span022。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span022，然后将DNA片段I电转至带有pKD46的大肠杆菌Span022。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物XZ-ldhA-up/XZ-ldhA-down进行验证，正确的PCR产物应该3415bp，包括ldhA上游同源臂380bp，cat-sacB片段2619bp和ldhA下游同源臂416bp。挑选一个正确的单菌落，命名为Span023。
第二步，以大肠杆菌MG1655(来自ATCC，编号700926)的基因组DNA为模板，用引物ldhA-panE-up/ldhA-panE-down扩增出1012bp的DNA片段II。DNA片段II包括ldhA上游同源臂50bp，panE基因912bp和ldhA下游同源臂50bp。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span023。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方 法一致。菌落PCR对克隆进行验证，所用引物为XZ-ldhA-up/XZ-ldhA-down，正确的菌落扩增产物为1708bp的片段，包括ldhA上游同源臂380bp，panE基因912bp和ldhA下游同源臂416bp。挑选一个正确的单菌落，将其命名为Span024。
Span024是将2-脱氢泛酸酯-2-还原酶基因panE(编码的蛋白质序列为NCBI QPA14304.1，coded_by＝CP032679.1:443607..444518)整合到大肠杆菌Span022的ldhA位点得到的重组菌，该重组菌中乳酸脱氢酶基因ldhA(编码的蛋白质序列为NCBI ACA77911.1，coded_by＝CP000946.1:2508048..2509037)同时被敲除。
实施例13 2-脱氢泛酸酯-2-还原酶基因panE的表达调控
从Span024出发，使用人工调控元件调控整合在乳酸脱氢酶基因ldhA位点的2-脱氢泛酸酯-2-还原酶基因panE的表达，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物ldhA-csin-up/panE-ProCS-down扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括ldhA上游同源臂50bp，cat-sacB片段2619bp和panE下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Span024。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span024，然后将DNA片段I电转至带有pKD46的大肠杆菌Span024。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物XZ-ldhA-up/panE-YZ245-down进行验证，正确的PCR产物应该3244bp，包括ldhA上游同源臂380bp，cat-sacB片段2619bp和panE下游同源臂245bp。挑选一个正确的单菌落，命名为Span025。
第二步，以M1-93(Lu,et al.,Appl Microbiol Biotechnol,2012,93:2455-2462)的基因组DNA为模板，用引物panE-Pro-up/panE-Pro-down扩增出189bp的DNA片段II。DNA片段II包括ldhA上游同源臂50bp，人工启动子RBSL2序列89bp和panE下游同源臂50bp。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span025。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为XZ-ldhA-up/panE-YZ245-down，正确的菌落扩增产物为714bp的片段，包括ldhA上游同源臂380bp，人工启动子RBSL2序列89bp和panE下游同源臂245bp。挑选一个正确的单菌落，将其命名为Span026。
Span026是将RBSL2启动子(核苷酸序列是序列表中序列8)整合到大肠杆菌Span024的panE基因上游得到的重组菌，该重组菌中RBSL2启动子驱动panE基因的表达。
实施例14甘氨酸羟甲基转移酶基因glyA在甲基乙二醛合酶基因mgsA位点 的整合和mgsA位点的敲除
从Span026出发，将甘氨酸羟甲基转移酶基因glyA在甲基乙二醛合酶基因mgsA位点，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物mgsA-cs-up/mgsA-cs-down扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括mgsA上游同源臂50bp，cat-sacB片段2619bp和mgsA下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Span026。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span026，然后将DNA片段I电转至带有pKD46的大肠杆菌Span026。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物XZ-mgsA-up/XZ-mgsA-down进行验证，正确的PCR产物应该3646bp，包括mgsA上游同源臂516bp，cat-sacB片段2619bp和mgsA下游同源臂511bp。挑选一个正确的单菌落，命名为Span027。
