WO2023056699A1 - 一种生产l-丙氨酸的基因工程菌株及其构建方法和应用 - Google Patents

一种生产l-丙氨酸的基因工程菌株及其构建方法和应用 Download PDF

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WO2023056699A1
WO2023056699A1 PCT/CN2021/135643 CN2021135643W WO2023056699A1 WO 2023056699 A1 WO2023056699 A1 WO 2023056699A1 CN 2021135643 W CN2021135643 W CN 2021135643W WO 2023056699 A1 WO2023056699 A1 WO 2023056699A1
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alanine
gene
fermentation
genetically engineered
seq
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French (fr)
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许平
韩笑
陶飞
穆晓玲
陈思弘
李维理
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安徽丰原生物技术股份有限公司
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12R2001/07—Bacillus
    • C12R2001/10—Bacillus licheniformis

Definitions

  • the invention relates to the field of bioengineering, in particular to a genetically engineered bacterial strain for producing L-alanine and its construction method and application.
  • L-alanine is one of the 20 basic amino acids and one of the smallest chiral molecules. It is widely used in food, medicine, daily chemical and other fields. In the food industry, L-alanine can be added to food as a preservative, sweetener, etc. In medicine and health care, L-alanine can not only be used in amino acid nutritional supplements, but also play an important role in the treatment of acute and chronic renal failure, liver disease, diabetes, obesity and other diseases. In daily chemical products, L-alanine can be used to synthesize amino acid surfactants with high bioaffinity.
  • the production methods of L-alanine mainly include chemical synthesis, enzyme catalysis and fermentation.
  • the chemical synthesis method mainly adopts the propionic acid chlorination process, with liquid chlorine and propionic acid as the main raw materials. Due to the long synthetic route, high cost and low yield of chemical synthesis, it is basically eliminated at present.
  • the enzyme-catalyzed method mainly utilizes immobilized L-aspartic acid- ⁇ -decarboxylase to catalyze the decarboxylation of aspartic acid to generate L-alanine.
  • the enzymatic method to generate a three-carbon product from a four-carbon substrate has a low carbon utilization rate, and the price of aspartic acid is high, so it is difficult to meet the needs of large-scale production.
  • the fermentation method mainly uses glucose as the substrate, and constructs strains to ferment and produce L-alanine by means of genetic engineering. It has the advantages of cheap and easy-to-obtain raw materials, high product yield, and low cost, so it has attracted widespread attention.
  • high temperature fermentation can significantly reduce the risk of pollution, improve the conversion efficiency of raw materials and reduce the cost of heat exchange, so it has a high application value in large-scale fermentation production.
  • Alanine fermentation usually uses genetically engineered Escherichia coli as the production strain, but the optimum growth temperature of this strain is 37°C, so it cannot be fermented at high temperature.
  • the technical problem to be solved by the present invention is to provide a genetically engineered strain capable of producing L-alanine at a high temperature above 42°C and its construction method and application.
  • the first aspect of the present invention provides a method for constructing a genetically engineered strain producing L-alanine, comprising the following steps:
  • S400 The genome of the starting bacterium contains the alanine racemase gene, and this step includes inactivating or deleting the alanine racemase gene.
  • the starting bacterium also has a lactic acid synthesis pathway, and the genome of the starting bacterium contains a lactate dehydrogenase gene; the construction method also includes the following steps:
  • the application of the genetic engineering strain described in the second aspect of the present invention in the production of L-alanine is provided.
  • a method for producing L-alanine is provided, wherein the genetic engineering strain described in the second aspect of the present invention is fermented and cultivated, wherein the fermentation temperature is 42°C-55°C.
  • the step of fermenting and cultivating includes: inoculating the activated seeds into a fermentation medium, the fermentation medium contains a carbon source, and the fermentation culture temperature is 42°C-55°C,
  • the fed-batch fermentation is carried out under the condition that the stirring speed is 50rpm-350rpm, and cultured to the set fermentation density.
  • the genetically engineered bacterial strain provided by the present invention realizes high-temperature production of L-alanine (for example, 42°C to 55°C), has the advantages of low energy consumption and anti-pollution during the fermentation process, and effectively reduces production costs.
  • thermophilic bacteria as the starting bacteria to construct genetically engineered strains can realize the fermentation and production of L-alanine under high temperature conditions (for example, 42° C. to 55° C., specifically 50° C.). Further, by enhancing the glycolytic pathway or/and introducing thermostable alanine dehydrogenase, the production of alanine can be significantly improved. Production of optically pure L-alanine can be achieved by knocking out the alanine racemase gene.
  • the combination of the above means can realize the synergistic effect of high-yield optically pure L-alanine at high temperature, the temperature of fermentation production can be as high as 42°C-55°C, and the optical purity of L-alanine can generally achieve more than 98%, and some can achieve More than 99%, in some embodiments, the output of optically pure L-alanine can reach more than 95g/L, and some can reach more than 100g/L.
  • the invention solves the problems of low fermentation temperature and high cost in producing L-alanine in the prior art, realizes the high-temperature fermentation production of L-alanine, and has high industrial application value.
  • the proportion of pyruvate synthesis pathway can be increased, thereby increasing the production of L-alanine.
  • the ratio of the alanine synthesis pathway can be increased, and further, by enhancing the glycolysis pathway and introducing thermostable alanine Amino acid dehydrogenase can be used to construct genetically engineered strains with high alanine production. Knockout of the alanine racemase gene enables the production of optically pure L-alanine.
  • Fig. 1 is in one embodiment of the present invention, the synthetic and metabolic pathway schematic diagram of alanine in the engineering bacterium taking Bacillus licheniformis as example;
  • Fig. 2 is in one embodiment of the present invention, a kind of genetically engineered bacterial strain BLA-1 that produces L-alanine in 5L fermentor 50 °C the fermentation graph ( ⁇ , OD600; , glucose; , alanine);
  • Figure 3 is a structural diagram of the PKVM vector used in some embodiments of the present invention, which includes two antibiotic resistance genes ampR (ampicillin resistance gene) and ermC (erythromycin resistance gene), and encoding galactoside
  • ampR ampicillin resistance gene
  • ermC erythromycin resistance gene
  • the bgaB gene of the enzyme can be used as a selection marker; the plasmid has a heat-sensitive replication origin site oriTpE194ts, as well as replication origin sites ori and oriT in Escherichia coli and Bacillus licheniformis. .
  • the percentage concentration involved in the present invention means the final concentration in the relevant system unless otherwise specified or specified.
  • starting bacteria and “starting bacteria” have the same meaning and can be used interchangeably.
  • the present invention involves temperature control, which is allowed to be a constant temperature, and also allows a certain range of temperature fluctuations.
  • high-temperature production of L-alanine is relative to normal temperature (referring to below 42°C, usually below 37°C).
  • the high-temperature production herein refers to the conditions of 42°C to 55°C unless otherwise specified.
  • examples include but are not limited to 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 52°C, 53°C, 54°C, 55°C.
  • pyruvate synthesis pathway refers to the pathway in which carbon sources synthesize pyruvate through glycolysis.
  • having a pyruvate synthesis pathway refers to having the ability to synthesize pyruvate endogenously.
  • the "synthetic route of D-alanine” means a route for synthesizing D-alanine from L-alanine. "Having a synthetic pathway for D-alanine” means having the ability to synthesize D-alanine endogenously.
  • the gene cluster fragment refers to at least two related gene units, and the related gene units can be the same or different genes. If it is a gene cluster fragment composed of the same gene, "repetition" should be understood in a broad sense, allowing adjacent sequences to appear, and an interval sequence can also exist between any two gene units.
  • Inactivation and “deletion” in the present invention may refer to partial or complete inactivation, partial or complete deletion unless otherwise specified.
  • SEQ ID NO.3 CCTCGCGTCGGGCGATATCGGATCCGAAGGGGAAAGTCTTCGATTTCT
  • SEQ ID NO.6 CCATGGTACCCGGGAGCTCGAATTCCATAAGACCGCTGATGACAAGC
  • SEQ ID NO.8 AATAGGCGTCACCTTGACTCATCATTCCTTTGCCGTT
  • SEQ ID NO.12 CTTCATGGTGTTCAGTTACAATACAGTCGCATGGCC
  • SEQ ID NO.14 ACTAGACAGATCTATCGATGCATGCTTTCCCTTATTCCTTTAAACCCG
  • SEQ ID NO.15 AATAGGCGTCACCTTGCTGAAGAGGTCAAAAAGCC
  • SEQ ID NO. 16 TTTGACCTCTTTCAGCAAGGTGACGCCTATTTCACTTTCT
  • SEQ ID NO. 17 AGAGGGCTTTTTCATTTAGCCATGCAGCAGGCTATG
  • SEQ ID NO.20 CTAATTCATCAATTTGACACTTCCTGTTCCTTGTTTCACT
  • SEQ ID NO.21 GGAACAGGAAGTGTCAAATTGATGAATTAGCGGAAAAAC
  • SEQ ID NO.22 CCATGGTACCCGGGAGCTCGAATTCCGGAGTCTCTTTCAAAACCGTAG
  • SEQ ID NO.23 CCTCGCGTCGGGCGATATCGGATCCAAAATCATGTAAGCCCATTCCG
  • SEQ ID NO.24 GTGAGTATGGGAAAACAACGCTCCCTTCTTTCTTGTC
  • SEQ ID NO.26 CCATGGTACCCGGGAGCTCGAATTCTAAAATGAAGGTGGTCCGGGAT
  • the first aspect of the present invention provides a method for constructing a genetically engineered bacterial strain producing L-alanine, comprising the steps of:
  • thermostable alanine dehydrogenase gene preferably overexpressed thermostable alanine dehydrogenase gene GSald
  • S400 The genome of the starting bacterium contains an alanine racemase gene, and this step includes inactivating or deleting the alanine racemase gene.
  • the S200, S300, and S400 can be independently implemented in the following ways:
  • S400 partially or completely inactivate the alanine racemase gene, or partially or completely delete the alanine racemase gene; for example, knock out the alanine racemase gene.
  • the L-alanine-producing genetic engineering strain constructed in the first aspect of the present invention can ferment and produce L-alanine at a high temperature above 42°C (eg, 42°C-55°C).
  • step S200 can realize the overexpression of 6-phosphofructokinase and pyruvate kinase in the glycolysis pathway, thereby enhancing the glycolysis pathway, increasing the supply of pyruvate, and promoting the increase of L-alanine production.
  • a thermostable alanine dehydrogenase gene can be introduced, which can enhance the synthesis pathway from pyruvate to L-alanine, and effectively increase the production of L-alanine.
  • Step S400 can inactivate or delete the alanine racemase gene, which is beneficial to realize the production of optically pure L-alanine.
  • the adoption of any one of these steps is conducive to improving the output of L-alanine, and when two or three of the steps are used in combination, it can play a synergistic effect .
  • the starting bacterium also has a lactic acid synthesis pathway, and the genome of the starting bacterium contains a lactate dehydrogenase gene; the construction method also includes the following steps:
  • FIG. 1 a schematic diagram of alanine synthesis and metabolic pathways in a genetically engineered bacterium producing L-alanine is shown in FIG. 1 .
  • the carbon source is converted into pyruvate through the glycolytic pathway, and then pyruvate consumes NADH and ammonium ions are catalyzed by alanine dehydrogenase to generate L-alanine.
  • a part of the generated L-alanine is directly secreted out of the cell, and the other part is catalyzed by alanine racemase to form D-alanine, which is then secreted out of the cell.
  • the lactic acid synthesis pathway occupies the main carbon flux downstream of pyruvate in the starter bacteria
  • blocking the lactic acid synthesis pathway can make the carbon flux flow to the alanine synthesis pathway as much as possible.
  • overexpression of 6-phosphofructokinase and pyruvate kinase in the glycolytic pathway can increase the glycolytic pathway and increase the supply of pyruvate, thereby increasing the production of alanine.
  • Further knockout of alanine racemase can block the D-alanine synthesis pathway, thereby obtaining an optically pure L-alanine product.
  • the starting bacterium also has a D-lactic acid synthesis pathway, and the genome of the starting bacterium contains the D-lactate dehydrogenase gene ldh Ti ; the construction method also includes the following Step S500: inactivating or deleting the D-lactate dehydrogenase gene ldh Ti contained in the genome of the starting bacterium.
  • the step S500 includes: knocking out the D-lactate dehydrogenase gene ldh Ti contained in the genome of the starting bacterium.
  • the sequence of the D-lactate dehydrogenase gene ldh Ti is shown in SEQ ID NO.31.
  • the sequence of the 6-phosphofructokinase gene pfk is shown in SEQ ID NO.27, and the sequence of the pyruvate kinase gene pyk is shown in SEQ ID NO.28.
  • sequence of the alanine dehydrogenase gene is shown in SEQ ID NO.1.
