WO2015154209A1 - 一种高产l-丙氨酸且耐受自来水的菌株及其构建方法 - Google Patents

一种高产l-丙氨酸且耐受自来水的菌株及其构建方法 Download PDF

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WO2015154209A1
WO2015154209A1 PCT/CN2014/000522 CN2014000522W WO2015154209A1 WO 2015154209 A1 WO2015154209 A1 WO 2015154209A1 CN 2014000522 W CN2014000522 W CN 2014000522W WO 2015154209 A1 WO2015154209 A1 WO 2015154209A1
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gene
protein
ion
alanine
sequence
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张学礼
郭恒华
张冬竹
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安徽华恒生物技术股份有限公司
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    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/48Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
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    • C12P13/06Alanine; Leucine; Isoleucine; Serine; Homoserine

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a strain which is highly resistant to L-alanine and which is resistant to tap water and a method for constructing the same.
  • L-alanine is a non-essential amino acid in the human body and is transferred from the amino group of glycine to pyruvic acid in the living body.
  • L-alanine is a white crystalline or crystalline powder with a sweet taste and is easily soluble in water, and has a wide range of uses in the food and pharmaceutical industries.
  • L-alanine can increase the nutritional value of foods, and the addition of L-alanine can significantly improve protein utilization in foods and beverages.
  • L-alanine improves the taste of synthetic sweeteners and makes them like natural sweeteners.
  • L-alanine can also improve the sour taste of organic acids, making them closer to natural taste.
  • L-alanine is often used as an amino acid nutrient, and L-alanine is also an important raw material for the production of vitamin B6, synthetic calcium pantothenate and other organic compounds.
  • L-alanine production methods mainly include chemical synthesis and biosynthesis.
  • chemical synthesis methods mainly include propionic acid chlorination, ex-bromopropionic acid chlorination and cyanohydrin.
  • These methods all require petroleum-based raw materials such as propionic acid, ex-bromopropionic acid, acetaldehyde and hydrocyanic acid, so the cost is trapped in crude oil prices. As the price of oil increases, the cost will become higher and higher.
  • these methods are completed by complex chemical catalysis, heavy pollution, high separation and extraction costs, and are not suitable for sustainable development.
  • L-alanine by biological method is mainly carried out by using L-aspartic acid as a raw material and decarboxylation under the catalysis of aspartic acid- ⁇ -decarboxylase to produce L-alanine.
  • This method is currently the main production technology used by domestic L-alanine manufacturers.
  • the production of the raw material aspartic acid in the method is based on maleic anhydride
  • the production cost of L-alanine still depends on the petroleum price. With the shortage of petroleum resources and the increase in prices, the shortage of maleic anhydride resources and rising prices will lead to huge hidden dangers in the supply of aspartic acid, which will affect the production and cost of L-alanine.
  • L-alanine With the development of synthetic biology and metabolic engineering, research on the production of L-alanine by microbial fermentation in recent years has received increasing attention.
  • the microbial fermentation method can produce L-alanine from sugars such as glucose.
  • Glucose is a renewable biomass resource that can be obtained by degradation of lignocellulose that is widely found in nature. Therefore, using it as a raw material enables L-
  • the production cost of alanine is maintained at a stable level and has a long-term economic advantage.
  • strains capable of producing L-alanine Smi th et al. constructed a strain of E.
  • coli ALS929 in which the alanine dehydrogenase AlaD expressed in the plasmid was able to convert the intracellular pyruvate into L-alanine. After 48 hours of fermentation, the strain was able to produce 88 g/L of L-alanine.
  • Lee et al. constructed a strain of A coli ALA887 (pTrc99A-a ⁇ ) which produced 32 g/L of L-alanine in 27 hours.
  • alanine dehydrogenase is a key step in the production of L-alanine, and among the reported strains, the gene encoding the protein is generally expressed by a high copy plasmid.
  • the fermentation of L-alanine by the strains uses a medium configured by distilled water, which increases the cost of production in industrial production.
  • the development of a strain capable of directly producing a L-alanine by directly using a culture medium of a tap water configuration and production thereof will greatly reduce the production cost of L-alanine.
  • the method for constructing the recombinant strain A (XZ-A43) provided by the present invention comprises the steps of: replacing the Ion protein-encoding gene on the chromosome of the bacterium with the gene encoding the Ion* protein, and obtaining the recombinant strain A;
  • the amino acid sequence of the Ion* protein is to mutate the alanine A at position 437 of the amino acid sequence of the Ion protein to aspartic acid 0.
  • the Ion* protein is a protein encoded by nucleotides 1-2355 from the 5' end of the sequence 2 in the sequence listing.
  • the Ion* protein-encoding gene is a gene obtained by mutating the base at position 1310 of the nucleotide sequence of the Ion protein-encoding gene to C.
  • nucleotide sequence of the Ion* protein-encoding gene is the sequence 2 in the sequence listing. Nucleotide 1-2355 from the 5'end;
  • the nucleotide sequence of the DNA fragment II is particularly specifically the sequence 2 in the sequence listing.
  • the starting bacteria are obtained by integrating the L-alanine dehydrogenase gene on the chromosome of Bacillus stearothermophilus on the lactate dehydrogenase of the chromosome of Escherichia coli ATCC8739, and sequentially knocking out the obtained Escherichia coli chromosome a pyruvate formate lyase gene, an alcohol dehydrogenase gene, an acetate kinase gene, a fumarate reductase gene, and an alanine racemase gene, and then serially subcultured in a fermentor;
  • the starting bacteria is specifically Escherichia coli XZ-A26 CGMCC No. 4036.
  • the recombinant A prepared by the above method is also within the scope of protection of the present invention.
  • Another object of the present invention is to provide a method of constructing recombinant B (XZ-A47).
  • the method for constructing recombinant B according to the present invention comprises the steps of: replacing the Ion-encoding protein gene on the chromosome of the above-mentioned fungus with the gene encoding the Ion* protein, and mutating the gene encoding the clpA protein on the chromosome of the starting bacterium a recombinant gene B obtained as a gene encoding the clpA* protein;
  • the amino acid sequence of the Ion* protein is a mutation of the 437th alanine A of the amino acid sequence of the Ion protein to aspartic acid D;
  • the amino acid sequence of the clpA* protein is a mutation of the 632th isoleucine I of the amino acid sequence of the clpA protein to serine S.
  • the clpA* protein is a protein encoded by nucleotides 1-2272 from the 5' end of the sequence 4 in the sequence listing.