第二步，以野生型大肠杆菌ATCC8739的基因组DNA为模板，用引物mgsA-glyA-up/mgsA-glyA-down扩增出1354bp的DNA片段II。DNA片段II包括mgsA上游同源臂50bp，glyA片段1254bp和mgsA下游同源臂50bp。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span027。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为XZ-mgsA-up/XZ-mgsA-down，正确的菌落扩增产物为2281bp的片段，包括mgsA上游同源臂516bp，glyA片段1254bp和mgsA下游同源臂511bp。挑选一个正确的单菌落，将其命名为Span028。
Span028是将甘氨酸羟甲基转移酶基因glyA(编码的蛋白质序列为NCBI ACA76793.1，coded_by＝CP000946.1:1227416..1228669)整合到大肠杆菌Span026的mgsA位点得到的重组菌，该重组菌中mgsA基因(编码的蛋白质序列为NCBIACA78263.1，coded_by＝CP000946.1:2883345..2883803)同时被敲除。
实施例15甘氨酸羟甲基转移酶基因glyA的调控
从Span028出发，使用人工调控元件调控整合在mgsA位点的甘氨酸羟甲基转移酶基因glyA的表达，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物mgsA-cs-up/glyA-ProCS-down扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括mgsA上游同源臂50bp，cat-sacB片段2619bp和glyA下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Span028。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span028，然后将DNA片段I电转至带有pKD46的大肠杆菌Span028。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物XZ-mgsA-up/glyA-YZ364-down进行验证，正确的PCR产物应该3499bp，包括mgsA上游同源臂516bp，cat-sacB片段2619bp和glyA下游同源臂364bp。挑选一个正确的单菌落，命名为Span029。
第二步，以M1-46(Lu,et al.,Appl Microbiol Biotechnol,2012,93:2455-2462)的基因组DNA为模板，用引物glyA-Pro-up/glyA-Pro-down扩增出188bp的DNA片段II。DNA片段II包括mgsA上游同源臂50bp，M1-46启动子88bp和glyA下游同源臂50bp。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span029。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为XZ-mgsA-up/glyA-YZ364-down，正确的菌落扩增产物为968bp的片段，包括mgsA上游同源臂516bp，M1-46启动子88bp和glyA下游同源臂364bp。挑选一个正确的单菌落，将其命名为Span030。
Span030是将M1-46启动子(核苷酸序列是序列表中序列6)整合到Span028的glyA基因上游得到的重组菌，该重组菌中M1-46启动子驱动glyA基因的表达。
实施例16大肠杆菌野生型氨甲基转移酶基因gcvT的调控
从Span030出发，使用人工调控元件调控大肠杆菌野生型氨甲基转移酶基因gcvT的表达，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物gcvT-Pcat-up/gcvT-PsacB-down扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括gcvT基因上游同源臂50bp，cat-sacB片段2619bp和gcvT下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Span030。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span030，然后将DNA片段I电转至带有pKD46的大肠杆菌Span030。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物gcvT-up-500/gcvT-350-down进行验证，正确的PCR产物应该3197bp，包括gcvT基因上游同源臂228bp，cat-sacB片段2619bp和gcvT下游同源臂350bp。挑选一个正确的单菌落，命名为Span031。
第二步，以M1-93(Lu,et al.,Appl Microbiol Biotechnol,2012,93:2455-2462)的基因组DNA为模板，用引物gcvT-M93-up/gcvT-M93-down 扩增出188bp的DNA片段II。DNA片段II包括gcvT基因上游同源臂50bp，M1-93启动子88bp和gcvT下游同源臂50bp。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span031。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为gcvT-up-500/gcvT-350-down，正确的菌落扩增产物为666bp的片段，包括gcvT基因上游同源臂228bp，M1-93启动子88bp和gcvT下游同源臂350bp。挑选一个正确的单菌落，将其命名为Span032。
Span032是将M1-93启动子(核苷酸序列是序列表中序列3)整合到大肠杆菌Span030的氨甲基转移酶基因gcvT(编码的蛋白质序列为NCBI ACA76476.1，coded_by＝CP000946.1:862077..863171)的上游得到的重组菌，该重组菌中，M1-93启动子可以驱动gcvT基因的表达。