  • the types of the inactivated or deleted alanine racemase genes are 1, 2 or more.
  • the way to delete the relevant gene is to knock out the gene.
  • the step S400 includes: inactivating or deleting the alanine racemase gene alr1 or the alanine racemase gene alr2. In some embodiments of the present invention, the step S400 includes: inactivating or deleting the alanine racemase gene alr1 and the alanine racemase gene alr2. In some preferred embodiments of the present invention, the step S400 includes: knocking out the alanine racemase gene alr1 and the alanine racemase gene alr2.
  • modifying the genome of the starting bacterium includes step S100 and step S200.
  • the lactic acid synthesis pathway can be blocked by inactivation or deletion of the lactate dehydrogenase gene, and the glycolysis pathway can be enhanced by the overexpression of the 6-phosphofructokinase gene pfk and the pyruvate kinase gene pyk.
  • modifying the genome of the starting bacterium further includes step S300, at this time, inserting a thermostable alanine dehydrogenase gene can further increase the production of L-alanine.
  • modifying the genome of the starting bacterium includes step S100, step S200, step S300 and step S400.
  • the lactate dehydrogenase gene in the starter bacteria can block the lactic acid synthesis pathway; overexpression of the 6-phosphofructokinase gene and pyruvate kinase gene in the starter bacteria can enhance the glycolysis pathway, thereby increasing the acetone Acid supply; introduction of overexpressed alanine dehydrogenase gene can enhance the synthetic pathway from pyruvate to L-alanine; further inactivate or delete the alanine racemase gene in the starting bacteria, synergistically Achieve high yields of optically pure L-alanine.
  • modifying the genome of the starting bacterium includes step 500 and step 200, at this time, the lactic acid synthesis pathway is blocked by the inactivation or deletion of the D-lactate dehydrogenase gene ldh Ti , the glycolytic pathway was enhanced by overexpression of the 6-phosphofructokinase gene pfk and the pyruvate kinase gene pyk.
  • the construction method includes the following steps:
  • the lactate dehydrogenase gene is D-lactate dehydrogenase gene ldh Ti (can realize blocking lactic acid synthetic pathway);
  • S200 Overexpression of a copy of the 6-phosphofructokinase gene pfk and the pyruvate kinase gene pyk (can enhance the glycolysis pathway and increase the supply of pyruvate);
  • the construction method includes the following steps:
  • the lactate dehydrogenase gene is D-lactate dehydrogenase gene ldh Ti (which can block the lactic acid synthesis pathway), More preferably, the sequence of the D-lactate dehydrogenase gene ldh Ti is shown in SEQ ID NO.31;
  • S200 Insert a copy of the 6-phosphofructokinase gene pfk and a copy of the pyruvate kinase gene pyk (the 6-phosphofructokinase pfk and pyruvate kinase pyk in the starter bacteria can be overexpressed, thereby enhancing the glycolytic pathway and increasing the acetone acid supply); preferably, the sequence of the 6-phosphofructokinase gene pfk is shown in SEQ ID NO.27, and the sequence of the pyruvate kinase gene pyk is shown in SEQ ID NO.28;
  • S400 Knockout of alanine racemase gene alr1 and alanine racemase gene alr2 (can block the conversion of L-alanine to D-alanine, which is beneficial to the realization of optically pure L-alanine production); preferably, the sequence of the alanine racemase gene alr1 is as shown in SEQ ID NO.29, and the sequence of the alanine racemase gene alr2 is as shown in SEQ ID NO.30;
  • the method of inserting the relevant gene is to increase the copy of the gene on the chromosome (the increased copy number can be 1 or more), and insert Front tandem promoter.
  • the promoter is any one of P als , Plac, Ptrc, Ptac, Pc P43 and the like.
  • the starting bacterium is a thermophilic bacterium.
  • the starting bacterium is Bacillus.
  • the starting bacterium is Bacillus licheniformis.
  • Bacillus licheniformis is a facultative anaerobic, Gram-positive, endospore-type bacterium with fast growth rate and wide substrate spectrum, and can carry out high-temperature fermentation at 50°C.
  • the genetic manipulation of this strain is convenient and stable, and it is also a GRAS (generally regarded as safe) strain recognized by the US Food and Drug Administration, which has the potential to become an ideal platform strain.
  • the starting bacterium is Bacillus licheniformis, Bacillus coagulans, Bacillus methylotroph, Bacillus thermoinulinus or Geobacillus stearothermophilus.
  • the starting bacterium is Bacillus licheniformis ATCC 14580 and derivative strains thereof.
  • the derivative strains include, but are not limited to: Bacillus licheniformis MW3, Bacillus licheniformis BN11 and the like.
  • the starting bacterium is Bacillus licheniformis BN11, the sequence of the 6-phosphofructokinase gene pfk is shown in SEQ ID NO.27; the sequence of the pyruvate kinase gene pyk The sequence is shown in SEQ ID NO.28.
  • the starting bacterium is Bacillus licheniformis BN11, and the sequence of the alanine dehydrogenase gene GSald is shown in SEQ ID NO.1.
  • the starting bacterium is Bacillus licheniformis BN11, and its endogenous alanine racemase genes include alr1 and alr2, wherein the sequence of the alanine racemase gene alr1 As shown in SEQ ID NO.29, the sequence of the alanine racemase gene alr2 is shown in SEQ ID NO.30.
  • the starting bacterium is Bacillus licheniformis BN11, and the sequence of the D-lactate dehydrogenase gene ldh Ti is as shown in SEQ ID NO.31 shown.
  • the third aspect of the present invention there is provided the application of the genetically engineered strain described in the second aspect of the present invention in the production of L-alanine, which can be used to ferment and produce L-alanine under high temperature conditions, which can significantly increase the yield, Further, a high yield of optically pure L-alanine can be achieved under high temperature conditions.
  • a method for producing L-alanine wherein the genetic engineering strain described in the second aspect of the present invention is fermented and cultivated, wherein the fermentation temperature is 42°C-55°C.
  • fermentation culture temperatures include but are not limited to 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C .
  • the step of fermenting and cultivating comprises: inoculating the activated seeds into a fermentation medium containing a carbon source, at a fermentation temperature of 42°C to 55°C and a stirring speed of Under the condition of 50rpm ⁇ 350rpm, the fed-batch fermentation is carried out, and cultured to the set fermentation density.
  • the inoculum amount for inoculation of the activated seeds may be 3%-5% by volume percentage, for example, 3%, 4%, or 5%.
  • the production method further performs seed culture to obtain activated seeds before the fermentation culture, and then inoculates the activated seeds into a fermentation medium to perform the fermentation culture.
  • the seed cultivation step includes: inoculating the genetically engineered strain into the seed medium, and culturing for 12h to 16h at a seed cultivation temperature of 42°C to 55°C to obtain The seed culture solution is the activated seed.
  • the seed medium comprises the following components: peptone, yeast powder and sodium chloride.
  • the seed medium consists of the following components: peptone, yeast powder and sodium chloride.
  • described production method comprises the following steps:
  • Seed culture inoculate the genetically engineered strain into the seed culture medium, and cultivate it under the condition that the seed culture temperature is 42°C-55°C to obtain activated seeds;
  • Fermentation culture inoculate the activated seeds into the fermentation medium, which contains carbon source, and carry out fed-batch fermentation under the condition of the fermentation temperature of 42°C-55°C, and cultivate to the set The fermentation density; wherein, the inoculum size can be 3% to 5% by volume.
  • described production method comprises the following steps:
  • Seed cultivation inoculate the genetically engineered strain into the seed culture medium, and cultivate for 12h to 16h at a seed cultivation temperature of 42°C to 55°C to obtain activated seeds;
  • Fermentation culture inoculate the activated seeds into the fermentation medium, the fermentation medium contains a carbon source, and carry out batch supplementation at a fermentation temperature of 42°C to 55°C and a stirring speed of 50rpm to 350rpm.
  • the material is fermented and cultivated to a set fermentation density; wherein, the inoculum amount can be 3% to 5% by volume.
  • the carbon source is glucose, glycerol, xylose or arabinose.
  • the fermentation medium in the fermentation culture step, comprises the following components: glucose, yeast powder, ammonium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, ferrous sulfate and manganese sulfate.
  • the fermentation medium is composed of the following components: glucose, yeast powder, ammonium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, ferrous sulfate and manganese sulfate.
  • the set fermentation density can be detected by OD 600 , for example, when the OD 600 reaches 6.5-8.0, the fermentation is stopped.
  • the fermentation and cultivation step includes: adjusting the pH of ammonia water to about 7.0, carrying out fermentation and cultivation under aeration and stirring conditions (the first stage of fermentation cultivation) to When the OD600 value reaches 6.5-8.0, stop the ventilation, reduce the stirring speed and continue the fermentation culture (second stage fermentation culture) to the set fermentation density, and end the fermentation; during the fermentation process, when the concentration of the carbon source drops to a certain value, it can be The carbon source is supplemented.
  • the concentration of ammonia water used to adjust the pH may be 20%-30% (v/v), specifically for example 20%, 25%, 30%, calculated by volume percentage.
  • the ventilation condition is preferably air.
  • the ventilation volume of the ventilation can be 0.8vvm-1.2vvm, for example, 0.9vvm, 1.0vvm, 1.1vvm, 1.2vvm and so on.
  • Fermentation culture is carried out in stages. It can be carried out in two stages, including the first stage of fermentation culture under aeration conditions, and the second stage of fermentation culture after stopping the aeration.
  • the bacteria can grow rapidly, and through the second stage of fermentation culture, the efficient production of L-alanine can be realized.
  • the stirring speed during the first stage of fermentation culture is preferably 280rpm-320rpm, for example, 280rpm, 290rpm, 300rpm, 310rpm, 320rpm, etc.
  • the time of the first stage of fermentation culture is determined according to the OD600 value, for example, it can be 6 hours to 8 hours, specifically for example 6h, 6.5h, 7h, 7.5h, 8h.
  • the stirring speed in the second stage of fermentation culture is preferably 60rpm-100rpm, for example, 60rpm, 70rpm, 80rpm, 90rpm, 100rpm, etc.
  • the time of the second stage of fermentation culture is also determined according to the OD600 value (such as 1.2), for example, it can be 50 hours to 70 hours, specifically for example 50h, 55h, 60h, 65h, 70h.
  • the number of supplementary carbon sources in the fermentation process can be one time, two times, etc.
  • the carbon source is replenished once, and for example, when the carbon source concentration drops to 20g/L-40g/L, it is still possible Add another carbon source.
  • the carbon source is glucose.
  • the concentration of the carbon source is reduced to 30g/L ⁇ 50g/L (for example, 30g/L, 35g/L, 40g/L, 45g/L L, 50g/L)
  • the carbon source concentration can be supplemented to 80g/L-120g/L (for example, 80g/L, 90g/L, 100g/L, 110g/L, 120g/L).
  • the carbon source is glucose.
  • the carbon source is supplemented 2 times.
  • the concentration of the carbon source drops to 30g/L-50g/L (for example, 30g/L, 35g/L, 40g/L, 45g/L, 50g/L)
  • the concentration of the carbon source can be supplemented to 80g/L-120g/L (for example, 80g/L, 90g/L, 100g/L, 110g/L, 120g/L).
  • the concentration of the carbon source drops to 20g/L-40g/L (such as 20g/L, 25g/L, 30g/L, 35g/L, 40g/L).
  • the concentration of the carbon source can be supplemented to 60g/L-80g/L (for example, 60g/L, 65g/L, 70g/L, 75g/L, 80g/L).
  • the carbon source is glucose.
  • the fermentation and cultivation step comprises: using 20% to 30% (v/v) ammonia water to adjust the pH to 7.0 ⁇ 0.2, and the ventilation rate to be 0.8vvm to 1.2vvm, the initial stirring speed is 280rpm-320rpm, carry out the fermentation culture for 6-8 hours until the OD600 value reaches 6.5-8.0, stop the ventilation, adjust the stirring speed to 60rpm-100rpm, and continue the fermentation until 50-70 hours (for example, continue to ferment Cultivate to 60 hours), end the fermentation; further, supplement the carbon source when the carbon source concentration drops to a certain value during the fermentation process, it can be supplemented once, twice, etc.
  • the carbon source is supplemented only once during the fermentation process, and the carbon source is supplemented for the first time when the concentration of the carbon source drops to 30g/L-50g/L, until the concentration of the carbon source is 80g/L ⁇ 120g/L.
  • the carbon source is supplemented twice during the fermentation process, and the carbon source is supplemented for the first time when the concentration of the carbon source drops to 30g/L-50g/L, until the concentration of the carbon source is 80g/L ⁇ 120g/L, then, when the concentration of the carbon source drops to 20g/L ⁇ 40g/L, supplement the carbon source for the second time until the concentration of the carbon source is 60g/L ⁇ 80g/L.
  • the carbon source is glucose.