  • the above method comprises the steps of: first replacing the Ion-encoding protein gene on the chromosome of the starting bacterium with the gene encoding the Ion* protein, obtaining the recombinant bacterium A, and then the recombinant bacterium on the A chromosome
  • the clpA protein-encoding gene is replaced with the coding gene of the c lpA* protein, and the obtained recombinant bacteria B; in the above method, the Ion* protein-encoding gene is the base of the 13th nucleotide sequence of the Ion protein-encoding gene. a gene obtained by mutating C to A;
  • the c lpA* protein-encoding gene is a gene obtained by mutating the base 1895 of the nucleotide sequence of the c lpA protein-encoding gene into T;
  • the nucleotide sequence of the Ion* protein-encoding gene is specifically nucleotides 1-2355 from the 5' end of sequence 2 in the sequence listing;
  • nucleotide sequence of the clpA* protein-encoding gene is specifically nucleotides 1-2272 from the 5' end of the sequence 2 in the sequence listing;
  • the above method includes the following steps:
  • the ⁇ fragment III containing the gene encoding the Ion* protein is homologously recombined into the starting bacterium;
  • the coding gene for replacing the clpA protein-encoding gene on the chromosome of the intermediate bacteria with the clpA* protein is a homologous recombination of the ⁇ fragment IV containing the gene encoding the clpA* protein into the recombinant bacterium A;
  • the nucleotide sequence of the DNA fragment III is specifically the sequence 3 in the sequence listing;
  • the nucleotide sequence of the DNA fragment IV is specifically the sequence 4 in the sequence listing.
  • the recombinant B prepared by the above method is also within the scope of protection of the present invention.
  • the production and/or enhancement of L-alanine is specifically produced by fermentation of the recombinant A or the recombinant B in a fermentation medium prepared by using tap water as a solvent.
  • a method for producing L-alanine comprising the steps of: fermenting recombinant A or the recombinant B in a fermentation medium prepared by using tap water as a solvent, collecting the fermentation product, thereby obtaining L- Alanine.
  • Escherichia coli engineering strain XZ- A26 CGMCC No. 4036 deposited on July 26, 2010 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC, Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing) ), the deposit number is CGMCC NO. 4036, and the classification is named Escherichia coli.
  • This strain is capable of fermenting L-alanine in an inorganic salt medium.
  • Escherichia coli engineered bacteria XZ-A26 CGMCC No. 4036 capable of fermenting glucose to produce L-alanine in an inorganic salt medium in distilled water.
  • Table 1 for specific characteristics
  • Seed medium The following solute is dissolved in solvent distilled water to obtain a seed medium: glucose 120g/L, ammonium chloride 5g/L, NaH 2 P0 4 5g/L, Na 2 HP0 4 5g/L, MgS0 4 ⁇ 73 ⁇ 40 5 ⁇ The lg / L, CaCl 2 2H 2 0 0. lg / L, a trace of inorganic salts 5ml / L, the medium pH 6. 5 .
  • composition of the trace inorganic salt is: FeCl 3 ⁇ 6H 2 01. 5mg, CoCl 2 ⁇ 6H 2 0 0. lmg, CuCl 2 ⁇ 2H 2 0
  • composition of the fermentation medium I is: same as the seed medium.
  • composition of the fermentation medium ⁇ is: Same as the seed medium, except that tap water is used instead of distilled water as a solvent.
  • the seed medium in a 250 ml flask was 150 ml and sterilized at 121 °C for 15 min. After cooling, it was connected to XZ-A26, and the culture was incubated at 30 °C for 30 h at 30 °C for fermentation medium inoculation.
  • the volume of the fermentation medium in the 3L fermenter was 2.4 L and sterilized at 121 °C for 15 min.
  • the inoculation amount was 0.1% (V/V).
  • the fermentation temperature was 30 ° C, the stirring speed was 100 rpm, and the fermentation was carried out for 48 h.
  • the neutralizing agent is ammonia water, and the pH of the fermenter is controlled at 6.5.
  • Analytical method The components in the fermentation broth were determined using Agilent (Agi lent-1200) high performance liquid chromatography. The concentration of glucose and organic acids in the fermentation broth was determined by Biorad's Aminex HPX-87H organic acid analytical column. The quantitative and chirality of L-alanine was determined by Dac iel's ligand exchange chiral isomer LC column (Chiralpak MA (+)).
  • the results are shown in Table 2.
  • the strain XZ-A26 was fermented in the fermentation medium I of distilled water for 48 hours, and the L-alanine production reached 1 15 g/L. Fermentation in fermentation medium I I in tap water for 48 hours resulted in a yield of 80 g/L for L-alanine and a 30% reduction in yield.
  • the fermentation medium was 2.4 L.
  • the neutralizing agent used was ammonia water, and the pH of the fermenter was controlled at 6.5.
  • b L-alanine production was defined as 100% in the fermentation medium II of the XZ-A26 strain in tap water.
  • the engineering strain XZ-A26 was continuously passaged in the medium of tap water configuration to improve its ability to withstand tap water.
  • the fermentation medium used for evolutionary metabolism is the same as that of the fermentation medium I I described in the above 1.
  • the evolutionary metabolic process uses a 500 ml fermenter.
  • the volume of the fermentation medium II is 250 ml, and the mixture is sterilized at 121 °C for 15 min. After cooling, it is connected to XZ-A26, and the inoculum is 0.1% (V/V).
  • the fermentation temperature was 30 ° C and the stirring speed was 100 rpm.
  • Ammonia is used as a neutralizing agent in the fermentation process to control the pH of the fermenter to 6.5.
  • the engineered bacteria were continuously subcultured in a fermenter, and the bacterial liquid in the fermenter was transferred to a new fermenter at a ratio of 1:1000 every 24 hours. After 820 generations of transfer, the strain XZ-A41 was finally obtained (Table 1).
  • the fermentation yield in the base was basically the same (Table 2).
  • a single clone of the engineered strain XZ-A41 was inoculated into 4 ml of LB liquid medium, and cultured overnight at a culture temperature of 37 ° C and a rotation speed of 250 rpm, and three parallels were set. The cultured three parallel cells were mixed and the cells were collected, and the bacterial genomic DNA was extracted using Wizard® Genomic DNA Purificati on Ki t (promega). Detection of DNA concentration was quantified by Qubi t Fluorometer and agarose gel electrophoresis.
  • Genomic resequencing was completed by Shenzhen Huada Gene Technology Co., Ltd.
  • the whole genome shotgun method was used to construct the Paired-End fragment library for sequencing.
  • the overall sequencing depth was over 100 times, and the expected data volume was 500 Mbp.
  • the technical methods and routes used for sequencing are as follows: DNA sample preparation, one-on-one sequencing, one data processing, and one biological information analysis.
  • the reference sequence for sequence analysis is E. coli
  • the ATP-dependent molecular chaperone protein ClpA GenBank No. ADT74495. 1
  • the nucleotide sequence at position 1895 has a T mutation of G, and the corresponding amino acid at position 632 is mutated to S, and the gene is named as c ⁇ *, the encoded protein is clpA*.
  • the tap water resistance may be caused by mutation of the gene and the M gene, and the two genes of the starting bacteria are mutated in the next step, thereby obtaining a tap water resistant strain.
  • Example 2 M gene mutation to obtain L-alanine-producing and tap water resistant strain XZ-A43
  • M* was subjected to two-step homologous recombination.
  • Method XZ-A26 was introduced to obtain XZ-A43 (Table 1). Specific steps are as follows:
  • the first step is to use pXZ-CS plasmid (Tan et al., Appl Environ Microbiol. 2013, 79: 4838-4844; public can be obtained from Anhui Huaheng Biotechnology Co., Ltd.;) Sa is a template, using primer XZ-lon *cat-up / XZ- lon*sacB- down amplified 2719 bp DNA fragment I (sequence 1).
