Genetically modified bacterial strains producing L-alanine, methods for constructing the same, and their use.

Abstract

The present invention discloses a genetically engineered strain for producing L-alanine, and its construction and use, and relates to the field of biotechnology. According to the present invention, a genetically engineered strain capable of producing alanine at a high yield under high temperature conditions of 42°C to 55°C can be constructed by strengthening the glycolysis pathway and / or introducing a thermostable alanine dehydrogenase gene. Furthermore, the optical purity of L-alanine can be significantly increased by knocking out the alanine racemase gene. When the original strain has a lactic acid synthesis pathway, blocking this lactic acid synthesis pathway can increase the proportion of the pyruvic acid synthesis pathway, and the yield of L-alanine can be further increased. The present invention overcomes the problems of fermentation at low temperatures and high costs caused by the use of conventional L-alanine production techniques, and can produce L-alanine at a yield of 95g / L or more by fermentation at a high temperature of 42°C to 55°C, which has high industrial utility.

Description

[Technical Field]

[0001] This invention relates to the field of biotechnology, and more particularly to genetically modified bacterial strains that produce L-alanine, as well as methods for constructing and using the same. [Background technology]

[0002] As one of the 20 basic amino acids and one of the smallest chiral molecules, L-alanine is widely used in food, pharmaceuticals, general chemicals, and other fields. In the food industry, L-alanine can be added to food as a preservative, sweetener, etc. In the fields of pharmaceuticals and healthcare, L-alanine can be used not only in amino acid-based nutritional supplements but also plays an important role in the treatment of acute and chronic renal failure, liver disease, diabetes, obesity, and other diseases. In the field of general chemicals, L-alanine can be used to synthesize amino acid-based surfactants that offer high biocompatibility.

[0003] Methods for producing L-alanine primarily include chemical synthesis, enzymatic catalysis, and fermentation. The current major chemical synthesis approach is the propionic acid chlorination process, using liquid chlorine and propionic acid as the main raw materials. However, due to its lengthy synthesis pathway, high cost, and low yield, the chemical synthesis approach has been virtually abandoned. Enzymatic catalysis of L-alanine essentially involves decarboxylation using an aspartic acid-catalyzed L-aspartic acid-β-decarboxylase. However, the enzymatic catalysis approach is unsuitable for large-scale production due to the low carbon utilization of the 4-carbon substrate in the 3-carbon product and the relatively high price of aspartic acid. L-alanine can also be produced by fermentation using genetically engineered strains, primarily with glucose as the substrate. This method has gained widespread attention due to its advantages such as inexpensive and readily available raw materials, high product yield, and low cost.

[0004] Compared with fermentation at a common temperature of 42°C or lower, high-temperature fermentation can significantly reduce the risk of contamination, improve the conversion efficiency of raw materials, and lower heat exchange costs, thus being highly valuable for large-scale production by fermentation. In the production of alanine by fermentation, genetically engineered Escherichia coli strains are often used as production strains. However, since the optimal growth temperature of these strains is 37°C, they are not suitable for use in high-temperature fermentation.

[0005] Accordingly, those skilled in the art are working towards the development of strains suitable for large-scale production of L-alanine by fermentation under high-temperature conditions. SUMMARY OF THE INVENTION

[0006] In view of the above, the problem to be solved by the present invention is to provide a genetically engineered strain capable of producing L-alanine under high-temperature conditions of 42°C or higher, as well as the construction method and use thereof.

[0007] To achieve the above objective, in a first aspect of the present invention, there is provided a method for constructing a genetically engineered strain that produces L-alanine, the method comprising: a step of providing an original strain having a pyruvate synthesis pathway; a step of manipulating the genome of the original strain via any one, any two or all three of the following steps S200, S300 and S400; S200: a step of inserting a copy of the 6-phosphofructokinase gene pfk and a copy of the pyruvate kinase gene pyk; S300: a step of inserting the gene GSald of alanine dehydrogenase that is thermostable at 42°C to 55°C; S400: a step of inactivating or deleting the alanine racemase gene contained in the genome of the original strain There is provided a method comprising the above.