  • the carbon source is glucose
  • the fermentation and cultivation step includes: 25% ammonia water to adjust the pH to maintain 7.0, the ventilation rate is 1vvm, and the initial stirring speed is 300rpm.
  • the OD 600 of Bacillus licheniformis reaches 6.5 to 8.0, the aeration is stopped and the stirring speed is adjusted to 80 rpm, and the fermentation is continued until the end of 60 hours.
  • glucose concentration drops to 300g/L-50g/L
  • glucose is supplemented once so that the glucose concentration reaches 100g/L.
  • the glucose concentration drops to 20g/L-40g/L again supplement the glucose for the second time until the glucose concentration reaches 70g/L.
  • Determination method of alanine concentration use high performance liquid chromatography, weigh 0.54g of sodium octane sulfonate, add it to 800mL ultrapure water, adjust the pH to 2.1 with phosphoric acid, add 100mL methanol, and use ultrapure water to make up to volume to 1 L volume, as the mobile phase.
  • the chromatographic column is ZORBAX Eclipse XDB-C18 (150mm ⁇ 4.6mm, 5 ⁇ m), the column temperature is 30°C, the flow rate is 0.8mL/min, the injection volume is 10 ⁇ L, and the detector is a DAD detector with a wavelength of 210nm.
  • the sample to be tested is a fermented sample. First, it needs to be boiled for 10 minutes, then centrifuged at 8000rpm for 5 minutes, and the supernatant is taken and diluted to an appropriate multiple until the final concentration of alanine is 0.2g/L to 1g/L.
  • Alanine optical purity determination method use high performance liquid chromatography, mobile phase is 2mM copper sulfate aqueous solution.
  • the chromatographic column is SUMICHIRAL OA-5000 (150mm ⁇ 4.6mm, 5 ⁇ m), the column temperature is 30°C, the flow rate is 0.5mL/min, the injection volume is 10 ⁇ L, and the detector is a DAD detector with a wavelength of 254nm.
  • the sample to be tested is a fermented sample. First, it needs to be boiled for 10 minutes, then centrifuged at 8000rpm for 5 minutes, and the supernatant is taken and diluted to an appropriate multiple until the final concentration of alanine is 0.2g/L to 1g/L.
  • Glucose concentration determination method use SBA-40D biosensor analyzer (Shandong Academy of Sciences Institute of Biology) to measure glucose concentration.
  • Bacillus licheniformis was used as a starting bacterium to construct a genetically engineered strain capable of producing L-alanine under high temperature conditions. It should be understood that the construction method of the genetically engineered strain producing L-alanine in the following examples can also be used for other bacterial strains (examples include but are not limited to Bacillus coagulans, Bacillus methylotroph, Bacillus thermophilus, Bacillus Geobacillus stearothermii etc.) are used as the starting bacteria to obtain the genetically engineered bacterial strain that can produce L-alanine under high temperature conditions in the present invention.
  • PKVM vector its structure is shown in Figure 3, and it comprises ampR (ampicillin resistance gene) and ermC (erythromycin resistance gene) two antibiotic resistance genes, and the bgaB gene of coding galactosidase, can be As a selection marker; the plasmid has a heat-sensitive origin of replication site oriT pE194ts, as well as the origin of replication sites ori and oriT in Escherichia coli and Bacillus licheniformis.
  • Bacillus licheniformis BN11 is a lactic acid producing strain constructed on the basis of ATCC 14580. Its preservation number is CCTCC NO: M2016026. It was preserved in the China Center for Type Culture Collection on January 8, 2016.
  • Inoculation amount by volume percentage.
  • the newly inserted gene copy number is 1.
  • Example 1 Construction of Bacillus licheniformis knockout D-lactic acid synthesis pathway
  • 1.2 Purify the PCR product obtained in the above step 1.1. Then use PKVM-XPDdldh-UpF and PKVM-XPDdldh-DownR as primers for recombinant PCR amplification. After the PCR product is purified, it is connected to the PKVM vector by seamless cloning, Transformed into Escherichia coli S17 to obtain S17-PKVM ⁇ ldh Ti .
  • the picked transformants were cultured in LB medium containing erythromycin at 37°C, then transferred to LB medium and cultured at 50°C, diluted and spread on LB plates containing erythromycin, and cultured at 50°C to screen for single exchange transformants.
  • Single-crossover transformants were continuously cultured in LB medium at 30°C for two generations, then diluted and spread on LB plates, cultured overnight at 37°C, and double-crossover transformants were reverse-selected according to erythromycin resistance.
  • Example 2 Construction of Bacillus licheniformis overexpressing 6-phosphofructokinase and pyruvate kinase
  • PKVM-XPDldh-UpF SEQ ID NO.7
  • P als -XPDldh-UpR SEQ ID NO.8
  • XPDldh-P als -F SEQ ID NO.9
  • PFYAK-P als -R SEQ ID NO.10
  • P als -PFYAK-F SEQ ID NO.11
  • XPDldh-PFYAK-R SEQ ID NO.11
  • PKVM-XPDldh-DownR SEQ ID NO.14
  • the Escherichia coli S17-PKVM-PFYAK and Bacillus licheniformis BN11 ⁇ ldh Ti obtained in the above step 2.2 were subjected to conjugation transfer and homologous recombination experiments according to the method in Example 1, and a strain overexpressing the pfk and pyk genes was obtained, denoted as BN11 ⁇ ldh Ti -PFYAK.
  • Example 3 Construction of strains overexpressing heterologous alanine dehydrogenase
  • a potential thermostable alanine dehydrogenase (the amino acid sequence of which is shown in SEQ ID NO.32, protein The sequence number corresponds to WP_033014465.1), the alanine dehydrogenase gene derived from Geobacillus stearothermophilus is named as GSald, and the codon optimization has been carried out according to the Bacillus licheniformis genome (the optimized sequence is shown in SEQ ID NO.1 shown), and synthesized together with the P als promoter (SEQ ID NO.2), the combined sequence from 5' to 3' is P als -GSald.
  • Primers were designed according to the genome information of Bacillus licheniformis ATCC 14580 (GenBank No.NC_006270.3), respectively PKVM-XPDdldh-UpF (SEQ ID NO.3) and P als -XPDdldh-UpR (SEQ ID NO.15) , XPDdldh-P als -F (SEQ ID NO.16) and XPDdldh-GSald-R (SEQ ID NO.17), GSald-XPDdldh-DownF (SEQ ID NO.18) and PKVM-XPDdldh-DownR (SEQ ID NO .6) Perform PCR amplification for the upstream and downstream primers to obtain the upstream homology arm fragment of the ldh Ti gene promoter, the P als -GSald gene fragment and the downstream homology arm fragment of the ldh Ti gene.
  • PCR product obtained in the above step 3.2 Purify the PCR product obtained in the above step 3.2. Then use PKVM-XPDdldh-UpF and PKVM-XPDdldh-DownR as primers for recombinant PCR amplification. After the PCR product is purified, connect it to the PKVM vector by seamless cloning , transformed into Escherichia coli S17 to obtain S17-PKVM-GSald.
  • Escherichia coli S17-PKVM-GSald and Bacillus licheniformis BN11 ⁇ ldh Ti -PFYAK were subjected to conjugative transfer and homologous recombination experiments according to the method in Example 1 to obtain a strain expressing heterologous GSald, which was designated as BA-1.
  • Example 4 Knockout of the alanine racemase of Bacillus licheniformis
  • step 4.1 Purify the PCR product obtained in the above step 4.1, then perform recombinant PCR amplification with PKVM-XPDalr1-UpF and PKVM-XPDalr1-DownR as primers, purify the PCR product, and then connect it to the PKVM vector through seamless cloning , transformed into Escherichia coli S17 to obtain S17-PKVM ⁇ alr1.
  • Escherichia coli S17-PKVM ⁇ alr1 and Bacillus licheniformis BA-1 were subjected to conjugation transfer and homologous recombination experiments according to the method in Example 1 to obtain a strain that knocked out the alr1 gene (SEQ ID NO.29), denoted as BA- 1 ⁇ alr1.
  • Escherichia coli S17-PKVM ⁇ alr2 and Bacillus licheniformis BA-1 ⁇ alr1 were subjected to conjugative transfer and homologous recombination experiments according to the method in Example 1, and the alr1 gene (SEQ ID NO.29) and alr2 gene (SEQ ID NO.29) and alr2 gene (SEQ ID NO.30) strain, denoted as BLA-1.
  • Example 5 Recombinant Bacillus licheniformis BLA-1 carries out fed-batch fermentation (5L fermenter) at 42°C
  • Seed medium formula LB medium, yeast powder 5g/L, tryptone 10g/L, sodium chloride 10g/L.
  • Fermentation medium formula: glucose 100g/L, yeast powder 5g/L, ammonium sulfate 5g/L, dipotassium hydrogen phosphate trihydrate 1.3g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate heptahydrate 0.5g/L , ferrous sulfate heptahydrate 20mg/L, manganese sulfate tetrahydrate 20mg/L.
  • the glucose is sterilized separately, and sterilized at 115° C. for 20 minutes.
  • the seed liquid obtained in step 5.2. was transferred to a 5L fermenter equipped with a fermentation medium according to an inoculum amount of 5% by volume, so that the liquid capacity reached 3L.
  • the OD 600 of Bacillus licheniformis reached 6.5-8.0.
  • the aeration was stopped and the stirring speed was adjusted to 80 rpm, and the fermentation was continued until 60 hours.
  • sugar supplementation that is, carbon source supplementation, specifically glucose supplementation in this embodiment
  • sugar supplementation is carried out once to make the glucose concentration reach 100g/L.
  • concentration of L-alanine in the fermentation broth reached 93.7g/L
  • the conversion rate of sugar and acid was 74.4%
  • the optical purity of L-alanine reached 98.9% through detection and analysis.
  • Example 6 Recombinant Bacillus licheniformis BLA-1 carries out fed-batch fermentation (5L fermenter) at 50°C
  • the seed solution obtained in step 6.2. was transferred to a 5L fermenter equipped with a fermentation medium according to an inoculum amount of 5% by volume, so that the liquid content reached 3L.
  • 25% (v/v) ammonia water was used to control the fermentation pH to 7.0, the ventilation rate was 1.0vvm, the temperature was 50°C, and the stirring speed was 300rpm.
  • the OD 600 of Bacillus licheniformis reaches 6.5-8.0.
  • the aeration is stopped and the stirring speed is adjusted to 80 rpm, and the fermentation is continued until 60 hours.
  • sugar is added to make the glucose concentration reach 100g/L.
  • the fermentation curve is shown in Figure 2. After 60 hours of fermentation, the concentration of alanine in the fermentation liquid reached 96.8g/L, the conversion rate of sugar and acid was 76.8%, and the optical purity of L-alanine reached 98.9% through detection and analysis.
  • Example 7 Recombinant Bacillus licheniformis BLA-1 carries out fed-batch fermentation at 55°C (5L fermenter)
  • the seed solution obtained in step 7.2. was transferred to a 5L fermenter with a fermentation medium at an inoculum volume of 5% by volume, so that the liquid volume reached 3L.
  • 25% (v/v) ammonia water was used to control the fermentation pH to 7.0, the ventilation rate was 1.0vvm, the temperature was 55°C, and the stirring speed was 300rpm.
  • the OD 600 of Bacillus licheniformis reaches 3.0-4.5.
  • the aeration is stopped and the stirring speed is adjusted to 80 rpm, and the fermentation is continued until 60 hours.
  • the concentration of alanine in the fermentation broth reached 21.4g/L
  • the conversion rate of sugar and acid was 63.8%
  • the optical purity of L-alanine reached 97.2% through detection and analysis.
  • Example 8 Recombinant Bacillus licheniformis BLA-1 carries out fed-batch fermentation at 42°C (50L fermenter)
  • Example 9 Recombinant Bacillus licheniformis BLA-1 carried out fed-batch fermentation at 50°C, 50L fermenter
  • the seed solution obtained in step 9.2. was transferred to a 50 L fermenter equipped with a fermentation medium according to an inoculum size of 5%, so that the liquid content reached 30 L.
  • 25% (v/v) ammonia water was used to control the fermentation pH to 7.0, the ventilation rate was 1.0vvm, the temperature was 50°C, and the stirring speed was 300rpm.
  • the OD 600 of Bacillus licheniformis reaches 6.5-8.0.
  • the aeration is stopped and the stirring speed is adjusted to 80 rpm, and the fermentation is continued until 60 hours.
  • a sugar supplement is carried out so that the glucose concentration reaches 100g/L.
  • the concentration of alanine in the fermentation broth reached 108.2g/L
  • the conversion rate of sugar and acid was 73.3%
  • the optical purity of L-alanine reached 99.1% through detection and analysis.