实施例17大肠杆菌野生型甘氨酸脱羧酶基因gcvP的调控
从Span032出发，使用人工调控元件调控大肠杆菌野生型甘氨酸脱羧酶基因gcvP的表达，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物gcvP-Pcat-up/gcvP-PsacB-down扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括gcvP基因上游同源臂50bp，cat-sacB片段2619bp和gcvP下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Span032。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span032，然后将DNA片段I电转至带有pKD46的大肠杆菌Span032。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物gcvH-up/gcvP-390-down进行验证，正确的PCR产物应该3399bp，包括gcvP基因上游同源臂390bp，cat-sacB片段2619bp和gcvP下游同源臂390bp。挑选一个正确的单菌落，命名为Span033。
第二步，以M1-93(Lu,et al.,Appl Microbiol Biotechnol,2012,93:2455-2462)的基因组DNA为模板，用引物gcvP-M93-up/gcvP-M93-down扩增出188bp的DNA片段II。DNA片段II包括gcvP基因上游同源臂50bp，M1-93启动子88bp和gcvP下游同源臂50bp。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span033。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为gcvH-up/gcvP-390-down，正确的菌落扩增产物为868bp的片段，包括gcvP基因上游同源臂390bp，M1-93启动子88bp和gcvP下游同源臂390bp。挑选一个正确的单菌落，将其命名为 Span034。
Span034是将M1-93启动子(核苷酸序列是序列表中序列3)整合到大肠杆菌Span032的甘氨酸脱羧酶基因gcvP(编码的蛋白质序列为NCBI ACA76478.1，coded_by＝CP000946.1:863703..866576)的上游得到的重组菌，该重组菌中，M1-93启动子可以驱动gcvP基因的表达。
实施例18来自谷氨酸棒杆菌的3-甲基-2-氧代丁酸羟甲基转移酶基因panB在磷酸乙酰转移酶编码基因pta和乙酸激酶编码基因ackA位点的整合及ackA-pta位点的敲除
从Span034出发，将来自谷氨酸棒杆菌的3-甲基-2-氧代丁酸羟甲基转移酶基因panB在磷酸乙酰转移酶编码基因pta和乙酸激酶编码基因ackA位点，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物ackA-cs-up/pta-cs-down扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括ackA-pta基因上游同源臂50bp，cat-sacB片段2619bp和ackA-pta下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Span034。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span034，然后将DNA片段I电转至带有pKD46的大肠杆菌Span034。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物XZ-ackA-up/XZ-pta-down进行验证，正确的PCR产物应该3350bp，包括ackA-pta基因上游同源臂320bp，cat-sacB片段2619bp和ackA-pta下游同源臂411bp。挑选一个正确的单菌落，命名为Span035。
第二步，以谷氨酸棒杆菌ATCC13032(ATCC产品)的基因组DNA为模板，用引物ackA-panBC-up/ackA-panBC-down扩增出916bp的DNA片段II。DNA片段II包括ackA-pta基因上游同源臂50bp，来自谷棒的panB基因816bp和ackA-pta下游同源臂50bp。用于第二次同源重组。将DNA片段II电转至菌株Span035。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为XZ-ackA-up/XZ-pta-down，正确的菌落扩增产物为1547bp的片段，包括ackA-pta基因上游同源臂320bp，来自谷棒的panB基因816bp和ackA-pta下游同源臂411bp。挑选一个正确的单菌落，将其命名为Span036。
Span036是将谷氨酸棒杆菌的3-甲基-2-氧代丁酸羟甲基转移酶基因panB(panB基因，核苷酸序列是序列表中序列9，编码序列10所示的panB蛋白质)整合到大肠杆菌Span034的磷酸乙酰转移酶编码基因pta和乙酸激酶编码基因ackA位点得到的重组菌，该重组菌中pta基因(编码的蛋白质序列为NCBI ACA77021.1， coded_by＝CP000946.1:1484032..1486176)和ackA基因(编码的蛋白质序列为NCBI ACA77022.1，coded_by＝CP000946.1:1486251..1487453)同时被敲除。
实施例19谷氨酸棒杆菌来源的3-甲基-2-氧代丁酸羟甲基转移酶基因panB的调控