  • the amplification system was: NewEnglandBiolabs Phusion 5X Buffer 10 ⁇ l, dNTP (10 mM each for each dNTP) 1 ⁇ 1 , DNA template 20 ng, bow
  • 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 30 seconds (30 cycles); extension at 72 ° C for 5 minutes ( 1 cycle).
  • DNA fragment I contains 50 bases of the homologous arm upstream of the M gene encoding ATP-dependent protease La (sequence 1 from nucleotides 1 to 5 of the 5' end), ⁇ - ⁇ DM fragment (sequence 1 from 5' The nucleotides 5 to 5669 at the end) and the 50-base homolog of the M gene encoding the ATP-dependent protease La (sequence 1 from the 5' end of the 2670-2719 nucleotide).
  • DNA fragment I was used for the first homologous recombination.
  • the pKD46 plasmid (derived from Hefei Baimai Biotechnology Co., Ltd.) was transformed into Escherichia coli engineering strain XZ-A26 by calcium chloride transformation to obtain the large intestine with pKD46.
  • Bacillus engineered strain XZ-A26, and then DNA fragment I was electroporated into E. coli engineered strain XZ-A26 with pKD46.
  • the electroporation conditions were as follows: First, electroporation competent cells of Escherichia coli engineering strain XZ-A26 carrying PKD46 plasmid were prepared; 50 ⁇ l of competent cells were placed on ice, 50 ng of DNA fragment I was added, and placed on ice for 2 minutes, transferred to 0.2 Cm Bio-Rad electric shock cup. Using a MicroPulser (Bio-Rad) electroporator, the electric shock parameter was 2.5 kV. Immediately after the electric shock, the lml LB medium was transferred to the electric shock cup, and after 5 times of blowing, it was transferred to a test tube, 75 rpm, and incubated at 3 CTC for 2 hours.
  • the genomic DNA of the engineered strain XZ-A41 obtained in Example 1 was used as a template, and PCR amplification was carried out using the primer XZ-Ion*-up/XZ-Ion*-down to obtain a 2355 bp DNA fragment II (the The nucleotide sequence is the sequence 2) in the sequence listing, and the DNA fragment II is used for the second homologous recombination.
  • the PKD46 plasmid was first transformed into XZ-A42 by calcium chloride conversion to obtain E. coli engineered strain XZ-A42 carrying PKD46, and then DNA fragment II was electrotransformed into XZ-A42 carrying the pKD46 plasmid.
  • DNA fragment II contains the M* gene.
  • the nucleotide sequence of the M* gene is sequence 2 from the 5' end of the 1-2355 nucleotide, and the JOTJ* gene is the 13th C of the M gene.
  • the C mutation is A, the Ion* gene.
  • the encoded protein is a mutation of the alanine A at position 437 of the protein encoded by the M gene to aspartic acid D.
  • the electroporation conditions were as follows: First, electroporation competent cells of Escherichia coli engineering strain XZ-A42 carrying PKD46 plasmid were prepared; 50 ⁇ l of competent cells were placed on ice, 50 ng DNA fragment II was added, and placed on ice for 2 minutes, transferred to 0. 2 cm Bi o-Rad electric shock cup. Using a MicroPulser (Bio-Rad) electroporator, the shock parameter was 2. 5kv. Immediately after the electric shock, transfer the lml LB medium to the electric shock cup, pipette 5 times, transfer to the test tube, 75 rpm, and incubate for 3 hours at 3CTC.
  • the bacterial solution was transferred to LB liquid medium (50 ml medium in 250 ml flask) containing 10% sucrose without sodium chloride, and cultured for 24 hours, and then LB solid medium containing 6% sucrose containing no sodium chloride Line culture.
  • the primer used was XZ-lon*-up/XZ-lon*-down, and the correct colony amplification product was a fragment of 2355 bp. Pick a correct single colony and name it XZ-A43.
  • the primers used for M* integration are shown in Table 3.
  • Example 1 Using the method described in Example 1, the engineered strain XZ-A43 obtained by fermentation in the fermentation medium II configured with tap water, the yield of L-alanine reached 106 g/L after 48 hours, relative to the starting strain XZ- A26 increased by 33%.
  • Example 3 Gene and M gene mutations were obtained to produce L-alanine and resistant to tap water strain XZ-A47
  • the recombinant strain XZ-A43 obtained in Example 2 was introduced by the method of two-step homologous recombination of *(T1895G) to obtain XZ-A47 (Table 1). Specific steps are as follows:
  • DNA fragment III contains 50 bases of the homologous arm upstream of the gene (sequence 3 from the 5th end of nucleotides 1-50), ca t-sacB DNA fragment (sequence 3 from the 5' end 51-2669 nucleus Glycosyl) and the cpA gene downstream of the homology arm 50 bases (sequence 3 from the 5' end of the 2670-2719 nucleotides).
  • the PKD46 plasmid was transformed into the recombinant strain XZ-A43 obtained in Example 2 by calcium chloride conversion to obtain Escherichia coli engineering strain XZ-A43 with pKD46; then the DNA fragment III was electroporated to E. coli engineering with PKD46.
  • the strain XZ-A43 obtained recombinant bacteria.
  • the recombinant strain was PCR-tested with the primer XZ-clpA*-up I XZ- clpA*-down, and the correct colony amplification product was a 3419 bp fragment. Pick a correct single colony and name it XZ - A46.
  • the genomic DNA of the engineered strain XZ-A41 obtained in Example 1 was used as a template, and PCR amplification was carried out using the primer XZ-clpA*-up I XZ- clpA*-down to obtain a 2272 bp DNA fragment IV (sequence 4), DNA fragment IV was used for the second homologous recombination.
  • the DNA fragment IV contains the ⁇ / ⁇ * gene, the nucleotide sequence of the ⁇ / ⁇ * gene is the sequence 4 from the 5' end of the 1-2272, the c ⁇ * gene is the 1895th of the gene T mutation to G, clpA *
  • the gene-encoded protein is a mutation of the 632th isoleucine I of the protein encoded by the gene to serine S.
  • the PKD46 plasmid was first transformed into XZ-A46 by calcium chloride conversion to obtain XZ-A46 carrying the pKD46 plasmid; then the DNA fragment IV was electrotransformed into XZ-A46 carrying the pKD46 plasmid to obtain a recombinant strain.
  • the recombinant strain was subjected to PCR verification using the primer XZ- clpA*-up I XZ- clpA*-down, and the correct colony amplification product was a 2272 bp fragment. Pick a correct single colony and name it XZ-A47.
  • Example 2 Using the method described in Example I, the engineered bacteria XZ-A47 obtained by fermentation in the fermentation medium II configured with tap water, the L-alanine yield reached 114 g/L after 48 hours, relative strain XZ-A26 Increased by 43% (Table 2).
  • Example 4 Effect of medium of distilled water and tap water on the production of L-alanine by Escherichia coli engineering bacteria XZ-A43 and XZ-A47
  • Example 2 Using the method described in Example 1, the XZ-A43 strain was fermented separately in a fermentation medium configured with distilled water and tap water.
  • the XZ-A43 strain was able to produce 114 g/L of L-alanine in the fermentation medium I in distilled water configuration, and was able to produce it when fermented in medium II using tap water. 106 g/L of L-alanine.