  • Example 10 Fed-batch fermentation of recombinant Bacillus licheniformis BLA-1 at 55°C (50L fermenter)
  • Example 11 Fermentation results of the BN11 ⁇ ldh Ti -PFYAK strain constructed in Example 2
  • Example 6 the BN11 ⁇ ldh Ti -PFYAK strain obtained in Example 2 was fermented and cultured.
  • Embodiment 12 The result of the fermentation culture of the BA-1 bacterial strain that embodiment 3 constructs
  • Example 6 The method and experimental parameters of Example 6 were used to ferment and culture the BA-1 strain obtained in Example 3.
  • the yield of alanine was 129g/L; 2.
  • the obtained product was a mixed product of L-type and D-type, and the optical purity of L-type alanine was 70.8%, which was difficult to meet the requirements.

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Abstract

公开了一种生产L-丙氨酸的基因工程菌株及其构建方法和应用，通过增强糖酵解途径或/和引入热稳定性的丙氨酸脱氢酶基因，可构建得到能够在42℃～55℃高温条件下高产丙氨酸的基因工程菌株；通过敲除丙氨酸消旋酶基因还能够显著提升L-丙氨酸的光学纯度；当起始菌中存在乳酸合成途径时，通过阻断乳酸合成途径，可增加丙酮酸合成途径的比例，进一步提高L-丙氨酸的产量，实现了L-丙氨酸在42℃～55℃条件下的高温发酵生产，丙氨酸产量能够达到95g/L以上。

Description

一种生产L-丙氨酸的基因工程菌株及其构建方法和应用 技术领域
本发明涉及生物工程领域，尤其涉及一种生产L-丙氨酸的基因工程菌株及其构建方法和应用。
背景技术
L-丙氨酸是20种基本氨基酸之一，也是最小的手性分子之一，在食品、医药、日化等领域具有广泛应用。在食品工业中，L-丙氨酸可以作为防腐剂、甜味剂等添加到食品中。在医药保健中，L-丙氨酸不仅可用于氨基酸类营养补充剂中，还在急慢性肾衰竭、肝脏疾病、糖尿病、肥胖症等疾病治疗中有重要作用。在日化用品中，L-丙氨酸可以用于合成氨基酸表面活性剂，具有较高的生物亲和性。
L-丙氨酸的生产方法主要包括化学合成法、酶催化法和发酵法。化学合成法主要采用丙酸氯化法工艺，以液氯和丙酸作为主要原料。由于化学合成法的合成路线长、成本高、收率低，目前基本被淘汰。酶催化法主要利用固定化的L-天冬氨酸-β-脱羧酶催化天冬氨酸脱羧生成L-丙氨酸。但是酶催化法由四碳底物生成三碳产物的碳利用率较低，并且天冬氨酸的价格偏高，因此很难满足大规模生产需求。发酵法主要以葡萄糖为底物，以基因工程手段构建菌株发酵生产L-丙氨酸，具有原料廉价易得、产品得率高、成本低等优势，因而受到广泛关注。
与42℃以下的常温发酵相比，高温发酵能够显著降低污染风险、提高原料转化效率并降低热交换成本，因此在大规模发酵生产中有较高的应用价值。丙氨酸发酵通常以基因工程改造的大肠杆菌为生产菌株，但是该菌株的最适生长温度为37℃，无法进行高温发酵。
因此，本领域的技术人员致力于开发一种能够在高温条件下发酵生产L-丙氨酸的菌株，以满足大规模生产的需求。
发明内容
有鉴于此，本发明所要解决的技术问题是提供一种可以在42℃以上的高温条件下生产L-丙氨酸的基因工程菌株及其构建方法和应用。
为实现上述目的，本发明的第一方面提供了一种生产L-丙氨酸的基因工程菌株的构建方法，包括如下步骤：
提供具有丙酮酸合成途径的起始菌；
进行S200、S300、S400中的任一种步骤、任两种步骤或三种步骤对所述起始菌 的基因组进行改造：
S200：插入6-磷酸果糖激酶基因pfk的拷贝和丙酮酸激酶基因pyk的拷贝；
S300：插入具有42℃～55℃热稳定性的丙氨酸脱氢酶基因GSald；
S400：所述起始菌的基因组中含有丙氨酸消旋酶基因，该步骤包括失活或缺失丙氨酸消旋酶基因。
本发明的第一方面构建得到的生产L-丙氨酸的基因工程菌株可以在42℃～55℃的高温条件下发酵生产L-丙氨酸。
在本发明的一些实施方式中，所述起始菌还具有乳酸合成途径，且所述起始菌的基因组中含有乳酸脱氢酶基因；所述的构建方法还包括如下步骤：
S100：失活或缺失所述起始菌的基因组中的乳酸脱氢酶基因。
在本发明的第二方面，提供了本发明的第一方面所述的构建方法构建得到的基因工程菌株。
在本发明的第三方面，提供了本发明的第二方面所述的基因工程菌株在生产L-丙氨酸中的应用。
在本发明的第四方面，提供了一种L-丙氨酸的生产方法，对本发明的第二方面所述的基因工程菌株进行发酵培养，其中，发酵培养温度为42℃～55℃。
在本发明的一些实施方式中，所述发酵培养步骤包括：将所述活化的种子接种到发酵培养基中，所述发酵培养基中包含碳源，在发酵培养温度为42℃～55℃、搅拌速度为50rpm～350rpm的条件下进行分批补料发酵，培养至设定的发酵密度。
本发明公开的技术方案所带来的有益效果包括：
1、本发明提供的基因工程菌株，实现了L-丙氨酸的高温生产(例如42℃～55℃)，在发酵过程中具有能耗低、抗污染等优点，有效降低了生产成本。
2、以嗜热菌作为起始菌构建基因工程菌株，可实现在高温条件下(例如42℃～55℃，具体地如50℃)进行L-丙氨酸的发酵生产。进一步地，通过增强糖酵解途径或/和引入热稳定性的丙氨酸脱氢酶，可显著提升丙氨酸的产量。通过敲除丙氨酸消旋酶基因可实现光学纯L-丙氨酸的生产。上述手段的组合可实现高温下高产光学纯L-丙氨酸的协同增效，发酵生产的温度可高达42℃～55℃，L-丙氨酸光学纯度可普遍实现98％以上，部分可实现99％以上，一些实施例中光学纯L-丙氨酸产量能够达到95g/L以上，部分可达到100g/L以上。本发明解决了现有技术中生产L-丙氨酸发酵温度低、成本高等问题，实现了L-丙氨酸的高温发酵生产，具有较高的工业化利用价值。
3、对于存在乳酸合成途径的起始菌，通过阻断乳酸合成途径，可增加丙酮酸合成途径的比例，进而实现L-丙氨酸的增产。采用同时产乳酸和丙酮酸的起始菌，通过阻断起始菌中的乳酸合成途径，可提高丙氨酸合成途径的比例，进一步地，通过增强糖酵解途径并引入热稳定性的丙氨酸脱氢酶，可构建得到高产丙氨酸的基因工程菌株。敲除丙氨酸消旋酶基因可实现光学纯L-丙氨酸的生产。
4、以能够50℃高温发酵的地衣芽孢杆菌为例，从一株高产D-乳酸的地衣芽孢杆菌BN11出发，通过消除其D-乳酸合成途径，增强糖酵解途径的通量并引入热稳定的异源丙氨酸脱氢酶，还消除D-丙氨酸的合成途径，可实现在42℃～55℃高温条件下高产光学纯L-丙氨酸，L-丙氨酸光学纯度可普遍实现98％以上，部分可实现99％以上；一些实施例中产量可实现95g/L以上，部分可实现100g/L以上。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明，以充分地了解本发明的目的、特征和效果。
附图说明
通过阅读参照以下附图，对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会阐述得更清晰：
图1是本发明的一个实施例中，以地衣芽孢杆菌为例的工程菌中丙氨酸的合成和代谢途径示意图；
图2是本发明的一个实施例中，一种生产L-丙氨酸的基因工程菌株BLA-1在5L发酵罐50℃分批补料发酵生产L-丙氨酸的发酵曲线图(▲，OD600；■，葡萄糖；●，丙氨酸)；
图3是本发明的一些实施例中所用的PKVM载体的结构图，其包含ampR(氨苄青霉素抗性基因)和ermC(红霉素抗性基因)两个抗生素抗性基因，以及编码半乳糖苷酶的bgaB基因，均可作为筛选标记；该质粒具有一个热敏型复制起始位点oriTpE194ts，还有在大肠杆菌和地衣芽孢杆菌的复制起始位点ori和oriT。。
具体实施方式
下面将结合附图对本发明提供的具体事实方式作详细说明。
以下参考说明书附图介绍本发明的多个优选实施方式和优选实施例，使其技术内容更加清楚和便于理解。本发明可以通过许多不同形式的实施方式和实施例来得以体现，本发明的保护范围并非仅限于文中提到的实施方式和实施例。
应理解，本发明的上述各技术特征和在下文(包括但不限于实施方式和实施例)中具体描述的各技术特征之间都可以通过任意合适的方式互相组合，从而构成新的或优选的技术方案，只要能够用于实施本发明的即可。
本发明中，“优选”、“特别”、“具体”、“进一步”、“更进一步”等词语仅表示效果较佳的或者较为具体的实施方式或实施例，应当理解，并不构成对本发明保护范围的限制。
本发明中，“和/或”、“或/和”均表示所列项目中的任一种或任意组合。
本发明中，“其组合”、“任何组合”、“任意组合”、“任意组合方式”，均指所列相 关项目的任意合适的组合方式，以能够顺利实施本发明为准。构成组合的相关项目的数量等于2或大于2。
本发明中涉及百分比浓度，如无特别限定或说明，表示在相关体系中的终浓度。
本发明中的数值范围，如无特别说明，均包括两个端点。
本发明中，“以上”、“以下”，如无特别限定，均包括本数。
本发明上文及下文中涉及的各技术特征可以以任意合适的方式进行组合，只要不存在矛盾，且组合后的技术方案能够顺利实施并能够解决本发明的技术问题。所述任意合适的方式，以能够实现本发明的技术方案，解决本发明的技术问题，且能够实现相应的技术效果为准。
在本发明中，“出发菌”和“起始菌”具有相同含义，可以互换使用。
本发明中涉及温度控制，允许为恒温，也允许存在一定的温度波动范围。
在本发明中，高温生产L-丙氨酸，是相对于常温(指低于42℃，通常为37℃以下)，本文中的高温生产，如无特别限定，指在42℃～55℃条件下进行发酵生产，举例包括但不限于42℃、43℃、44℃、45℃、46℃、47℃、48℃、49℃、50℃、52℃、53℃、54℃、55℃。
本发明中，如无特别说明，“丙酮酸合成途径”是指碳源经过糖酵解合成丙酮酸的途径。本发明中，“具有丙酮酸合成途径”是指具有内源性合成丙酮酸的能力。
本发明中，如无特别说明，“乳酸合成途径”是指：从丙酮酸转化为乳酸的合成途径。本发明中，如无特别说明，具有“乳酸合成途径”是可通过乳酸合成途径生产乳酸，也即，“具有乳酸合成途径”指具有内源性合成乳酸的能力。
本发明中，如无特别说明，“丙氨酸合成途径”是指：从丙酮酸转化为丙氨酸的合成途径。本发明中，如无特别说明，具有“丙氨酸合成途径”是可通过丙氨酸合成途径生产丙氨酸，换言之，指具有内源性合成丙氨酸的能力。本发明中，具有丙氨酸合成途径时，如无特别说明，同时具有从丙酮酸合成L-丙氨酸的途径，以及从L-丙氨酸合成D-丙氨酸的途径。
本发明中，如无特别说明，“D-丙氨酸的合成途径”是指，从L-丙氨酸合成D-丙氨酸的途径。“具有D-丙氨酸的合成途径”指具有内原性合成D-丙氨酸的能力。
本发明中，基因簇片段是指，相关基因单元的数量至少2个，相关基因单元可以为相同或不同的基因。如果为相同基因构成的基因簇片段，“重复”应进行广义理解，允许相邻地依次出现，也可以在任两个基因单元之间存在间隔序列。
本发明中，缺失相关基因的典型方式之一为基因敲除。
本发明中的“失活”、“缺失”，如无特别限定，可以为部分或全部的失活、部分或全部的缺失。
表1.部分术语的简写和中英文全称
Figure PCTCN2021135643-appb-000001