从Span036出发，使用人工调控元件调控整合在ackA-pta位点的3-甲基-2-氧代丁酸羟甲基转移酶基因panB的表达，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物ackA-cs-up/panBC-ProCS-down扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括ackA-pta基因上游同源臂50bp，cat-sacB片段2619bp和来自谷棒的panB基因下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Span036。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span036，然后将DNA片段I电转至带有pKD46的大肠杆菌Span036。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物XZ-ackA-up/panBC-YZ425-down进行验证，正确的PCR产物应该3364bp，包括ackA-pta基因上游同源臂320bp，cat-sacB片段2619bp和panB下游同源臂425bp。挑选一个正确的单菌落，命名为Span037。
第二步，以M1-93(Lu,et al.,Appl Microbiol Biotechnol,2012,93:2455-2462)的基因组DNA为模板，用引物panBC-Pro-up/panBC-Pro-down扩增出188bp的DNA片段II。DNA片段II包括ackA-pta基因上游同源臂50bp，M1-93启动子88bp和panB下游同源臂50bp。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span037。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为XZ-ackA-up/panBC-YZ425-down，正确的菌落扩增产物为833bp的片段，包括ackA-pta基因上游同源臂320bp，M1-93启动子88bp和panB下游同源臂425bp。挑选一个正确的单菌落，将其命名为Span038。
Span038是将M1-93启动子(核苷酸序列是序列表中序列3)整合到大肠杆菌Span036的panB基因上游得到的重组菌，该重组菌中M1-93启动子驱动panB基因的表达。
实施例20支链氨基酸氨基转移酶基因ilvE的表达弱化
从Span038出发，将支链氨基酸氨基转移酶基因ilvE的表达弱化，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物ilvE-cat-up/ilvE-sacB-down扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片 段I包括ilvE基因上游同源臂50bp，cat-sacB片段2619bp和ilvE基因下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Span038。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span038，然后将DNA片段I电转至带有pKD46的大肠杆菌Span038。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物ilvM-up/ilvE-down进行验证，正确的PCR产物应该3832bp，包括ilvE基因上游同源臂283bp，cat-sacB片段2619bp和ilvE基因下游同源臂930bp。挑选一个正确的单菌落，命名为Span039。
第二步，以野生型大肠杆菌ATCC 8739的基因组DNA为模板，用引物ilvEGTG-up/ilvE-down扩增出980bp的DNA片段II。DNA片段II包括将起始密码子ATG更改为GTG的ilvE基因。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span039。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为ilvM-up/ilvE-down，正确的菌落扩增产物为1213bp的片段，包括ilvE基因上游同源臂283bp和将起始密码子ATG更换为GTG的ilvE共930bp。挑选一个正确的单菌落，将其命名为Span040。
Span040是将Span038的ilvE的起始密码子ATG突变为GTG得到的重组菌，将突变后的基因记为ilvE*基因(其序列为序列表中序列11)，ilvE*基因编码ilvE*蛋白质(其序列为序列表中序列12)。
实施例21来自谷氨酸棒杆菌的磷酸甘油酸脱氢酶基因serA在核糖激酶ara位点的整合及ara位点的敲除
从Span040出发，将来自谷氨酸棒杆菌的磷酸甘油酸脱氢酶基因serA整合在核糖激酶ara位点，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物araBCD-CS-up/araBCD-CS-down扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括ara位点上游同源臂50bp，cat-sacB片段2619bp和ara位点下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Span041。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span041，然后将DNA片段I电转至带有pKD46的大肠杆菌Span041。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证， 使用引物araBCD-YZ300-up/araBCD-YZ468-down进行验证，正确的PCR产物应该3378bp，包括ara位点上游同源臂291bp，cat-sacB片段2619bp和ara位点下游同源臂468bp。挑选一个正确的单菌落，命名为Span041。