  • the L-alanine yield was increased by 32.5% when the XZ-A43 strain was fermented in a medium containing tap water.
  • the XZ-A47 strain was fermented separately in a fermentation medium configured with distilled water and tap water. It was found that after 48 h of fermentation, the XZ-A47 strain was able to produce 114 g/L of L-alanine in the fermentation medium I in distilled water configuration, and was able to produce when fermented in medium II using tap water. 114 g/L of L-alanine. The L-alanine production was increased by 42.5% when the XZ-A47 strain was fermented in the medium I I of the tap water configuration.
  • the experiment of the present invention proves that the present invention constructs two kinds of recombinant bacteria, one is a recombinant strain XZ-A43 obtained by mutating the M gene in the Escherichia coli engineering strain XZ-A26, and the other is the Escherichia coli engineering.
  • the Ion gene and the clpA gene in the strain XZ-A26 are mutated to obtain the recombinant strain XZ-A47; these two recombinant bacteria can not only increase the production of L-alanine, but also can be used in tap water.
  • the high yield of L-alanine in the fermentation medium can save costs by using a tap water configuration.

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Abstract

本发明公开了一种高产L-丙氨酸且耐受自来水的菌株及其构建方法,所述方法为突变出发菌中的 lon基因和 clpA基因,得到重组菌;所述突变出发菌中的 lon基因为将所述出发菌中的 lon基因核苷酸序列自5'末端第1310位的C突变为A;所述突变出发菌中的 clpA基因为将所述出发菌中的 clpA基因核苷酸序列自5'末端第1895位的T突变为G。本发明通过将大肠杆菌工程菌XZ-A26中的 lon基因和 clpA基因进行突变,得到了重组菌XZ-A47。

Description

-种高产 L-丙氨酸且耐受自来水的菌株及其构建方法 技术领域
本发明涉及生物技术领域, 尤其涉及一种高产 L-丙氨酸且耐受自来水 的菌株及其构建方法。
背景技术
L-丙氨酸作为人体非必需氨基酸, 在生物体内由甘氨酸的氨基转移至 丙酮酸而成。 L-丙氨酸是一种白色结晶或结晶性粉末, 带有甜味, 易溶于 水, 在食品及医药工业领域具有广泛的用途。 在食品工业领域, L-丙氨酸 可提高食品的营养价值,加入 L-丙氨酸后能够明显提高食品及饮料中蛋白 质利用率。 L-丙氨酸能够改善人工合成甜味剂的味感, 使其如同天然甜味 剂。 另外, L-丙氨酸还能够改善有机酸的酸味, 使其更接近自然味道。 在 医药领域, L-丙氨酸常被用作氨基酸类营养药, 同时 L-丙氨酸也是制造维 生素 B6、 合成泛酸钙和其他有机化合物的重要原料。
目前, L-丙氨酸的生产方法主要有化学合成法和生物合成法。 其中化 学合成法主要有丙酸氯化氨化法、 ex-溴丙酸氯化法和氰醇法。 这些方法都 需要石油基原材料, 如丙酸、 ex-溴丙酸、 乙醛和氢氰酸等, 因此成本受困 于原油价格。 随着石油价格的提升, 成本会越来越高。 另外, 这些方法都 是经过复杂的化学催化完成的, 污染重, 分离提取成本高, 不适合可持续 发展的需要。
生物法生产 L-丙氨酸目前主要是以 L-天冬氨酸为原料, 在天冬氨酸 -β-脱羧酶的催化下进行脱羧反应生产 L-丙氨酸。 该方法是目前国内 L-丙 氨酸生产厂家主要使用的生产技术。 但是由于该方法中的原材料天冬氨酸 的生产是以顺酐为原料的, 因此 L-丙氨酸的生产成本依旧依赖于石油价 格。 随着石油资源的匮乏和价格的提升, 顺酐资源的紧张和价格上涨将导 致天冬氨酸的供给存在巨大的隐患, 从而会影响到 L-丙氨酸的生产及成 本。
随着合成生物学和代谢工程的发展, 近年来使用微生物发酵法生产 L- 丙氨酸的研究越来越受到重视。 微生物发酵法能够实现以葡萄糖等糖类为 原料生产 L-丙氨酸。 葡萄糖属于可再生的生物质资源, 可以通过广泛存在 于自然界中的木质纤维素降解获得。 因此, 使用其作为原材料, 能够使 L- 丙氨酸的生产成本保持在稳定的水平, 具有长远的经济优势。 目前, 已经 有多株能够生产 L-丙氨酸的菌株的报道。 Smi th 等构建了一株 E. coli ALS929 (pTrc99A-a^^)菌株, 其中在质粒中表达的丙氨酸脱氢酶 AlaD能 够将胞内生成的丙酮酸转化为 L-丙氨酸, 该菌株经过 48小时发酵后, 能 够生产 88 g/L 的 L-丙氨酸。 Lee 等构建了一株 A coli ALA887 (pTrc99A-a^^)菌株, 发酵生产时能够在 27小时内生产 32 g/L的 L-丙氨 酸。 在这些菌株中, 丙氨酸脱氢酶是生产 L-丙氨酸的关键步骤, 在报道的 菌株中, 编码该蛋白的基因一般是通过高拷贝质粒进行表达的。 在进行菌 株培养基发酵过程中, 需要添加抗生素来维持质粒的稳定遗传。 这些因素 将导致发酵过程工艺复杂并提高 L-丙氨酸生产的成本。 因此, 随着合成生 物学和代谢工程的发展,构建遗传稳定的 L-丙氨酸生产菌株并经过微生物 发酵法生产 L-丙氨酸将是未来的发展趋势。
目前菌株发酵生产 L-丙氨酸使用的都是由蒸馏水配置的培养基, 蒸馏 水在工业生产中提高了生产的成本。 开发能够直接使用自来水配置的培养 基进行发酵生产 L-丙氨酸的菌株并用于生产, 将极大的降低 L-丙氨酸的生 产成本。 但是相对于蒸馏水, 自来水中存在大量的离子并且浓度较高。 为 了获得能够直接使用自来水配置的培养基进行发酵的菌株, 需要提高菌株 对高离子浓度的耐受力。