表2部分术语的中外译文对照
英文 中文
Bacillus licheniformis 地衣芽孢杆菌
Bacillus coagulans 凝结芽孢杆菌
Glucose 葡萄糖
Alanine 丙氨酸
本发明中涉及的氨基酸和核苷酸序列汇总于表3。
表3.本发明中涉及的一些氨基酸和核苷酸序列。
Figure PCTCN2021135643-appb-000002
Figure PCTCN2021135643-appb-000003
SEQ ID NO.1(GSald)：
Figure PCTCN2021135643-appb-000004
Figure PCTCN2021135643-appb-000005
SEQ ID NO.2(P als启动子)：
Figure PCTCN2021135643-appb-000006
SEQ ID NO.3：CCTCGCGTCGGGCGATATCGGATCCGAAGGGGAAAGTCTTCGATTTCT
SEQ ID NO.4：AGAGGGCTTTTTCATGCTGAAGAGGTCAAAAAGAGCC
SEQ ID NO.5：TTTGACCTCTTCAGCATGAAAAAGCCCTCTTTGAAAAG
SEQ ID NO.6：CCATGGTACCCGGGAGCTCGAATTCCATAAGACCGCTGATGACAAGC
SEQ ID NO.7：TCCAGCCTCGCGTCGGGCGATATCGTCCCCATAACAACGGAATCATC
SEQ ID NO.8：AATAGGCGTCACCTTGACTCATCATTCCTTTGCCGTT
SEQ ID NO.9：AAGGAATGATGAGTCAAGGTGACGCCTATTTCACTTTC
SEQ ID NO.10：TCCAATTCGCTTCATAGCCCTCACTCCTCCATTTTC
SEQ ID NO.11：GGAGGAGTGAGGGCTATGAAGCGAATTGGAGTATTGACA
SEQ ID NO.12：CTTCATGGTGTTCAGTTACAATACAGTCGCATGGCC
SEQ ID NO.13：GCGACTGTATTGTAACTGAACACCATGAAGATACTAACATCA
SEQ ID NO.14：ACTAGACAGATCTATCGATGCATGCTTTCCCTTATTCCTTTAAACCCG
SEQ ID NO.15：AATAGGCGTCACCTTGCTGAAGAGGTCAAAAAGAGCC
SEQ ID NO.16：TTTGACCTCTTCAGCAAGGTGACGCCTATTTCACTTTCT
SEQ ID NO.17：AGAGGGCTTTTTCATTTAGCCATGCAGCAGGCTATG
SEQ ID NO.18：CTGCTGCATGGCTAAATGAAAAAGCCCTCTTTGAAAAG
SEQ ID NO.19：CCTCGCGTCGGGCGATATCGGATCCAAAATATGACGCTGTCTCAAATTGA
SEQ ID NO.20：CTAATTCATCAATTTGACACTTCCTGTTCCTTGTTTCACT
SEQ ID NO.21：GGAACAGGAAGTGTCAAATTGATGAATTAGCGGAAAAAC
SEQ ID NO.22：CCATGGTACCCGGGAGCTCGAATTCCGGAGTCTCTTTCAAAACCGTAG
SEQ ID NO.23：CCTCGCGTCGGGCGATATCGGATCCAAAATCATGTAAGCCCATTCCG
SEQ ID NO.24：GTGAGTATGGGAAAACAACGCTCCCTTCTTTCTTGTC
SEQ ID NO.25：AAGAAGGGAGCGTTGTTTTCCCATACTCACAGGCCG
SEQ ID NO.26：CCATGGTACCCGGGAGCTCGAATTCTAAAATGAAGGTGGTCCGGGAT
SEQ ID NO.27(Pfk)：
Figure PCTCN2021135643-appb-000007
SEQ ID NO.28(Pyk)：
Figure PCTCN2021135643-appb-000008
Figure PCTCN2021135643-appb-000009
SEQ ID NO.29(Alr1):
Figure PCTCN2021135643-appb-000010
SEQ ID NO.30(Alr2):
Figure PCTCN2021135643-appb-000011
Figure PCTCN2021135643-appb-000012
SEQ ID NO.31(No):
Figure PCTCN2021135643-appb-000013
SEQ ID NO.32(GSald)：
Figure PCTCN2021135643-appb-000014
本发明的第一方面提供了一种生产L-丙氨酸的基因工程菌株的构建方法，包括如下步骤：
提供具有丙酮酸合成途径的起始菌；
进行S200、S300、S400中的任一种步骤、任两种步骤或三种步骤对所述起始菌的基因组进行改造：
S200：引入过表达的6-磷酸果糖激酶基因pfk的拷贝和丙酮酸激酶基因pyk；
S300：引入过表达的热稳定的丙氨酸脱氢酶基因(优选过表达热稳定的丙氨酸脱氢酶基因GSald)；
S400：所述起始菌的基因组中含有丙氨酸消旋酶基因，该步骤包括失活或缺失所述丙氨酸消旋酶基因。
在本发明的一些实施方式中，所述S200、S300、S400可各自独立地通过以下方式实现：
S200：插入6-磷酸果糖激酶基因pfk的拷贝和丙酮酸激酶基因pyk的拷贝；
S300：插入具有42℃～55℃热稳定性的丙氨酸脱氢酶基因GSald；
S400：部分或全部地失活所述丙氨酸消旋酶基因，或者部分或全部地缺失所述丙氨酸消旋酶基因；例如敲除所述丙氨酸消旋酶基因。
本发明的第一方面构建得到的生产L-丙氨酸的基因工程菌株可以在42℃以上(如42℃～55℃)的高温条件下发酵生产L-丙氨酸。其中，步骤S200可实现过表达糖酵解途径的6-磷酸果糖激酶和丙酮酸激酶，从而增强糖酵解途径，提升丙酮酸的供给，对提高L-丙氨酸产量具有促进作用。步骤S300可引入热稳定性的丙氨酸脱氢酶基因，可增强从丙酮酸到L-丙氨酸的合成途径，有效提高L-丙氨酸产量。步骤S400可失活或缺失丙氨酸消旋酶基因，有利于实现光学纯L-丙氨酸的生产。上述步骤S200、S300、S400中，采用了其中的任一种步骤均有利于提高L-丙氨酸的产量，采用其中的两种或三种步骤的组合时，可起到协同增效的作用。
在本发明的一些实施方式中，所述起始菌还具有乳酸合成途径，且所述起始菌的基因组中含有乳酸脱氢酶基因；所述的构建方法还包括如下步骤：
S100：失活或缺失所述起始菌的基因组中的乳酸脱氢酶基因。
在本发明的一些实施方式中，一种生产L-丙氨酸的基因工程菌中丙氨酸的合成和代谢途径示意图如图1所示。其中，碳源通过糖酵解途径转化为丙酮酸，随后丙酮酸消耗NADH和铵离子被丙氨酸脱氢酶催化生成L-丙氨酸。生成的L-丙氨酸一部分直接分泌到胞外，另一部分则被丙氨酸消旋酶催化形成D-丙氨酸，随后分泌到胞外。在起始菌中乳酸合成途径占据丙酮酸下游主要的碳通量情况下，阻断乳酸合成途径可使碳通量尽量流向丙氨酸合成途径。进一步地，过表达糖酵解途径的6-磷酸果糖激酶和丙酮酸激酶可以增加糖酵解途径，使丙酮酸的供给提升，从而提升了丙氨酸产量。进 一步敲除丙氨酸消旋酶可阻断D-丙氨酸合成途径，从而得到光学纯的L-丙氨酸产品。
在本发明的一些实施方式中，所述起始菌还具有D-乳酸合成途径，且所述起始菌的基因组中含有D-乳酸脱氢酶基因ldh Ti；所述的构建方法还包括如下步骤S500：失活或缺失所述起始菌的基因组中含有的D-乳酸脱氢酶基因ldh Ti。在本发明的一些优选的实施方式中，所述步骤S500包括：敲除所述起始菌的基因组中含有的D-乳酸脱氢酶基因ldh Ti。
在本发明的一些实施方式中，所述D-乳酸脱氢酶基因ldh Ti的序列如SEQ ID NO.31所示。
在本发明的一些实施方式中，所述6-磷酸果糖激酶基因pfk的序列如SEQ ID NO.27所示，所述丙酮酸激酶基因pyk的序列如SEQ ID NO.28所示。
在本发明的一些实施方式中，所述丙氨酸脱氢酶基因的序列如SEQ ID NO.1所示。
在本发明的一些实施方式中，所述步骤S400中，失活或缺失的所述丙氨酸消旋酶基因的种类为1种、2种或更多种。
在本发明的一些实施方式中，缺失相关基因的方式是敲除该基因。
在本发明的一些实施方式中，所述步骤S400包括：失活或缺失丙氨酸消旋酶基因alr1或丙氨酸消旋酶基因alr2。在本发明的一些实施方式中，所述步骤S400包括：失活或缺失丙氨酸消旋酶基因alr1和丙氨酸消旋酶基因alr2。在本发明的一些优选的实施方式中，所述步骤S400包括：敲除丙氨酸消旋酶基因alr1和丙氨酸消旋酶基因alr2。
在本发明的一些实施方式中，所述丙氨酸消旋酶基因alr1的序列如SEQ ID NO.29所示，所述丙氨酸消旋酶基因alr2的序列如SEQ ID NO.30所示。
在本发明的一些实施方式中，对所述起始菌的基因组进行改造包括步骤S100和步骤S200。此时，可以通过乳酸脱氢酶基因的失活或缺实现乳酸合成途径被阻断失，糖酵解途径通过6-磷酸果糖激酶基因pfk和丙酮酸激酶基因pyk的过表达被增强。优选地，对所述起始菌的基因组进行改造还包括步骤S300，此时，插入热稳定的丙氨酸脱氢酶基因，可实现L-丙氨酸的进一步增产。
在本发明的一些实施方式中，对所述起始菌的基因组进行改造包括步骤S100、步骤S200、步骤S300和步骤S400。此时，使起始菌中的乳酸脱氢酶基因可实现阻断乳酸合成途径；过表达起始菌的6-磷酸果糖激酶基因和丙酮酸激酶基因可实现增强糖酵解途径，从而提升丙酮酸的供给；引入过表达的丙氨酸脱氢酶基因可增强从丙酮酸到L-丙氨酸的合成途径；进一步使起始菌中的丙氨酸消旋酶基因失活或缺失，协同实现光学纯L-丙氨酸的高产量。
在本发明的一些实施方式中，对所述起始菌的基因组进行改造包括步骤500和步骤200，此时，乳酸合成途径通过D-乳酸脱氢酶基因ldh Ti的失活或缺失被阻断，糖 酵解途径通过6-磷酸果糖激酶基因pfk和丙酮酸激酶基因pyk的过表达被增强。
在本发明的一个较佳的实施例中，所述的构建方法包括如下步骤：
S500：全部失活或全部缺失所述起始菌的基因组中含有的乳酸脱氢酶基因，优选地，所述乳酸脱氢酶基因为D-乳酸脱氢酶基因ldh Ti(可实现阻断乳酸合成途径)；
S200：过表达6-磷酸果糖激酶基因pfk的拷贝和丙酮酸激酶基因pyk(可增强糖酵解途径，提高丙酮酸的供给)；
S300：过表达异源的丙氨酸脱氢酶基因GSald(可实现增加从丙酮酸到L-丙氨酸的合成途径)；和
S400：全部失活或全部缺失丙氨酸消旋酶基因alr1和丙氨酸消旋酶基因alr2(可阻断L-丙氨酸向D-丙氨酸的转变，有利于实现光学纯L-丙氨酸的高产)。
在本发明的一个较佳的实施例中，所述的构建方法包括如下步骤：
S500：敲除所述起始菌的基因组中含有的乳酸脱氢酶基因，优选地，所述乳酸脱氢酶基因为D-乳酸脱氢酶基因ldh Ti(可实现阻断乳酸合成途径)，更优选地，所述D-乳酸脱氢酶基因ldh Ti的序列如SEQ ID NO.31所示；
S200：插入6-磷酸果糖激酶基因pfk的拷贝和丙酮酸激酶基因pyk的拷贝(可过表达起始菌中的6-磷酸果糖激酶pfk和丙酮酸激酶pyk，从而增强糖酵解途径，提高丙酮酸的供给)；优选地，所述6-磷酸果糖激酶基因pfk的序列如SEQ ID NO.27所示，所述丙酮酸激酶基因pyk的序列如SEQ ID NO.28所示；
S300：插入异源的丙氨酸脱氢酶基因GSald(可过表达异源的丙氨酸脱氢酶基因GSald，实现增加从丙酮酸到L-丙氨酸的合成途径)；优选地，所述丙氨酸脱氢酶基因GSald的序列如SEQ ID NO.1所示；和