第二步，以谷氨酸棒杆菌ATCC13032(ATCC产品)的基因组DNA为模板，用引物araBCD-serA197-up/araBCD-serA197-down扩增出1102bp的DNA片段II。DNA片段II包括ara位点上游同源臂50bp，serA基因1002bp和ara位点下游同源臂50bp。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span041。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为araBCD-YZ300-up/araBCD-YZ468-down，正确的菌落扩增产物为1761bp的片段，包括包括ara位点上游同源臂291bp，serA基因1002bp和ara位点下游同源臂468bp。挑选一个正确的单菌落，将其命名为Span042。
Span042是将谷氨酸棒杆菌的磷酸甘油酸脱氢酶基因serA(serA基因，核苷酸序列是序列13，编码序列14所示的serA蛋白质)整合到大肠杆菌Span040的ara位点得到的重组菌，该重组菌中ara基因(编码的蛋白质序列为NCBI ACA79208.1，coded_by＝CP000946.1:3929533..3931233,编码的蛋白质序列为NCBI ACA79209.1，coded_by＝CP000946.1:3931244..3932746)同时被敲除。
实施例22来自谷氨酸棒杆菌的磷酸甘油酸脱氢酶基因serA的调控
从Span042出发，使用人工调控元件调控整合在ara位点的磷酸甘油酸脱氢酶基因serA的表达，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物araBCD-CS-up/serA197-ProCS-down扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括ara位点上游同源臂50bp，cat-sacB片段2619bp和serA位点下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Span042。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span042，然后将DNA片段I电转至带有pKD46的大肠杆菌Span042。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物araBCD-YZ300-up/SerA197-YZ358-down进行验证，正确的PCR产物应该3268bp，包括ara位点上游同源臂291bp，cat-sacB片段2619bp和serA位点下游同源臂358bp。挑选一个正确的单菌落，命名为Span043。
第二步，以M1-93(Lu,et al.,Appl Microbiol Biotechnol,2012,93:2455-2462)的基因组DNA为模板，用引物serA197-Pro-up/serA197-Pro-down扩增出188bp的DNA片段II。DNA片段II包括ara位点上 游同源臂50bp，M1-93启动子88bp和serA基因下游同源臂50bp。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span043。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为araBCD-YZ300-up/SerA197-YZ358-down，正确的菌落扩增产物为737bp的片段，包括ara位点上游同源臂291bp，M1-93启动子88bp和serA位点下游同源臂358bp。挑选一个正确的单菌落，将其命名为Span044。
Span044是M1-93启动子(核苷酸序列是序列表中序列3)整合到大肠杆菌Span042的serA基因上游得到的重组菌，该重组菌中M1-93启动子驱动serA基因的表达。
实施例23来自大肠杆菌的磷酸丝氨酸/磷酸羟基苏氨酸氨基转移酶基因serC和磷酸丝氨酸磷酸酶基因serB在缬氨酸-丙酮酸转氨酶基因avtA位点的整合及avtA位点的敲除
从Span044出发，将来自大肠杆菌的磷酸丝氨酸/磷酸羟基苏氨酸氨基转移酶基因serC和磷酸丝氨酸磷酸酶基因serB整合到缬氨酸-丙酮酸转氨酶基因avtA位点，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物avtA-CS-up/avtA-CS-down扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括avtA位点上游同源臂50bp，cat-sacB片段2619bp和avtA位点下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Span044。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span044，然后将DNA片段I电转至带有pKD46的大肠杆菌Span044。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物avtA-YZ-up/avtA-YZ-down进行验证，正确的PCR产物应该3454bp，包括avtA位点上游同源臂416bp，cat-sacB片段2619bp和avtA位点下游同源臂419bp。挑选一个正确的单菌落，命名为Span045。