发明公开
本发明的一个目的是提供构建重组菌 A的方法。
本发明提供的构建重组菌 A ( XZ-A43 ) 的方法, 包括如下步骤: 将出 发菌染色体上的 Ion蛋白编码基因替换为 Ion*蛋白的编码基因,得到的重 组菌 A;
所述 Ion*蛋白的氨基酸序列为将所述 Ion蛋白氨基酸序列的第 437位 丙氨酸 A突变为天冬氨酸0。
上述方法中,所述 Ion*蛋白为由序列表中序列 2 自 5 ' 末端第 1-2355 位核苷酸编码的蛋白。
上述方法中,所述 Ion*蛋白编码基因为将所述 Ion蛋白编码基因核苷 酸序列第 1310位的碱基为 C突变为 A得到的基因。
上述方法中, 所述 Ion*蛋白编码基因的核苷酸序列为序列表中序列 2 自 5 ' 末端第 1-2355位核苷酸;
所述将出发菌染色体上的 Ion蛋白编码基因替换为 Ion*蛋白编码基因 具体为将含有所述 Ion*蛋白编码基因的 DNA片段 II同源重组到所述出发菌 中;
所述 DNA片段 II的核苷酸序列尤其具体为序列表中序列 2。
上述方法中,所述出发菌为通过将嗜热脂肪地芽孢杆菌染色体上的 L- 丙氨酸脱氢酶基因整合在大肠杆菌 ATCC8739 染色体的乳酸脱氢酶处, 再 依次敲除所得大肠杆菌染色体的丙酮酸甲酸裂解酶基因、 乙醇脱氢酶基 因、 乙酸激酶基因、 富马酸还原酶基因和丙氨酸消旋酶基因, 然后在发酵 罐中连续传代培养而得的基因工程菌;
所述出发菌具体为大肠杆菌 XZ-A26 CGMCC No. 4036。
上述同源重组具体通过两步进行:
1 ) 将薩片段 I导入带有 pKD46的大肠杆菌工程菌株 XZ-A26中, 进 行第一次同源重组, 得到中间菌 XZ-A42 ;
2 ) 将 DNA片段 II导入所述中间菌 XZ-A42中, 进行第二次同源重组, 得到重组菌 XZ-A43 (重组菌 A) 。
由上述的方法制备的重组菌 A也是本发明保护的范围。
本发明的另一个目的是提供一种构建重组菌 B (XZ-A47 ) 的方法。 本发明提供的构建重组菌 B的方法, 包括如下步骤: 将上述的出发菌 染色体上的 Ion编码蛋白基因替换为 Ion*蛋白的编码基因,且将所述出发 菌染色体上的 clpA蛋白编码基因突变为 clpA*蛋白的编码基因,得到的重 组菌 B;
所述 Ion*蛋白的氨基酸序列为将所述 Ion蛋白氨基酸序列的第 437位 丙氨酸 A突变为天冬氨酸 D;
所述 clpA*蛋白的氨基酸序列为将所述 clpA蛋白氨基酸序列的第 632 位异亮氨酸 I突变为丝氨酸 S。
上述方法中,所述 clpA*蛋白为由序列表中序列 4自 5 ' 末端第 1-2272 位核苷酸编码的蛋白。
上述方法包括如下步骤: 先所述出发菌染色体上的 Ion编码蛋白基因 替换为 Ion*蛋白的编码基因, 得到重组菌 A, 再将所述重组菌 A染色体上 的 clpA蛋白编码基因替换为 c lpA*蛋白的编码基因, 得到的重组菌 B; 上 述方法中,所述 Ion*蛋白编码基因为将所述 Ion蛋白编码基因核苷酸序列 第 1310位的碱基为 C突变为 A得到的基因;
所述 c lpA*蛋白编码基因为将所述 c lpA 蛋白编码基因核苷酸序列第 1895位的碱基为 T突变为 G得到的基因;
所述 Ion*蛋白编码基因的核苷酸序列具体为序列表中序列 2 自 5 ' 末 端第 1-2355位核苷酸;
所述 clpA*蛋白编码基因的核苷酸序列具体为序列表中序列 2 自 5 ' 末端第 1-2272位核苷酸;
上述方法包括如下步骤:
所述将出发菌染色体上的 Ion编码蛋白基因替换为 Ion*蛋白的编码基 因为将含有所述 Ion*蛋白编码基因的 匪 片段 III同源重组到所述出发菌 中;
所述将所述中间菌染色体上的 clpA蛋白编码基因替换为 clpA*蛋白的 编码基因为将含有所述 clpA*蛋白编码基因的匪片段 IV同源重组到所述 重组菌 A中;
所述 DNA片段 III的核苷酸序列具体为序列表中序列 3 ;
所述 DNA片段 IV的核苷酸序列具体为序列表中序列 4。
上述方法具体通过两步同源重组进行:
1 ) 将 DNA片段 III导入带有 pKD46的大肠杆菌工程菌株 XZ-A43中, 进 行第一次同源重组, 得到中间菌 XZ-A46;
2 ) 将 DNA片段 IV导入所述中间菌 XZ-A46中, 进行第二次同源重组, 得到重组菌 XZ-A47 (重组菌 B ) 。
由上述的方法制备的重组菌 B也是本发明保护的范围。
上述的重组菌 A或上述的重组菌 B在产生和 /或提高 L-丙氨酸中的应 用也是本发明保护的范围;
所述产生和 /或提高 L-丙氨酸具体为将所述重组菌 A或所述重组菌 B 在自来水作为溶剂配制的发酵培养基中发酵生成。
或一种产生 L-丙氨酸的方法, 包括如下步骤: 在自来水作为溶剂配制 的发酵培养基中发酵重组菌 A或所述重组菌 B, 收集发酵产物, 即得到 L- 丙氨酸。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明, 均为常规方法。
下述实施例中所用的材料、 试剂等, 如无特殊说明, 均可从商业途径 得到。
大肠杆菌工程菌 XZ- A26 CGMCC No. 4036, 于 2010年 7月 26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(简称 CGMCC , 地址为: 北京市朝阳区北辰西路 1号院 3号) , 保藏编号为 CGMCC NO. 4036, 分类命 名为大肠埃希氏菌 Escherichia coli。 该菌株能够在无机盐培养基中发酵生 产 L-丙氨酸。
下述实施例中的自来水取自首创自来水公司山海关分公司 (硬度: 2- 3mmol/L、 pH=6. 5- 7. 5、 电导率: 400- 600 μ s/cm、 氯化物 100- 250 mg/L、 硫酸盐 100-250mg/L ) 。
下述实施例中的蒸馏水 (硬度 0、 pH=7. 0-8. 0、 电导率: 5 s/cm ) 实施例 1、 用自来水配置的培养基筛选产 L-丙氨酸且耐自来水菌株
1、 对比蒸馏水和自来水配置的培养基对大肠杆菌工程菌 XZ-A26发酵 生产 L-丙氨酸的影响
大肠杆菌工程菌 XZ-A26 CGMCC No. 4036 (具体特征见表 1 ) , 能够 在蒸馏水配置的无机盐培养基中发酵葡萄糖生产 L-丙氨酸。然而, 由于工 业发酵中使用蒸馏水成本太高, 因此希望能直接使用自来水配置培养基。
因此,对比了蒸馏水和自来水配置的培养基对大肠杆菌工程菌 XZ-A26 发酵生产 L-丙氨酸的影响。
表 1生产 L-丙氨酸的重组大肠杆菌
相关特征
XZ- -A26 E. coliATCC 8739 (ApflB, A frd, A aclhE, A ackA, A gsA,
Δ dadX, ldhA :: alaD from G. Stearo thermophilus), CGMCC No.