S400：敲除丙氨酸消旋酶基因alr1和丙氨酸消旋酶基因alr2(可阻断L-丙氨酸向D-丙氨酸的转变，有利于实现光学纯L-丙氨酸的生产)；优选地，所述丙氨酸消旋酶基因alr1的序列如SEQ ID NO.29所示，所述丙氨酸消旋酶基因alr2的序列如SEQ ID NO.30所示；
应当理解，如无特别说明，本发明各方案中的多个特征的优选各自独立，可以采用合适的方式进行组合。
在本发明的一些实施方式中，在步骤200和步骤300中，插入相关基因的方式为在染色体上增加该基因的拷贝(增加的拷贝数可以为1个或更多个)，并在该基因前面串联启动子。
在本发明的一些实施例中，所述启动子为P als、Plac、Ptrc、Ptac、Pc P43等中任一种。
在本发明的一些实施方式中，所述启动子为P als。
在本发明的一些实施方式中，所述启动子的序列如SEQ ID NO.2所示。
在本发明的一些实施方式中，所述起始菌为嗜热菌。
在本发明的一些优选的实施方式中，所述起始菌为芽孢杆菌。
在本发明的一些优选的实施方式中，所述起始菌为地衣芽孢杆菌。地衣芽孢杆菌是一种兼性厌氧、革兰氏阳性、内生孢子型的细菌，具有较快的生长速度和较广泛的底物谱，能够在50℃的条件下进行高温发酵。而且该菌株遗传操作方便稳定，还是美国食品和药物管理局公认的GRAS(generally regarded as safe)菌株，有潜力成为理想的平台菌株。
在本发明的一些优选的实施方式中，所述起始菌为地衣芽孢杆菌、凝结芽孢杆菌、甲基营养芽孢杆菌、嗜热菊糖芽孢杆菌或嗜热脂肪地芽孢杆菌。
在本发明的一些优选的实施方式中，所述起始菌为地衣芽孢杆菌ATCC 14580及其衍生菌株。所述衍生菌株的举例包括但不限于：地衣芽孢杆菌MW3、地衣芽孢杆菌BN11等。
在本发明的一些优选的实施方式中，所述起始菌为地衣芽孢杆菌BN11，保藏号为CCTCC NO:M2016026，于2016年1月8日保藏于中国典型培养物保藏中心。
在本发明的一些优选的实施方式中，所述起始菌为地衣芽孢杆菌BN11，所述6-磷酸果糖激酶基因pfk的序列如SEQ ID NO.27所示；所述丙酮酸激酶基因pyk的序列如SEQ ID NO.28所示。
在本发明的一些优选的实施方式中，所述起始菌为地衣芽孢杆菌BN11，所述丙氨酸脱氢酶基因GSald的序列如SEQ ID NO.1所示。
在本发明的一些优选的实施方式中，所述起始菌为地衣芽孢杆菌BN11，其内源的丙氨酸消旋酶基因包括alr1和alr2，其中，丙氨酸消旋酶基因alr1的序列如SEQ ID NO.29所示，丙氨酸消旋酶基因alr2的序列如SEQ ID NO.30所示。
在本发明的一些实施方式中，在本发明的一些优选的实施方式中，所述起始菌为地衣芽孢杆菌BN11，所述D-乳酸脱氢酶基因ldh Ti的序列如SEQ ID NO.31所示。
在本发明的第二方面，提供了本明的第一方面所述的构建方法制得的基因工程菌株。该基因工程菌株能够在42℃以上(如42℃～55℃)发酵生产L-丙氨酸，可实现产量的显著提升，进一步地，还可以实现高产光学纯L-丙氨酸。
在本发明的第三方面，提供了本发明的第二方面所述的基因工程菌株在生产L-丙氨酸中的应用，可用于高温条件发酵生产L-丙氨酸，可显著提高产量，进一步地，可在高温条件下实现光学纯产L-丙氨酸的高产。
在本发明的第四方面，提供了一种L-丙氨酸的生产方法，对本发明的第二方面所述的基因工程菌株进行发酵培养，其中，发酵培养温度为42℃～55℃。发酵培养的温度举例包括但不限于42℃、43℃、44℃、45℃、46℃、47℃、48℃、49℃、50℃、51℃、52℃、53℃、54℃、55℃。
在本发明的一些实施方式中，所述发酵培养步骤包括：将活化的种子接种到发酵培养基中，所述发酵培养基中包含碳源，在发酵培养温度为42℃～55℃、搅拌速度为 50rpm～350rpm的条件下进行分批补料发酵，培养至设定的发酵密度。进一步地，接种所述活化的种子的接种量，以体积百分比计，可以为3％～5％，举例如3％、4％、5％。
在本发明的一些实施方式中，所述的生产方法在所述发酵培养之前还进行种子培养得到活化的种子，然后将所述活化的种子接种到发酵培养基中进行所述发酵培养。
在本发明的一些实施方式中，所述种子培养步骤包括：将所述的基因工程菌株接种到种子培养基中，在种子培养温度为42℃～55℃的条件下培养12h～16h，得到的种子培养液即活化的种子。
在本发明的一些实施方式中，所述种子培养基包含以下成分：蛋白胨、酵母粉和氯化钠。
在本发明的一些实施方式中，所述种子培养基由以下组分构成：蛋白胨、酵母粉和氯化钠。
在本发明的一些实施方式中，所述的生产方法包括如下步骤：
种子培养：将所述的基因工程菌株接种到种子培养基中，在种子培养温度为42℃～55℃的条件下培养，得到活化的种子；和
发酵培养：将所述活化的种子接种到发酵培养基中，所述发酵培养基中包含碳源，在发酵培养温度为42℃～55℃的条件下进行分批补料发酵，培养至设定的发酵密度；其中，接种量可以为体积比3％～5％。
在本发明的一些实施方式中，所述的生产方法包括如下步骤：
种子培养：将所述的基因工程菌株接种到种子培养基中，在种子培养温度为42℃～55℃的条件下培养12h～16h，得到活化的种子；和
发酵培养：将所述活化的种子接种到发酵培养基中，所述发酵培养基中包含碳源，在发酵培养温度为42℃～55℃、搅拌速度为50rpm～350rpm的条件下进行分批补料发酵，培养至设定的发酵密度；其中，接种量可以为体积比3％～5％。
在本发明的一些实施方式中，在所述发酵培养步骤中，所述碳源为葡萄糖、甘油、木糖或阿拉伯糖。
在本发明的一些实施方式中，在所述发酵培养步骤中，所述发酵培养基包含以下成分：葡萄糖、酵母粉、硫酸铵、磷酸氢二钾、磷酸二氢钾、硫酸镁、硫酸亚铁和硫酸锰。在本发明的一些实施方式中，所述发酵培养基由以下成分组成：葡萄糖、酵母粉、硫酸铵、磷酸氢二钾、磷酸二氢钾、硫酸镁、硫酸亚铁和硫酸锰。
在本发明的一些实施方式中，所述设定的发酵密度可以通过OD 600进行检测，比如，当OD 600达到6.5～8.0时停止发酵。
在本发明的一些实施方式中，在所述发酵培养步骤中，所述发酵培养步骤包括：使氨水调节pH为7.0左右，通气条件下，搅拌条件下进行发酵培养(第一段发酵培养)至OD600值达到6.5～8.0，停止通气，降低搅拌速度继续发酵培养(第二段发酵培养)至设定的发酵密度，结束发酵；在发酵过程中，所述碳源的浓度降到一定值时可补充 所述碳源。
用于调节pH的氨水的浓度可以为20％～30％(v/v)，具体举例如20％、25％、30％，以体积百分比计算。
所述通气条件，优选为通入空气。
通气的通气量可以为0.8vvm～1.2vvm，举例如0.9vvm、1.0vvm、1.1vvm、1.2vvm等。
发酵培养分段进行。可以分两段进行，包括通气条件下的第一段发酵培养，停止通气后的第二段发酵培养。通过第一段发酵培养，可使菌体快速生长，通过第二段发酵培养，可以实现高效生产L-丙氨酸。其中，第一段发酵培养时的搅拌速度优选280rpm～320rpm，举例如280rpm、290rpm、300rpm、310rpm、320rpm等。第一段发酵培养的时间根据OD600值确定，例如可以为6小时至8小时，具体举例如6h、6.5h、7h、7.5h、8h。第二段发酵培养时的搅拌速度优选60rpm～100rpm，举例如60rpm、70rpm、80rpm、90rpm、100rpm等。第二段发酵培养的时间也根据OD600值(比如1.2)确定，例如可以为50小时至70小时，具体举例如50h、55h、60h、65h、70h。
发酵过程中补充碳源的次数可以为一次、两次等。
关于发酵过程中补充碳源时的时点，比如碳源浓度下降到30g/L～50g/L时补充一次碳源，又比如，碳源浓度降到20g/L～40g/L时，还可以再补充一次碳源。优选地，所述碳源为葡萄糖。
在本发明的一些实施方式中，仅补充一次碳源时，以在所述碳源的浓度降到30g/L～50g/L(举例如30g/L、35g/L、40g/L、45g/L、50g/L)时，可以将碳源的浓度补充至80g/L～120g/L(举例如80g/L、90g/L、100g/L、110g/L、120g/L)。优选地，所述碳源为葡萄糖。
在本发明的一些实施方式中，补充2次碳源。第一次补充碳源时，可以在所述碳源的浓度降到30g/L～50g/L(举例如30g/L、35g/L、40g/L、45g/L、50g/L)时，可以将碳源的浓度补充至80g/L～120g/L(举例如80g/L、90g/L、100g/L、110g/L、120g/L)。第二次补充碳源时，可以在所述碳源的浓度降到20g/L～40g/L(举例如20g/L、25g/L、30g/L、35g/L、40g/L)时，可以将碳源的浓度补充至60g/L～80g/L(举例如60g/L、65g/L、70g/L、75g/L、80g/L)。优选地，所述碳源为葡萄糖。
在本发明的一些实施方式中，在所述发酵培养步骤中，所述发酵培养步骤包括：使用20％～30％(v/v)的氨水调节pH为7.0±0.2，通气量为0.8vvm～1.2vvm，初始搅拌转速为280rpm～320rpm，进行发酵培养6～8小时至OD600值达到6.5～8.0，停止通气，调节搅拌转速为60rpm～100rpm，继续发酵培养到50小时～70小时(例如继续发酵培养到60小时)，结束发酵；进一步地，发酵过程中在碳源浓度降低至一定值时补充该碳源，可以补充一次、两次等。在一些实施方式中，在发酵过程中仅补充一次碳源，所述碳源的浓度降到30g/L～50g/L时进行第一次补充所述碳源，至所述碳源的浓度为 80g/L～120g/L。在另一些实施方式中，在发酵过程中补充两次碳源，所述碳源的浓度降到30g/L～50g/L时进行第一次补充所述碳源，至所述碳源的浓度为80g/L～120g/L，然后，当所述碳源的浓度降到20g/L～40g/L时，第二次补充所述碳源，至所述碳源的浓度为60g/L～80g/L。优选地，所述碳源为葡萄糖。
在本发明的一些实施方式中，在所述发酵培养步骤中，所述碳源为葡萄糖；所述发酵培养步骤包括：25％的氨水调节pH使其维持7.0，通气量为1vvm，初始搅拌转速为300rpm。发酵培养6小时～8小时后地衣芽孢杆菌的OD 600达到6.5～8.0，停止通气并调节搅拌转速为80rpm，继续发酵到60h结束。发酵过程中葡萄糖浓度降到300g/L～50g/L时进行一次补充葡萄糖，使得葡萄糖浓度达到100g/L。当葡萄糖浓度再次降低到20g/L～40g/L时进行第二次补充葡萄糖，至葡萄糖浓度达到70g/L。
以下结合本发明的实施例对本发明作进一步详细说明。下述实施例是说明性的，不是限定性的，不能以下述实施例来限定本发明的保护范围。本发明的实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。下述实施例中未注明具体条件的实验方法(比如PCR扩增、转化、基因敲除、基因插入等)，优选地按照上文的方法和条件进行；然后通常按照常规方法和条件，例如Sambrook等分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的方法和条件，或者按照制造厂商所建议的条件。
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。
丙氨酸浓度测定方法：使用高效液相色谱法，称取0.54g的辛烷磺酸钠，加入到800mL超纯水中，用磷酸调节pH为2.1，加入100mL甲醇，用超纯水定容到1L体积，作为流动相。色谱柱为ZORBAX Eclipse XDB-C18(150mm×4.6mm,5μm)，柱温30℃，流速0.8mL/min，进样量10μL，检测器为DAD检测器，波长210nm。待检样品为发酵样品，首先需要煮沸10min，随后8000rpm离心5min，吸取上清稀释到合适的倍数，至丙氨酸终浓度为0.2g/L到1g/L。
丙氨酸光学纯度测定方法：使用高效液相色谱法，流动相为2mM的硫酸铜的水溶液。色谱柱为SUMICHIRAL OA-5000(150mm×4.6mm,5μm)，柱温30℃，流速0.5mL/min，进样量10μL，检测器为DAD检测器，波长254nm。待检样品为发酵样品，首先需要煮沸10min，随后8000rpm离心5min，吸取上清稀释到合适的倍数，至丙氨酸终浓度为0.2g/L到1g/L。