第二步，以大肠杆菌MG1655(来自ATCC，编号700926)的基因组DNA为模板，用引物avtA-serCB-up/serC-down扩增出1181的片段II。以大肠杆菌MG1655(来自ATCC，编号700926)的基因组DNA为模板，使用引物serB-up/avtA-serCB-down扩增出1062bp的片段III。以引物avtA-serCB-up/avtA-serCB-down进行PCR扩增，模板为等摩尔的片段II和片段III，扩增体系和条件同实施例1中所述进行融合PCR获得片段IV。片段IV为2179bp的DNA片段，将其用于第二次同源重组。片段IV包括avtA上游同源臂50bp，serC基因1089bp，用于serB基因翻译起始的RBS序列21bp和serB基因969bp，以及avtA下游同源臂50bp。将DNA片段IV电转至菌株Span045。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为avtA-YZ-up/avtA-YZ-down，正确的菌落扩增产物为2914bp的片段，包括avtA位点上游同源臂416bp，serC基因1089bp，用于serB基因翻译起始的RBS序列21bp和serB基因969bp，以及avtA位点下游同源臂419bp。挑选一个正确的单菌落，将其命名为Span046。
Span046是将大肠杆菌的磷酸丝氨酸/磷酸羟基苏氨酸氨基转移酶基因serC和磷酸丝氨酸磷酸酶基因serB(serCB基因簇，核苷酸序列是序列表中序列15，serCB基因簇编码序列16所示的serC蛋白质和序列17所示的serB蛋白质)整合到大肠杆菌Span044的avtA位点得到的重组菌，该重组菌中avtA基因(编码的蛋白质序列为NCBI ACA75824.1，coded_by＝CP000946.1:153868..155121)同时被敲除。
序列15中，第1-88位为M1-93启动子序列，第89-1177位为serC基因序列，第1178-1198位为用于serB基因翻译起始的RBS序列，第1199-2167位为serB基因的序列。
实施例24整合在avtA位点的serCB基因簇的表达调控
从Span046出发，使用人工调控元件调控整合在avtA位点的来自大肠杆菌的磷酸丝氨酸/磷酸羟基苏氨酸氨基转移酶基因serC和磷酸丝氨酸磷酸酶基因serB基因簇的表达，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物avtA-CS-up/serCB-ProCS-down扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括avtA位点上游同源臂50bp，cat-sacB片段2619bp和serC基因下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Span046。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span046，然后将DNA片段I电转至带有pKD46的大肠杆菌Span046。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物avtA-YZ-up/serCB-YZ317-down进行验证，正确的PCR产物应该3456bp，包括avtA位点上游同源臂416bp，cat-sacB片段2619bp和serC基因下游同源臂421bp。挑选一个正确的单菌落，命名为Span047。
第二步，以M1-93(Lu,et al.,Appl Microbiol Biotechnol,2012,93:2455-2462)的基因组DNA为模板，用引物serCB-Pro-up/serCB-Pro-down扩增出188bp的DNA片段II。DNA片段II包括avtA位点上游同源臂50bp，M1-93启动子序列88bp和serC基因下游同源臂50bp。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span047。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为avtA-YZ-up/serCB-YZ317-down， 正确的菌落扩增产物为925bp的片段，包括avtA位点上游同源臂416bp，M1-93启动子序列88bp和serC基因下游同源臂421bp。挑选一个正确的单菌落，将其命名为Span048。
Span048是将M1-93启动子整合到大肠杆菌Span046的serCB基因簇上游得到的重组菌，含有序列15所示的serCB基因簇表达盒，该重组菌中M1-93启动子(核苷酸序列是序列表中序列15的第1-88位)驱动serCB基因簇中serC和serB基因的表达。
实施例25 L-丝氨酸脱氨酶I基因sdaA的敲除
从Span048出发，敲除L-丝氨酸脱氨酶I的编码基因sdaA，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物sdaA-delcat-up/sdaA-delsacB-down扩增出2719bp的DNA片段I，用于第一步同源重组。DNA片段I包括sdaA位点上游同源臂50bp，cat-sacB片段2619bp和sdaA基因下游同源臂50bp。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Span048。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Span048，然后将DNA片段I电转至带有pKD46的大肠杆菌Span048。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物sdaA-YZ-up/sdaA-YZ-down进行验证，正确的PCR产物应该3428bp，包括sdaA位点上游同源臂383bp，cat-sacB片段2619bp和sdaA基因下游同源臂426bp。挑选一个正确的单菌落，命名为Span049。
第二步，以大肠杆菌ATCC 8739的基因组DNA为模板，用引物sdaA-YZ-up/SdaAdel-down扩增出433bp的DNA片段II。DNA片段II包括sdaA上游同源臂383bp和下游同源臂50bp。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Span049。
电转条件和步骤同实施例1中所述用于alsS在tdcDE位点整合的第二步方法一致。菌落PCR对克隆进行验证，所用引物为sdaA-YZ-up/sdaA-YZ-down，正确的菌落扩增产物为809bp的片段，包括sdaA位点上游同源臂383bp和sdaA基因下游同源臂426bp。挑选一个正确的单菌落，将其命名为Span050。