4036
XZ- -A41 XZ-A26经过 820代进化后获得的菌株
XZ- -A43 XZ-A26,把野生型 M替换为来自 XZ-A41菌株中含有基因突变
( C 13 10A ) 的 lor^ XZ-A47 XZ-A43 ,把野生型 替换为来自 XZ-A41菌株中含有基因突 变 ( T1895G) 的 clpA
具体步骤如下:
种子培养基: 将如下溶质溶解在溶剂蒸馏水中, 得到种子培养基: 葡萄糖 120g/L, 氯化铵 5g/L, NaH2P04 5g/L, Na2HP04 5g/L, MgS04 · 7¾0 lg/L, CaCl2 2H20 0. lg/L, 微量无机盐 5ml/L, 培养基 pH6. 5。
微量无机盐组成为: FeCl3 · 6H201. 5mg, CoCl2 · 6H20 0. lmg, CuCl2 · 2H20
0. lmg, ZnCl20. lmg, Na2Mo04 · 2H20 0. lmg, MnCl2 · 4H20 0. 2mg, 蒸馏水定 容至 1L, 过滤除菌。
发酵培养基 I的组成为: 同种子培养基。
发酵培养基 Π 的组成为: 同种子培养基, 只是用自来水替代蒸馏水 作为溶剂。
250ml三角瓶中种子培养基为 150 ml , 121 °C灭菌 15min。 冷却后接入 XZ-A26, 在 30°C, 摇床转速为 50 rpm, 培养 18 h, 用于发酵培养基接种。
3L发酵罐中发酵培养基体积为 2 . 4 L, 121 °C灭菌 15min。 接种量为 0. 1% ( V/V) 。 发酵温度为 30°C, 搅拌转速为 100 rpm, 发酵 48 h。 中和 剂为氨水, 使发酵罐的 pH控制在 6. 5。
分析方法: 使用安捷伦 (Agi lent-1200 ) 高效液相色谱对发酵液中的 组分进行测定。 发酵液中的葡萄糖和有机酸浓度采用伯乐 (Biorad ) 公司 的 Aminex HPX-87H有机酸分析柱。 L-丙氨酸的定量和手性测定采用大赛 璐 (Dac iel ) 公司的配基交换型手性异构体液相色谱分离柱 (Chiralpak MA (+) ) 。
结果见表 2, 菌株 XZ-A26在蒸馏水配置的发酵培养基 I中发酵 48小 时, L-丙氨酸产量达到 1 15 g/L。 在自来水配置的发酵培养基 I I 中发酵 48小时, L-丙氨酸产量达 80 g/L, 产量降低了 30%。
表 2 重组大肠杆菌发酵生产 L-丙氨酸
菌株 a 培养基条件 L-丙氨酸 L-丙氨酸的相对
产量 (g/L) 产量
(%) b
XZ-A26 蒸馏水配置的发酵培养基 I 1 15 143. 75 自来水配置的发酵培养基 80 100
II
xz- -A41 自来水配置的发酵培养基 114 142. 5
II
xz- -A43 自来水配置的发酵培养基 106 132. 5
II
xz- -A43 蒸馏水配置的发酵培养基 I 114 142. 5
xz- -A47 自来水配置的发酵培养基 114 142. 5
II
xz- -A47 蒸馏水配置的发酵培养基 I 114 142. 5
a使用 3 L的发酵罐, 发酵培养基为 2. 4 L。 使用的中和剂为氨水, 使 发酵罐的 pH控制在 6. 5。
b以 XZ-A26菌株在自来水配置的发酵培养基 II中 L-丙氨酸产量定义 为 100%。
2、 采用适应进化提高工程菌耐受自来水的能力
采用适应进化技术,在自来水配置的培养基中连续传代工程菌 XZ-A26, 以提高其耐受自来水的能力。
进化代谢所使用的发酵培养基同上述 1中所述发酵培养基 I I的成分相 同。进化代谢过程使用 500 ml的发酵罐, 发酵培养基 II的体积为 250 ml , 121 °C灭菌 15min, 冷却后接入 XZ-A26, 接种量为 0. 1% (V/V) 。 发酵温度 为 30°C, 搅拌转速为 100 rpm。 发酵过程中使用氨水为中和剂, 使发酵罐 的 pH控制在 6. 5。 在发酵罐中连续传代培养工程菌, 每 24小时将发酵罐 中的菌液按照 1 : 1000的比例转接到一个新的发酵罐中。 经过 820代转接, 最终获得菌株 XZ-A41 (表 1 ) 。
使用同上述 1 中所述的方法, 在用自来水配置的发酵培养基 II 中发 酵获得的工程菌 XZ-A41 , 48小时后 L-丙氨酸产量达 114 g/L, 和在蒸馏 水配置的培养基中发酵产量基本一样 (表 2 ) 。
上述结果表明, 工程菌 XZ-A41不仅可以高产 L-丙氨酸, 而且耐受自 来水。 因此, 为了研究其耐受性是否是由基因突变引起的, 对其基因组进 行测序。 3、 工程菌 XZ-A41的基因组测序
(1) 发酵培养与基因组制备
挑取工程菌株 XZ-A41 的单克隆接种到 4 ml 的 LB液体培养基中, 在 培养温度为 37 °C,转速为 250 rpm的条件下震荡培养过夜,设定三个平行。 将培养好的三个平行中的细胞混匀并收集细胞,使用 Wi zard® Genomic DNA Purificati on Ki t (promega)抽提细菌基因组 DNA。 DNA浓度的检测通过 Qubi t Fluorometer和琼脂糖凝胶电泳定量完成。
(2) 基因组重测序
基因组重测序由深圳华大基因科技有限公司完成。 采用全基因组鸟枪 法, 构建 Paired-End片段库进行测序, 整体测序深度在 100倍以上, 期 望数据量为 500Mbp。 测序采用的技术方法和路线为: DNA样品制备一一上 机测序一一数据处理一生物信息分析。序列分析的参考序列为 E. coli隱 8
Figure imgf000009_0001
(编码 ATP依赖型分子伴侣蛋白 ClpA, GenBank No. ADT74495. 1 ) 的核苷 酸序列第 1895位的 T突变为 G, 其对应的氨基酸第 632位的 I突变成 S, 将该基因命名为 c^^*, 编码的蛋白为 clpA*。
另外, 发现 Ion 基因 (编码 ATP 依赖型蛋白酶 La, GenBank No. AFH10177. 1 )核苷酸序列第 1310位的 C突变为 A, 其对应的氨基酸序列第 437位的 A突变成 D。 将该基因命名为 M*, 编码的蛋白为 lon*。
因此, 认为耐自来水性可能是由于 基因和 M基因突变引起的, 下一步对出发菌的这两个基因进行突变, 从而获得耐自来水菌株。
实施例 2、 M基因突变获得产 L-丙氨酸且耐自来水菌株 XZ-A43 为了验证 M基因突变(C 1310A )对工程菌株耐受自来水能力的影响, 将 M*通过两步同源重组的方法引入 XZ-A26 , 获得 XZ-A43 (表 1 ) 。 具 体步骤如下:
第一步, 以 pXZ- CS质粒(Tan et al., Appl Environ Microbiol. 2013, 79 : 4838-4844; 公众可从安徽华恒生物科技股份有限公司获得; ) 薩为 模板, 使用引物 XZ-lon*cat-up / XZ- lon*sacB- down扩增出 2719 bp 的 DNA片段 I (序列 1) 。
扩增体系为: NewEnglandBiolabs Phusion 5X缓冲液 10 μ 1、 dNTP (每 种 dNTP各 10 mM) 1 μ 1、 DNA模板 20 ng、 弓 |物(10 μ M)各 2 μ 1、 Phusion High- Fidelity DNA聚合酶 (2·5 υ/μ 1) 0.5 μ 1、 蒸馏水 33· 5 μ 1, 总 体积为 50 μ 1。
扩增条件为 98°C预变性 2分钟(1个循环); 98°C变性 10秒、 56°C退 火 10秒、 72°C延伸 30秒(30个循环); 72°C延伸 5分钟(1个循环)。
DNA片段 I包含编码 ATP依赖型蛋白酶 La的 M基因上游同源臂 50 个碱基 (序列 1 自 5' 末端第 1-50位核苷酸) 、 ^-^^ DM片段 (序 列 1 自 5' 末端第 51-2669位核苷酸) 及编码 ATP依赖型蛋白酶 La的 M 基因下游同源臂 50个碱基 (序列 1 自 5' 末端第 2670-2719位核苷酸) 。
将 DNA片段 I用于第一次同源重组, 首先将 pKD46质粒 (来源于合肥 百迈生物技术有限公司) 通过氯化钙转化法转化至大肠杆菌工程菌株 XZ-A26, 得到带有 pKD46的大肠杆菌工程菌株 XZ-A26, 然后将 DNA片段 I 电转至带有 pKD46的大肠杆菌工程菌株 XZ-A26。
电转条件为: 首先准备带有 PKD46 质粒的大肠杆菌工程菌株 XZ-A26 的电转化感受态细胞; 将 50μ 1感受态细胞置于冰上, 加入 50ngDNA片段 I, 冰上放置 2分钟, 转移至 0.2 cm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 lml LB 培养基转移至电击杯中, 吹打 5次后转移至试管中, 75转, 3CTC孵育 2小 时。 取 200 μ 1菌液涂在含有氯霉素 (终浓度为 17ug/ml) 的 LB平板上, 37°C过夜培养后, 挑选 5个单菌落进行 PCR验证, 使用引物 XZ-lon*-up/ XZ-lon*-doWn进行验证, 正确的菌落扩增产物为 3419bp的片段。 挑选一 个正确的单菌落, 将其命名为 XZ-A42。
第二步, 以实施例 1得到的工程菌株 XZ-A41 的基因组 DNA为模板, 使用引物 XZ- Ion*- up/XZ- Ion*- down进行 PCR扩增, 获得 2355 bp 的 DNA 片段 II (其核苷酸序列为序列表中的序列 2) , DNA片段 II用于第二次同 源重组。 首先将 PKD46 质粒通过氯化钙转化法转化至 XZ-A42, 得到带有 PKD46的大肠杆菌工程菌株 XZ-A42,然后将 DNA片段 II电转化至带有 pKD46 质粒的 XZ-A42。 DNA片段 I I包含 M*基因, M*基因的核苷酸序列为序列 2 自 5 ' 末 端第 1-2355位核苷酸, JOTJ*基因为 M基因的第 1310位 C突变为 A, Ion* 基因编码的蛋白为将 M基因编码的蛋白的第 437位丙氨酸 A突变为天冬 氨酸 D。
电转条件为: 首先准备带有 PKD46 质粒的大肠杆菌工程菌株 XZ-A42 的电转化感受态细胞; 将 50 μ 1感受态细胞置于冰上, 加入 50ngDNA片段 I I,冰上放置 2分钟,转移至 0. 2 cm的 Bi o-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 lml LB 培养基转移至电击杯中, 吹打 5次后转移至试管中, 75转, 3CTC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-lon*-up/XZ-lon*-down, 正确的菌落扩增产物为 2355bp的片段。 挑选 一个正确的单菌落, 将其命名为 XZ-A43。
M*整合所用引物见表 3。
使用同实施例 1 中所述的方法, 在用自来水配置的发酵培养基 I I 中 发酵获得的工程菌 XZ-A43 , 48小时后 L-丙氨酸产量达 106 g/L, 相对出 发菌株 XZ-A26提高了 33%。
表 3 本发明中所使用的引物
Figure imgf000011_0001
XZ-clpA*sacB-down CGGAATTCCGGTGTAAAGATCTTCTTGATCTCCTCCATCGCAT
CGGTGCTTTATTTGTTAACTGTTAATTGTCCT
XZ-clpA*-up ATGCTCAATCAAGAACTGGAAC
XZ-clpA*-down GCGCTGCTTCCGCCTTGTGC
实施例 3、 基因和 M基因突变获得产 L-丙氨酸且耐自来水菌株 XZ-A47
将 * ( T1895G) 通过两步同源重组的方法引入实施例 2获得的重 组菌 XZ-A43 , 获得 XZ-A47 (表 1 ) 。 具体步骤如下:
第一步, 以 pXZ- CS 质粒 DNA 为模板, 使用引物 XZ- ClpA*cat- up /
XZ-clpA*sacB- down扩增出 2719 bp的 DNA片段 III (序列 3 ) 。
DNA片段 III包含 ^基因上游同源臂 50个碱基 (序列 3 自 5 ' 末端 第 1-50位核苷酸) 、 ca t-sacB DNA片段 (序列 3 自 5 ' 末端第 51-2669 位核苷酸) 及 clpA 基因下游同源臂 50 个碱基 (序列 3 自 5 ' 末端第 2670-2719位核苷酸) 。
首先将 PKD46 质粒通过氯化钙转化法转化至实施例 2 获得的重组菌 XZ-A43 , 得到带有 pKD46的大肠杆菌工程菌株 XZ-A43 ; 然后将 DNA片段 III 电转至带有 PKD46的大肠杆菌工程菌株 XZ-A43 , 得到重组菌。
将重组菌用引物 XZ-clpA*-up I XZ- clpA*- down进行 PCR验证, 正确 的菌落扩增产物为 3419 bp的片段。 挑选一个正确的单菌落, 将其命名为 XZ - A46。
第二步, 以实施例 1得到的工程菌株 XZ-A41 的基因组 DNA为模板, 使用引物 XZ-clpA*-up I XZ- clpA*- down进行 PCR扩增, 获得 2272 bp 的 DNA片段 IV (序列 4 ) , DNA片段 IV用于第二次同源重组。
DNA片段 IV包含 ^/^*基因, ^/^*基因的核苷酸序列为序列 4 自 5 ' 末端第 1-2272位, c^^*基因为 基因的第 1895位 T突变为 G, clpA* 基因编码的蛋白为将 基因编码的蛋白的第 632位异亮氨酸 I突变为 丝氨酸 S。