葡萄糖浓度测定方法：使用SBA-40D型生物传感分析仪(山东省科学院生物研究所)测定葡萄糖浓度。
以下实施例以地衣芽孢杆菌作为起始菌构建可以在高温条件下生产L-丙氨酸的基因工程菌株。应当理解，以下实施例中生产L-丙氨酸的基因工程菌株的构建方法也可以用于其它菌株(示例包括但不限于凝结芽孢杆菌、甲基营养芽孢杆菌、 嗜热菊糖芽孢杆菌、嗜热脂肪地芽孢杆菌等)作为起始菌，用来得到本发明中可以在高温条件下生产L-丙氨酸的基因工程菌株。
PKVM载体：其结构如图3所示，其包含ampR(氨苄青霉素抗性基因)和ermC(红霉素抗性基因)两个抗生素抗性基因，以及编码半乳糖苷酶的bgaB基因，均可作为筛选标记；该质粒具有一个热敏型复制起始位点oriT pE194ts，还有在大肠杆菌和地衣芽孢杆菌的复制起始位点ori和oriT。
地衣芽孢杆菌BN11，是ATCC 14580为基础构建的乳酸生产菌株，保藏号为CCTCC NO:M2016026，于2016年1月8日保藏于中国典型培养物保藏中心。
接种量：以体积百分比计。
如无特别说明，新插入的基因拷贝数为1个。
实施例1：敲除D-乳酸合成途径的地衣芽孢杆菌的构建
1.1.根据地衣芽孢杆菌ATCC 14580的基因组信息(GenBank No.NC_006270.3)设计引物，分别以PKVM-XPDdldh-UpF(SEQ ID NO.3)和XPDdldh-UpR(SEQ ID NO.4)、XPDdldh-DownF(SEQ ID NO.5)和PKVM-XPDdldh-DownR(SEQ ID NO.6)为上下游引物，分别进行PCR扩增，分别得到ldh Ti基因启动子上游同源臂片段和ldh Ti基因下游同源臂片段。
1.2.将上述步骤1.1.得到的PCR产物进行纯化，然后以PKVM-XPDdldh-UpF和PKVM-XPDdldh-DownR为引物进行重组PCR扩增，对PCR产物进行纯化后通过无缝克隆连接到PKVM载体，转化到大肠杆菌S17中得到S17-PKVMΔldh Ti。
1.3.将步骤1.2.得到的S17-PKVMΔldh Ti(大肠杆菌)和地衣芽孢杆菌BN11(起始菌)分别进行发酵培养，均至OD 600为1.2时结束发酵培养，然后6000rpm离心5min，用PBS洗涤两次。然后将肠杆菌的改造菌株S17-PKVMΔldh Ti：地衣芽孢杆菌BN11的按照相同OD 600(也即相同浓度)下的体积比7:1混合滴加在LB平板，30℃过夜培养后稀释涂在含有红霉素和多粘菌素的LB平板上，30℃培养。挑取转化子在含有红霉素的LB培养基37℃培养，随后转接到LB培养基50℃培养，稀释涂布在含有红霉素的LB平板上，50℃培养筛选单交换转化子。单交换转化子在LB培养基30℃连续培养两代后稀释涂布在LB平板，37℃过夜培养并根据红霉素抗性反向筛选双交换转化子。以PKVM-XPDdldh-UpF和PKVM-XPDdldh-DownR为引物进行PCR和测序验证，得到正确地敲除了D-乳酸合成途径的基因工程菌株，敲除了D-乳酸脱氢酶基因ldh Ti(SEQ ID NO.31)，记为BN11Δldh Ti。
实施例2：过表达6-磷酸果糖激酶和丙酮酸激酶的地衣芽孢杆菌的构建
2.1.根据地衣芽孢杆菌ATCC 14580的基因组信息(GenBank No.NC_006270.3)设计引物，分别以PKVM-XPDldh-UpF(SEQ ID NO.7)和P als-XPDldh-UpR(SEQ ID NO.8)、XPDldh-P als-F(SEQ ID NO.9)和PFYAK-P als-R (SEQ ID NO.10)、P als-PFYAK-F(SEQ ID NO.11)和XPDldh-PFYAK-R(SEQ ID NO.12)、PFYAK-XPDldh-DownF(SEQ ID NO.13)和PKVM-XPDldh-DownR(SEQ ID NO.14)为上下游引物，分别进行PCR扩增，分别得到ldh基因上游同源臂片段、P als启动子(SEQ ID NO.2)片段、pfk(SEQ ID NO.27)和pyk(SEQ ID NO.28)基因簇片段以及ldh基因下游同源臂片段。
2.2.将上述步骤2.1.得到的PCR产物进行纯化，然后以PKVM-XPDldh-UpF和PKVM-XPDldh-DownR为引物进行重组PCR扩增，对PCR产物进行纯化后通过无缝克隆连接到PKVM载体，转化到大肠杆菌S17中得到S17-PKVM-PFYAK。
2.3.将上述步骤2.2得到的大肠杆菌S17-PKVM-PFYAK和地衣芽孢杆菌BN11Δldh Ti按照实施例1中的方法进行接合转移和同源重组实验，得到过表达pfk和pyk基因的菌株，记为BN11Δldh Ti-PFYAK。
实施例3：过表达异源的丙氨酸脱氢酶的菌株的构建
3.1.根据丙氨酸脱氢酶的氨基酸序列通过BLAST方法在嗜热脂肪地芽孢杆菌基因组中挖掘潜在的热稳定性丙氨酸脱氢酶(其氨基酸序列如SEQ ID NO.32所示，蛋白序列编号对应WP_033014465.1)，嗜热脂肪地芽孢杆菌来源的丙氨酸脱氢酶基因被命名为GSald，根据地衣芽孢杆菌基因组进行了密码子优化(优化后的序列如SEQ ID NO.1所示)，并和P als启动子(SEQ ID NO.2)一起进行合成，合并序列从5'至3'为P als-GSald。
3.2.根据地衣芽孢杆菌ATCC 14580的基因组信息(GenBank No.NC_006270.3)设计引物，分别以PKVM-XPDdldh-UpF(SEQ ID NO.3)和P als-XPDdldh-UpR(SEQ ID NO.15)、XPDdldh-P als-F(SEQ ID NO.16)和XPDdldh-GSald-R(SEQ ID NO.17)、GSald-XPDdldh-DownF(SEQ ID NO.18)和PKVM-XPDdldh-DownR(SEQ ID NO.6)为上下游引物进行PCR扩增，得到ldh Ti基因启动子上游同源臂片段、P als-GSald基因片段以及ldh Ti基因下游同源臂片段。
3.3.将上述步骤3.2.得到的PCR产物进行纯化，然后以PKVM-XPDdldh-UpF和PKVM-XPDdldh-DownR为引物进行重组PCR扩增，对PCR产物进行纯化后分别通过无缝克隆连接到PKVM载体，转化到大肠杆菌S17中，得到S17-PKVM-GSald。然后，将大肠杆菌S17-PKVM-GSald和地衣芽孢杆菌BN11Δldh Ti-PFYAK按照实施例1中的方法进行接合转移和同源重组实验，得到表达异源GSald的菌株，记为BA-1。
实施例4：敲除地衣芽孢杆菌的丙氨酸消旋酶
4.1.根据地衣芽孢杆菌ATCC 14580的基因组信息(GenBank No.NC_006270.3)设计引物，分别以PKVM-XPDalr1-UpF(SEQ ID NO.19)和XPDalr1-UpR(SEQ ID NO.20)、XPDalr1-DownF(SEQ ID NO.21)和PKVM-XPDalr1-DownR(SEQ ID NO.22)为上下游引物进行PCR扩增，得到alr1 基因上游同源臂片段和alr1基因下游同源臂片段。
4.2.将上述步骤4.1.得到的PCR产物进行纯化，然后以PKVM-XPDalr1-UpF和PKVM-XPDalr1-DownR为引物进行重组PCR扩增，对PCR产物进行纯化，然后通过无缝克隆连接到PKVM载体，转化到大肠杆菌S17中，得到S17-PKVMΔalr1。
4.3.将大肠杆菌S17-PKVMΔalr1和地衣芽孢杆菌BA-1按照实施例1中的方法进行接合转移和同源重组实验，得到敲除alr1基因(SEQ ID NO.29)的菌株，记为BA-1Δalr1。
4.4.根据地衣芽孢杆菌ATCC 14580的基因组信息(GenBank No.NC_006270.3)设计引物，分别以PKVM-XPDalr2-UpF(SEQ ID NO.23)和XPDalr2-UpR(SEQ ID NO.24)、XPDalr2-DownF(SEQ ID NO.25)和PKVM-XPDalr2-DownR(SEQ ID NO.26)为上下游引物，进行PCR扩增，得到alr2基因上游同源臂片段和alr2基因下游同源臂片段。
4.5.将上述步骤4.4.得到的PCR产物进行纯化，然后以PKVM-XPDalr2-UpF和PKVM-XPDalr2-DownR为引物进行重组PCR扩增，对PCR产物进行纯化，然后通过无缝克隆连接到PKVM载体，转化到大肠杆菌S17中，得到S17-PKVMΔalr2。
4.6.将大肠杆菌S17-PKVMΔalr2和地衣芽孢杆菌BA-1Δalr1按照实施例1中的方法进行接合转移和同源重组实验，得到同时敲除alr1基因(SEQ ID NO.29)和alr2基因(SEQ ID NO.30)的菌株，记为BLA-1。
实施例5：重组地衣芽孢杆菌BLA-1在42℃进行分批补料发酵(5L发酵罐)
5.1.种子培养基和发酵培养基
种子培养基配方：LB培养基，酵母粉5g/L，胰蛋白胨10g/L，氯化钠10g/L。
发酵培养基配方：葡萄糖100g/L，酵母粉5g/L，硫酸铵5g/L，三水合磷酸氢二钾1.3g/L，磷酸二氢钾0.5g/L，七水合硫酸镁0.5g/L，七水合硫酸亚铁20mg/L，四水合硫酸锰20mg/L。其中，对葡萄糖单独灭菌，115℃灭菌20min。
5.2.种子培养
从甘油管转接BLA-1到装有5mL的LB培养基的摇管中，50℃200rpm振荡培养12h。随后按照体积比3％～5％的接种量转接到装有150mL的LB培养基的500mL三角瓶中，50℃200rpm振荡培养12h。得到种子液(也即活化的种子)。该步骤实现了种子的活化。
5.3.发酵培养
将步骤5.2.得到的种子液按照体积比5％的接种量转接到装有发酵培养基的5L发酵罐中，使得装液量达到3L。用25％(v/v)的氨水控制发酵pH为7.0，通气(通入空气)，通气量为1.0vvm，温度42℃，搅拌转速为300rpm。进行发酵培养6～8 小时后地衣芽孢杆菌的OD 600达到6.5～8.0，此时停止通气并调节搅拌转速为80rpm，继续发酵到60h结束。发酵过程中葡萄糖浓度降到30g/L～50g/L时进行一次补糖(也即补充碳源，本实施例中具体补充葡萄糖)，使得葡萄糖浓度达到100g/L。发酵60h后，经检测分析，发酵液中L-丙氨酸浓度达到93.7g/L，糖酸转化率为74.4％，L-丙氨酸光学纯度达到98.9％。
实施例6：重组地衣芽孢杆菌BLA-1在50℃进行分批补料发酵(5L发酵罐)
6.1.种子培养基和发酵培养基
同实施例5.1.。
6.2.种子培养
同实施例5.2.。
6.3.发酵培养
将步骤6.2.得到的种子液按照体积比5％的接种量转接到装有发酵培养基的5L发酵罐中，使得装液量达到3L。用25％(v/v)的氨水控制发酵pH为7.0，通气量为1.0vvm，温度50℃，搅拌转速为300rpm。进行发酵培养6～8小时后地衣芽孢杆菌的OD 600达到6.5～8.0，此时停止通气并调节搅拌转速为80rpm，继续发酵到60h结束。发酵过程中葡萄糖浓度降到30g/L～50g/L时进行补糖，使得葡萄糖浓度达到100g/L。发酵曲线如图2所示。发酵60h后，经检测分析，发酵液中丙氨酸浓度达到96.8g/L，糖酸转化率为76.8％，L-丙氨酸光学纯度达到98.9％。
实施例7：重组地衣芽孢杆菌BLA-1在55℃进行分批补料发酵(5L发酵罐)
7.1.种子培养基和发酵培养基
同实施例5.1.。
7.2.种子培养
同实施例5.2.。
7.3.发酵培养