Span050是将大肠杆菌Span048的L-丝氨酸脱氨酶I基因sdaA(编码的蛋白质序列为NCBI ACA77468.1，coded_by＝CP000946.1:2018393..2019757)敲除得到的重组菌，该重组菌中不含有sdaA基因。
Span050已于2021年01月22日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.21699。
实施例26使用Span050生产泛解酸
种子培养基由以下成分组成(溶剂为水)：
大量元素：葡萄糖20g/L、(NH 4) 2HPO 4 3.5g/L、KH 2PO 4 3.91g/L、K 2HPO 4 4.48g/L、MgSO 4·7H 2O 0.18g/L、甜菜碱-HCl 0.15g/L；
微量元素：FeCl 3·6H 2O 1.5μg/L、CoCl 2·6H 2O 0.1μg/L、CuCl 2·2H 2O 0.1μg/L、ZnCl 2 0.1μg/L、Na 2MoO 4·2H 2O 0.1μg/L、MnCl 2·4H 2O 0.2μg/L,H 3BO 3 0.05μg/L。
发酵培养基大部分和种子培养基相同，区别是葡萄糖浓度为50g/L，发酵培养基还含有5g/L丝氨酸。
Span050的发酵包括以下步骤：
(1)种子培养：将LB平板上新鲜的克隆接种到含有4ml种子培养基的试管中，37℃，250rpm振荡培养过夜。然后，按照2％(V/V)的接种量将培养物转接到含有30ml种子培养基的250ml三角瓶中，在37℃，250rpm振荡培养12小时得到种子培养液用于发酵培养基接种。
(2)发酵培养：250ml三角瓶中发酵培养基体积为25ml，将种子培养液按照终浓度OD550＝0.1的接种量接种于发酵培养基，37℃，250rpm，发酵60小时，得到发酵液。
分析方法：使用安捷伦(Agilent-1260)高效液相色谱仪对发酵3天的发酵液中的组分进行测定。发酵液中的葡萄糖和泛解酸浓度测定采用伯乐(Biorad)公司的Aminex HPX–87H有机酸分析柱。
结果发现：Span050菌株发酵3天，能够生产1.2g/L的泛解酸，由此可知，Span050菌株中泛解酸合成途径已打通并可以发酵过程中实现泛解酸的积累。
实施例27 Span050在5L罐中的发酵
种子培养基的组成和配制、分析方法同实施例26中所述相同。
发酵培养基：葡萄糖30g/L、硫酸镁5g/L、磷酸二氢钾10.5g/L、酵母粉20g/L、磷酸氢二铵6g/L、一水柠檬酸1.84g/L，微量元素同实施例26发酵培养基，溶剂为水。
补料培养基：600g/L葡萄糖，溶剂为水。
发酵在5L发酵罐(上海保兴，BIOTECH-5BG)中进行，包括以下步骤：
(1)种子培养：500mL三角瓶中种子培养基为50mL，115℃灭菌15min。冷却后将重组大肠杆菌Span050按照1％(V/V)的接种量接种于种子培养基，在37℃和250rpm的条件下培养12小时得到种子液，用于发酵培养基接种。
(2)发酵培养：5L发酵罐中发酵培养基体积为3L，115℃灭菌25min。将种子液按照终浓度OD550＝0.2的接种量接种于发酵培养基，溶氧维持30％，用氨水作中和剂pH维持7.0，通过补料培养基将罐内葡萄糖浓度控制在5g/L以下，37℃培养3天，得到发酵液。发酵液为发酵罐内所有物质。
结果发现：Span050发酵3天后，泛解酸产量达到22g/L，具有很好的工业应用潜力。
工业应用
本发明利用重组大肠杆菌的构建方法成功得到了可以生产泛解酸的菌株，该菌株的泛解酸合成途径已打通并可以发酵过程中实现泛解酸的积累，本发明的重组大肠杆菌的构建方法与所得重组大肠杆菌具有很好的工业应用潜力。

Claims (10)

  1. 一种重组大肠杆菌的构建方法，包括：对出发大肠杆菌进行如下A1-A25的改造，得到重组大肠杆菌：
    A1、导入乙酰乳酸合成酶基因alsS基因，并使所述alsS基因得到表达；
    A2、将驱动乙酰乳酸合成酶基因ilvB基因的启动子替换为M1-93启动子，所述M1-93启动子为下述任一种DNA分子：
    a1)一条链的核苷酸序列为序列表中序列3的DNA分子；
    a2)与a1)的DNA分子具有80％以上同一性且具有启动子功能的DNA分子；
    A3、将驱动乙酰乳酸合成酶基因ilvG基因的启动子替换为所述M1-93启动子；
    A4、将乙酰乳酸合成酶基因ilvH基因突变为ilvH突变基因，所述ilvH突变基因编码序列表中序列5所示的蛋白质；
    A5、导入乙酰羟基酸还原异构酶编码基因ilvC基因，并使所述ilvC基因得到表达；
    A6、导入二羟酸脱水酶编码基因ilvD基因，并使所述ilvD基因得到表达；
    A7、导入来源于大肠杆菌的3-甲基-2-氧代丁酸羟甲基转移酶基因panB基因，记为E-panB基因，并使所述E-panB基因得到表达；
    A8、导入2-脱氢泛酸酯-2-还原酶基因panE基因，并使所述panE基因得到表达；
    A9、导入甘氨酸羟甲基转移酶基因glyA基因，并使所述glyA基因得到表达；
    A10、将驱动氨甲基转移酶基因gcvT基因的启动子替换为所述M1-93启动子；
    A11、将驱动甘氨酸脱羧酶基因gcvP基因的启动子替换为所述M1-93启动子；
    A12、导入来源于谷氨酸棒杆菌的3-甲基-2-氧代丁酸羟甲基转移酶基因panB基因，记为C-panB基因，并使所述C-panB基因得到表达；
    A13、将支链氨基酸氨基转移酶基因ilvE基因突变为ilvE突变基因，所述ilvE突变基因编码序列表中序列12所示的蛋白质；
    A14、导入磷酸甘油酸脱氢酶基因serA基因，并使所述serA基因得到表达；
    A15、导入磷酸丝氨酸/磷酸羟基苏氨酸氨基转移酶基因serC基因和磷酸丝氨酸磷酸酶基因serB基因，并使所述serC基因和所述serB基因得到表达；
    A16、敲除L-丝氨酸脱氨酶I基因sdaA基因；