首先将 PKD46质粒通过氯化钙转化法转化至 XZ-A46 , 得到带有 pKD46 质粒的 XZ-A46; 然后将 DNA片段 IV电转化至带有 pKD46质粒的 XZ-A46 , 得到重组菌。 将重组菌用引物 XZ- clpA*- up I XZ- clpA*- down进行 PCR验证, 正确 的菌落扩增产物为 2272 bp的片段。 挑选一个正确的单菌落, 将其命名为 XZ-A47。
上述所用引物序列见表 3。
使用同实施例 I 中所述的方法, 在用自来水配置的发酵培养基 I I 中 发酵获得的工程菌 XZ-A47 , 48小时后 L-丙氨酸产量达 114 g/L, 相对菌 株 XZ- A26提高了 43% (表 2 ) 。
实施例 4、 对比蒸馏水和自来水配置的培养基对大肠杆菌工程菌 XZ-A43和 XZ-A47发酵生产 L-丙氨酸的影响
1、 对比蒸馏水和自来水配置的培养基对大肠杆菌工程菌 XZ-A43发酵 生产 L-丙氨酸的影响
使用同实施例 1中所述方法, 分别在用蒸馏水和自来水配置的发酵培 养基中发酵 XZ-A43菌株。
结果发现, 经过 48 h的发酵后, XZ-A43菌株在蒸馏水配置的发酵培 养基 I 中能够生产 114 g/L 的 L-丙氨酸, 而在使用自来水配置的培养基 I I中发酵时能够生产 106 g/L的 L-丙氨酸。 XZ-A43菌株和 XZ-A26相比, 在使用自来水配置的培养基 Π中发酵时, L-丙氨酸产量提高了 32. 5%。
2、 对比蒸馏水和自来水配置的培养基对大肠杆菌工程菌 XZ-A47发酵 生产 L-丙氨酸的影响
使用同实施例 1中所述方法, 分别在用蒸馏水和自来水配置的发酵培 养基中发酵 XZ-A47菌株。 结果发现, 经过 48 h的发酵后, XZ-A47菌株在 蒸馏水配置的发酵培养基 I中能够生产 114 g/L的 L-丙氨酸, 而在使用自 来水配置的培养基 I I中发酵时能够生产 114 g/L的 L-丙氨酸。 XZ-A47菌 株和 XZ-A26相比, 在使用自来水配置的培养基 I I中发酵时, L-丙氨酸产 量提高了 42. 5%。
工业应用
本发明的实验证明, 本发明构建了两种重组菌, 一种为将大肠杆菌工程 菌 XZ-A26中的 ^ M基因进行突变, 得到的重组菌 XZ-A43 , 另一种为将大肠 杆菌工程菌 XZ-A26 中的 Ion基因和 clpA 基因进行突变, 得到的重组菌 XZ-A47 ; 这两种重组菌不仅能够提高 L-丙氨酸产量, 而且还可以在自来水配 置的发酵培养基中高产 L-丙氨酸, 采用自来水配置可以节约成本。

Claims

权利要求
1、 一种构建重组菌 A 的方法, 包括如下步骤: 将出发菌染色体上的 Ion蛋白编码基因替换为 Ion*蛋白的编码基因, 得到的重组菌 A;
所述 Ion*蛋白的氨基酸序列为将所述 Ion蛋白氨基酸序列的第 437位 丙氨酸 A突变为天冬氨酸 D。
2、 根据权利要求 1所述的方法, 其特征在于:
所述 Ion*蛋白编码基因为将所述 Ion 蛋白编码基因核苷酸序列第 1310位的碱基为 C突变为 A得到的基因。
3、 根据权利要求 1或 2所述的方法, 其特征在于:
所述 Ion*蛋白编码基因的核苷酸序列为序列表中序列 2自 5 ' 末端第 1-2355位核苷酸;
所述将出发菌染色体上的 Ion蛋白编码基因替换为 Ion*蛋白编码基因 具体为将含有所述 Ion*蛋白编码基因的 DNA片段 II同源重组到所述出发菌 中;
所述匪片段 II的核苷酸序列尤其具体为序列表中序列 2。
4、 根据权利要求 1-3 中任一所述的方法, 其特征在于: 所述出发菌 为通过将嗜热脂肪地芽孢杆菌染色体上的 L-丙氨酸脱氢酶基因整合在大 肠杆菌 ATCC8739 染色体的乳酸脱氢酶处, 再依次敲除所得大肠杆菌染色 体的丙酮酸甲酸裂解酶基因、 乙醇脱氢酶基因、 乙酸激酶基因、 富马酸还 原酶基因和丙氨酸消旋酶基因, 然后在发酵罐中连续传代培养而得的基因 工程菌;
所述出发菌具体为大肠杆菌 XZ-A26 CGMCC No. 4036。
5、 由权利要求 1-4中任一所述的方法制备的重组菌 A。
6、 一种构建重组菌 B的方法, 包括如下步骤: 将权利要求 1-4中任 一所述的方法中所述的出发菌染色体上的 Ion 编码蛋白基因替换为 Ion* 蛋白的编码基因, 且将所述出发菌染色体上的 clpA 蛋白编码基因替换为 clpA*蛋白的编码基因, 得到的重组菌 B;
所述 Ion*蛋白的氨基酸序列为将所述 Ion蛋白氨基酸序列的第 437位 丙氨酸 A突变为天冬氨酸 D; 所述 clpA*蛋白的氨基酸序列为将所述 clpA蛋白氨基酸序列的第 632 位异亮氨酸 I突变为丝氨酸 S。
7、 根据权利要求 6所述的方法, 其特征在于: 所述方法包括如下步 骤:先所述出发菌染色体上的 Ion编码蛋白基因替换为 Ion*蛋白的编码基 因, 得到重组菌 A, 再将所述重组菌 A染色体上的 clpA蛋白编码基因替换 为 clpA*蛋白的编码基因, 得到的重组菌 B;
所述 Ion*蛋白编码基因为将所述 Ion 蛋白编码基因核苷酸序列第 1310位的碱基为 C突变为 A得到的基因;
所述 clpA*蛋白编码基因为将所述 clpA 蛋白编码基因核苷酸序列第 1895位的碱基为 T突变为 G得到的基因;
所述 Ion*蛋白编码基因的核苷酸序列具体为序列表中序列 2 自 5 ' 末 端第 1-2355位核苷酸;
所述 clpA*蛋白编码基因的核苷酸序列具体为序列表中序列 2 自 5 ' 末端第 1-2272位核苷酸。
8、 根据权利要求 6或 Ί所述的方法, 其特征在于: 所述方法包括如 下步骤:
所述将出发菌染色体上的 Ion编码蛋白基因替换为 Ion*蛋白的编码基 因为将含有所述 Ion*蛋白编码基因的 匪 片段 III同源重组到所述出发菌 中;
所述将所述中间菌染色体上的 clpA蛋白编码基因替换为 clpA*蛋白的 编码基因为将含有所述 clpA*蛋白编码基因的匪片段 IV同源重组到所述 重组菌 A中;
所述 DNA片段 III的核苷酸序列具体为序列表中序列 3 ;
所述 DNA片段 IV的核苷酸序列具体为序列表中序列 4。
9、 由权利要求 6-8中任一所述的方法制备的重组菌 B。
10、 权利要求 5所述的重组菌 A或权利要求 6所述的重组菌 B在产生 和 /或提高 L-丙氨酸中的应用;
所述产生和 /或提高 L-丙氨酸具体为将所述重组菌 A或所述重组菌 B 在自来水作为溶剂配制的发酵培养基中发酵生成。
或一种产生 L-丙氨酸的方法, 包括如下步骤: 在自来水作为溶剂配制 的发酵培养基中发酵重组菌 A或所述重组菌 B, 收集发酵产物, 即得到 L- 丙氨酸。
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