将步骤7.2.得到的种子液按照体积比5％的接种量转接到装有发酵培养基的5L发酵罐中，使得装液量达到3L。用25％(v/v)的氨水控制发酵pH为7.0，通气量为1.0vvm，温度55℃，搅拌转速为300rpm。进行发酵培养6～8小时后地衣芽孢杆菌的OD 600达到3.0～4.5，此时停止通气并调节搅拌转速为80rpm，继续发酵到60h结束。发酵60h后，经检测分析，发酵液中丙氨酸浓度达到21.4g/L，糖酸转化率为63.8％，L-丙氨酸光学纯度达到97.2％。
实施例8：重组地衣芽孢杆菌BLA-1在42℃进行分批补料发酵(50L发酵罐)
8.1.种子培养基和发酵培养基
同实施例5.1.。
8.2.种子培养
从甘油管转接BLA-1到装有5mL的LB培养基的摇管中，50℃、200rpm振荡 培养12h。随后按照体积比3％～5％的接种量转接到装有150mL的LB培养基的500mL三角瓶中，50℃、200rpm振荡培养12h。最后按照体积比3％～5％的接种量转接到装有1.5L的LB培养基的5L三角瓶中，50℃、200rpm振荡培养12h。
8.3.发酵培养
将步骤8.2.得到的种子液按照体积比5％的接种量转接到装有发酵培养基的50L发酵罐中，使得装液量达到30L。用25％(v/v)的氨水控制发酵pH为7.0，通气量为1.0vvm，温度42℃，搅拌转速为300rpm。进行发酵培养6～8小时后地衣芽孢杆菌的OD 600达到6.5～8.0，此时停止通气并调节搅拌转速为80rpm，继续发酵到60h结束。发酵过程中葡萄糖浓度降到30g/L～50g/L时进行一次补糖，使得葡萄糖浓度达到100g/L。发酵60h后，经检测分析，发酵液中丙氨酸浓度达到40.5g/L，糖酸转化率为70.7％，L-丙氨酸光学纯度达到96.6％。
实施例9：重组地衣芽孢杆菌BLA-1在50℃进行分批补料发酵，50L发酵罐
9.1.种子培养基和发酵培养基
同实施例5.1.。
9.2.种子培养
同实施例8.2.。
9.3.发酵培养
将步骤9.2.得到的种子液按照5％的接种量转接到装有发酵培养基的50L发酵罐中，使得装液量达到30L。用25％(v/v)的氨水控制发酵pH为7.0，通气量为1.0vvm，温度50℃，搅拌转速为300rpm。进行发酵培养6～8小时后，地衣芽孢杆菌的OD 600达到6.5～8.0，此时停止通气并调节搅拌转速为80rpm，继续发酵到60h结束。发酵过程中葡萄糖浓度降到30g/L～50g/L时进行一次补糖，使得葡萄糖浓度达到100g/L。发酵60h后，经检测分析，发酵液中丙氨酸浓度达到108.2g/L，糖酸转化率为73.3％，L-丙氨酸光学纯度达到99.1％。
实施例10：重组地衣芽孢杆菌BLA-1在55℃进行的分批补料发酵(50L发酵罐)
10.1.种子培养基和发酵培养基
同实施例5.1.。
10.2.种子培养
同实施例8.2.。
10.3.发酵培养
将步骤10.2得到的种子液按照5％的接种量转接到装有发酵培养基的50L发酵罐中，使得装液量达到30L。用25％(v/v)的氨水控制发酵pH为7.0，通气量为1.0vvm，温度55℃，搅拌转速为300rpm。进行发酵培养6-8小时后，地衣芽孢杆菌的OD 600达到4.0～6.0，此时停止通气并调节搅拌转速为80rpm，继续发酵到60h 结束。发酵60h后，经检测分析，发酵液中丙氨酸浓度达到26.6g/L，糖酸转化率为59.0％，L-丙氨酸光学纯度达到98.7％。
实施例11.实施例2构建的BN11Δldh Ti-PFYAK菌株的发酵培养结果
参考实施例6的方法和实验参数对实施例2得到的BN11Δldh Ti-PFYAK菌株进行发酵培养。
结果发现：1、-丙氨酸产量64g/L；2、得到的产物是L型和D型的混合产品，L-丙氨酸的光学纯度仅67.7％难以满足要求，光学纯要求一般为98％以上。
实施例12.实施例3构建的BA-1菌株的发酵培养结果
采用实施例6的方法和实验参数对实施例3得到的BA-1菌株进行发酵培养。
结果发现：1、丙氨酸产量129g/L；2、得到的产物是L型和D型的混合产品，L型丙氨酸的光学纯度为70.8％，难以满足要求。
综合实施例11和实施例12的结果表明，敲除地衣芽孢杆菌的丙氨酸消旋酶是实现L-丙氨酸的高光学纯度所必需。
实施例13.仅敲除一个丙氨酸消旋酶
参考实施例4的方法仅敲除丙氨酸消旋酶alr1(SEQ ID NO.29)、敲除丙氨酸消旋酶alr2(SEQ ID NO.30)中的一个，参考实施例6的方法对构建得到的菌株进行发酵培养。
结果发现，光学纯度难以满足要求。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术人员无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。

Claims (28)

  1. 一种生产L-丙氨酸的基因工程菌株的构建方法，其特征在于，包括如下步骤：
    提供具有丙酮酸合成途径的起始菌；
    进行S200、S300、S400中的任一种步骤、任两种步骤或三种步骤对所述起始菌的基因组进行改造：
    S200：插入6-磷酸果糖激酶基因pfk的拷贝和丙酮酸激酶基因pyk的拷贝；
    S300：插入具有42℃～55℃热稳定性的丙氨酸脱氢酶基因GSald；
    S400：所述起始菌的基因组中含有丙氨酸消旋酶基因，该步骤包括失活或缺失丙氨酸消旋酶基因。
  2. 如权利要求1所述的生产L-丙氨酸的基因工程菌株的构建方法，其特征在于，所述起始菌还具有乳酸合成途径，且所述起始菌的基因组中含有乳酸脱氢酶基因；
    所述的构建方法还包括如下步骤：
    S100：失活或缺失所述起始菌的基因组中的乳酸脱氢酶基因。
  3. 如权利要求2所述的生产L-丙氨酸的基因工程菌株的构建方法，所述起始菌还具有D-乳酸合成途径，且所述起始菌的基因组中含有D-乳酸脱氢酶基因ldh Ti；
    所述的构建方法还包括如下步骤S500：失活或缺失所述起始菌的基因组中含有的D-乳酸脱氢酶基因ldh Ti；
    优选地，所述D-乳酸脱氢酶基因ldh Ti的序列如SEQ ID NO.31所示。
  4. 如权利要求1所述的生产L-丙氨酸的基因工程菌株的构建方法，其特征在于，所述6-磷酸果糖激酶基因pfk的序列如SEQ ID NO.27所示，所述丙酮酸激酶基因pyk的序列如SEQ ID NO.28所示；和/或，
    所述丙氨酸脱氢酶基因GSald的序列如SEQ ID NO.1所示。
  5. 如权利要求1所述的生产L-丙氨酸的基因工程菌株的构建方法，其特征在于，所述步骤S400中，失活或缺失的所述丙氨酸消旋酶基因的种类为1种、2种或更多种；和/或，
    所述步骤S400包括：失活或缺失丙氨酸消旋酶基因alr1和丙氨酸消旋酶基因alr2；
    优选地，所述丙氨酸消旋酶基因alr1的序列如SEQ ID NO.29所示，所述丙氨酸消旋酶基因alr2的序列如SEQ ID NO.30所示。
  6. 如权利要求2所述的生产L-丙氨酸的基因工程菌株的构建方法，其特征在于，对所述起始菌的基因组进行改造包括步骤S100和步骤S200；
    优选地，对所述起始菌的基因组进行改造包括步骤S100、步骤S200和步骤S300。
  7. 如权利要求2所述的生产L-丙氨酸的基因工程菌株的构建方法，其特征在于， 对所述起始菌的基因组进行改造包括步骤S100、步骤S200、步骤S300和步骤S400。
  8. 如权利要求3所述的生产L-丙氨酸的基因工程菌株的构建方法，其特征在于，包括如下步骤：
    S500：敲除所述起始菌的基因组中含有的D-乳酸脱氢酶基因ldh Ti；
    S200：插入6-磷酸果糖激酶基因pfk的拷贝和丙酮酸激酶基因pyk的拷贝；
    S300：插入异源的丙氨酸脱氢酶基因GSald；和
    S400：敲除丙氨酸消旋酶基因alr1和丙氨酸消旋酶基因alr2。
  9. 如权利要求1所述的生产L-丙氨酸的基因工程菌株的构建方法，其特征在于，在步骤200和步骤300中，插入相关基因的方式为在染色体上增加该基因的拷贝，并在该基因前面串联启动子。
  10. 如权利要求9所述的生产L-丙氨酸的基因工程菌株的构建方法，其特征在于，所述启动子为P als。
  11. 如权利要求10所述的生产L-丙氨酸的基因工程菌株的构建方法，其特征在于，所述启动子的序列如SEQ ID NO.2所示。
  12. 如权利要求1～11中任一项所述的生产L-丙氨酸的基因工程菌株的构建方法，其特征在于，所述生产L-丙氨酸的基因工程菌株能够在42℃～55℃发酵生产L-丙氨酸。
  13. 如权利要求1～11中任一项所述的生产L-丙氨酸的基因工程菌株的构建方法，其特征在于，所述起始菌为嗜热菌。
  14. 如权利要求1～11中任一项所述的生产L-丙氨酸的基因工程菌株的构建方法，其特征在于，所述起始菌为芽孢杆菌。
  15. 如权利要求14所述的生产L-丙氨酸的基因工程菌株的构建方法，其特征在于，所述起始菌为地衣芽孢杆菌、凝结芽孢杆菌、甲基营养芽孢杆菌、嗜热菊糖芽孢杆菌或嗜热脂肪地芽孢杆菌。
  16. 如权利要求14所述的生产L-丙氨酸的基因工程菌株的构建方法，其特征在于，所述起始菌为地衣芽孢杆菌ATCC 14580及其衍生菌株。
  17. 如权利要求14所述的生产L-丙氨酸的基因工程菌株的构建方法，其特征在于，所述起始菌为地衣芽孢杆菌BN11，保藏号为CCTCC NO:M2016026，于2016年1月8日保藏于中国典型培养物保藏中心。
  18. 根据权利要求1～17中任一项所述的构建方法制得的基因工程菌株。
  19. 如权利要求18所述的基因工程菌株在生产L-丙氨酸中的应用。
  20. 一种L-丙氨酸的生产方法，对权利要求18所述的基因工程菌株进行发酵培养，其中，发酵培养温度为42℃～55℃。
  21. 如权利要求20所述的生产方法，其特征在于，所述发酵培养步骤包括：将活化的种子接种到发酵培养基中，所述发酵培养基中包含碳源，在搅拌速度为50rpm～350rpm的条件下进行分批补料发酵，培养至设定的发酵密度。
  22. 如权利要求21所述的生产方法，其特征在于，在所述发酵培养步骤中，所述碳源为葡萄糖、甘油、木糖或阿拉伯糖。
  23. 如权利要求21所述的生产方法，其特征在于，在所述发酵培养步骤中，所述发酵培养基包含以下成分：葡萄糖、酵母粉、硫酸铵、磷酸氢二钾、磷酸二氢钾、硫酸镁、硫酸亚铁和硫酸锰。
  24. 如权利要求21所述的生产方法，其特征在于，所述发酵培养步骤包括：使用20％～30％(w/v)的氨水调节pH为7.0±0.2，通气量为0.8vvm～1.2vvm，初始搅拌转速为280rpm～320rpm，进行发酵培养6～8小时至OD600值达到6.5～8.0，停止通气，调节搅拌转速为60rpm～100rpm，继续发酵培养到50小时～70小时，结束发酵；在发酵过程中，所述碳源的浓度降到一定值时补充所述碳源；
    优选地，在发酵过程中，所述碳源的浓度降到30g/L～50g/L时，补充一次所述碳源，至所述碳源的浓度为80g/L～120g/L；
    优选地，在发酵过程中，所述碳源的浓度降到30g/L～50g/L时进行第一次补充所述碳源，至所述碳源的浓度为80g/L～120g/L，然后，当所述碳源的浓度降到20g/L～40g/L时，第二次补充所述碳源，至所述碳源的浓度为60g/L～80g/L。
  25. 如权利要求24所述的生产方法，其特征在于，在所述发酵培养步骤中，所述碳源为葡萄糖。
  26. 如权利要求20～25中任一项所述的生产方法，其特征在于，所述的生产方法在所述发酵培养步骤之前还进行种子培养得到活化的种子，然后将所述活化的种子接种到发酵培养基中进行所述发酵培养。
  27. 如权利要求26所述的生产方法，其特征在于，
    所述种子培养步骤包括：将所述的基因工程菌株接种到种子培养基中，在种子培养温度为42℃～55℃的条件下培养12h～16h，得到所述活化的种子。
  28. 如权利要求26所述的生产方法，其特征在于，在所述种子培养步骤中，所述种子培养基包含以下成分：蛋白胨、酵母粉和氯化钠。
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