    A17、敲除丙酸激酶编码基因tdcD基因和甲酸乙酰转移酶编码基因tdcE基因；
    A18、敲除醇脱氢酶基因adhE基因；
    A19、敲除丙酮酸甲酸裂解酶编码基因pflB基因；
    A20、敲除富马酸还原酶编码基因frd基因；
    A21、敲除乳酸脱氢酶基因ldhA基因；
    A22、敲除甲基乙二醛合酶基因mgsA基因；
    A23、敲除乙酸激酶编码基因pta基因和ackA基因；
    A24、敲除核糖激酶基因ara基因；
    A25、敲除缬氨酸-丙酮酸转氨酶基因avtA基因。
  2. 根据权利要求1所述的方法，其特征在于：
    所述alsS基因来源于枯草芽孢杆菌(Bacillus subtilis)；
    和/或，所述ilvC基因来源于大肠杆菌(Escherichia coli)；
    和/或，所述ilvD基因来源于大肠杆菌；
    和/或，所述panE基因来源于大肠杆菌；
    和/或，所述glyA基因来源于大肠杆菌；
    和/或，所述serA基因来源于谷氨酸棒杆菌；
    和/或，所述serC基因和所述serB基因来源于大肠杆菌。
  3. 根据权利要求2所述的方法，其特征在于：
    所述alsS基因编码序列表中序列2所示的alsS蛋白质；
    和/或，所述C-panB基因编码序列表中序列10所示的C-panB蛋白质；
    和/或，所述serA基因编码序列表中序列14所示的serA蛋白质；
    和/或，所述serC基因编码序列表中序列16所示的serC蛋白质；
    和/或，所述serB基因编码序列表中序列17所示的serB蛋白质。
  4. 根据权利要求1-3中任一所述的方法，其特征在于：
    所述alsS基因的序列为序列表中序列1；
    和/或，所述ilvH突变基因的序列为序列表中序列4；
    和/或，所述C-panB基因的序列为序列表中序列9；
    和/或，所述ilvE突变基因的序列为序列表中序列11；
    和/或，所述serA基因的序列为序列表中序列13；
    和/或，所述serC基因的序列为序列表中序列15的第89-1177位；
    和/或，所述serB基因的序列为序列表中序列15的第1199-2167位。
  5. 根据权利要求1-3中任一所述的方法，其特征在于：
    A1通过向所述受体大肠杆菌中导入alsS基因表达盒实现，所述alsS基因表达盒含有启动子和由所述启动子驱动的所述alsS基因；
    和/或，A5通过向所述受体大肠杆菌中导入ilvC基因表达盒实现，所述ilvC基因表达盒含有启动子和由所述启动子驱动的所述ilvC基因；
    和/或，A6通过向所述受体大肠杆菌中导入ilvD基因表达盒实现，所述ilvD基因表达盒含有启动子和由所述启动子驱动的所述ilvD基因；
    和/或，A7通过向所述受体大肠杆菌中导入E-panB基因表达盒实现，所述 E-panB基因表达盒含有启动子和由所述启动子驱动的所述E-panB基因；
    和/或，A8通过向所述受体大肠杆菌中导入panE基因表达盒实现，所述panE基因表达盒含有启动子和由所述启动子驱动的所述panE基因；
    和/或，A9通过向所述受体大肠杆菌中导入glyA基因表达盒实现，所述glyA基因表达盒含有启动子和由所述启动子驱动的所述glyA基因；
    和/或，A12通过向所述受体大肠杆菌中导入C-panB基因表达盒实现，所述C-panB基因表达盒含有启动子和由所述启动子驱动的所述C-panB基因；
    和/或，A14通过向所述受体大肠杆菌中导入serA基因表达盒实现，所述serA基因表达盒含有启动子和由所述启动子驱动的所述serA基因；
    和/或，A15通过向所述受体大肠杆菌中导入serCB基因表达盒实现，所述serCB基因表达盒含有启动子和由所述启动子驱动的所述serC基因和所述serB基因。
  6. 根据权利要求5所述的方法，其特征在于：
    A1、A7、A12、A14或A15中所述启动子为所述M1-93启动子；
    A5或A9中所述启动子为M1-46启动子，所述M1-46启动子为下述任一种DNA分子：
    1)一条链的核苷酸序列为序列表中序列6的DNA分子；
    2)与1)的DNA分子具有80％以上同一性且具有启动子功能的DNA分子；
    A6中所述启动子为RBSL1启动子，所述RBSL1启动子为下述任一种DNA分子：
    a1)一条链的核苷酸序列为序列表中序列7的DNA分子；
    a2)与a1)的DNA分子具有80％以上同一性且具有启动子功能的DNA分子；
    A8中所述启动子为RBSL2启动子，所述RBSL2启动子为下述任一种DNA分子：
    c1)一条链的核苷酸序列为序列表中序列8的DNA分子；
    c2)与c1)的DNA分子具有80％以上同一性且具有启动子功能的DNA分子；
    和/或，所述出发大肠杆菌为大肠杆菌ATCC 8739。
  7. 利用权利要求1-6中任一所述的方法得到的重组大肠杆菌。
  8. 根据权利要求7所述的重组大肠杆菌，其特征在于：所述重组大肠杆菌为在中国微生物菌种保藏管理委员会普通微生物中心保藏中心登记入册编号为CGMCC No.21699的菌株。
  9. 生产泛解酸的方法，包括：包括培养权利要求7或8所述重组大肠杆菌，得到发酵产物；从所述发酵产物中得到泛解酸。
  10. 下述任一应用：
    X1、权利要求1-6中任一所述的方法在生产泛解酸中的应用；
    X2、权利要求1-6中任一所述的方法在生产泛酸钙中的应用；
    X3、权利要求7或8所述的重组大肠杆菌在生产泛解酸中的应用；
    X4、权利要求7或8所述的重组大肠杆菌在制备生产泛解酸产品中的应用；
    X5、权利要求7或8所述的重组大肠杆菌在生产泛酸钙中的应用；
    X6、权利要求7或8所述的重组大肠杆菌在制备生产泛酸钙产品中的应用；
    X7、权利要求9所述的方法在生产泛酸钙中的应用。
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