[0008] The genetically engineered strain producing L-alanine constructed according to the first aspect of the present invention is capable of producing L-alanine by fermentation under high-temperature conditions of 42°C to 55°C.

[0009] In some embodiments of the present invention, the original strain further has a lactic acid synthesis pathway, and the genome of the original strain contains a lactate dehydrogenase gene. In these cases, the present method S100: a step of inactivating or deleting the lactate dehydrogenase gene in the genome of the original strain is further included.

[0010] In a second aspect of the present invention, a genetically engineered strain constructed according to the method of the first aspect of the present invention is provided.

[0011] In a third aspect of the present invention, there is provided use of the genetically engineered strain according to the second aspect of the present invention for producing L-alanine.

[0012] In a fourth aspect of the present invention, there is provided a method for producing L-alanine, which comprises subjecting the genetically engineered strain according to the second aspect of the present invention to fermentation culture, wherein the fermentation culture is at a temperature of 42°C to 55°C.

[0013] In some embodiments of the present invention, the fermentation culture step comprises inoculating an activated seed strain into a fermentation culture medium containing a carbon source, and performing fed-batch fermentation at a fermentation culture temperature of 42°C to 55°C and a stirring speed of 50 rpm to 350 rpm until a predetermined fermentation density is reached.

[0014] Advantages provided by the technology disclosed in the present invention include the following.

[0015] 1. The genetically engineered strain of the present invention is capable of producing L-alanine at a high temperature (for example, 42°C to 55°C) through a fermentation process, which has advantages including low energy consumption and contamination resistance, and effectively reduces production costs.

[0016] 2. Using a thermophilic strain as the starter strain, a genetically modified strain capable of producing L-alanine by fermentation at high temperatures of 42°C to 55°C (especially, 50°C, for example) can be constructed. Furthermore, by enhancing the glycolysis pathway and / or introducing a heat-stable alanine dehydrogenase, a significantly increased alanine yield can be obtained. Knocking out the alanine racemase gene enables the production of optically pure L-alanine. By synergistically combining these strategies, high-yield production of optically pure L-alanine by fermentation at high temperatures of 42°C to 55°C is possible, and the resulting L-alanine typically exhibits an optical purity of 98% or more, and even 99% or more. In some examples, the yield of optically pure L-alanine can be as high as 95 g / L or more, and even 100 g / L or more. This invention overcomes the problems of low-temperature fermentation and high costs associated with the use of conventional L-alanine production technologies, enabling L-alanine production by fermentation at high temperatures, thus having high industrial value.

[0017] 3. In the case of a strain possessing a lactate synthesis pathway, blocking the lactate synthesis pathway can increase the proportion of the pyruvate synthesis pathway and thus increase the yield of L-alanine. If the strain produces both lactate and pyruvate, blocking the lactate synthesis pathway in the strain can increase the proportion of the alanine synthesis pathway. Furthermore, by enhancing the glycolysis pathway and introducing a heat-stable alanine dehydrogenase, genetically modified strains capable of producing alanine in high yield can be constructed. Knockout of the alanine racemase gene enables the production of optically pure L-alanine.

[0018] 4. For example, Bacillus licheniformis, which can be used for fermentation at high temperatures of 50°C, can be used for this purpose. Bacillus licheniformis strain BN11, which can produce D-lactic acid in high yield, can be used as a starting strain, and the D-lactic acid synthesis pathway within it may be eliminated to enhance the glycolysis pathway. Furthermore, a heat-stable heterologous alanine dehydrogenase may be introduced to eliminate the D-alanine synthesis pathway. In this way, high-yield production of optically pure L-alanine can be achieved at high temperatures of 42°C to 55°C. The resulting L-alanine typically exhibits an optical purity of 98% or more, and even 99% or more. In some examples, the yield of optically pure L-alanine can be as high as 95 g / L or more, and even 100 g / L or more.

[0019] Hereinafter, in order to provide a complete understanding of the object, features, and effects of the present invention, the concepts, structural details, and resulting technical effects of this application will be further described with reference to the accompanying drawings.

[0020] Other features, purposes, and advantages of the present invention will become more apparent upon reading the following detailed description of non-limiting examples in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0021] [Figure 1] The alanine synthesis and metabolic pathways in an exemplary genetically modified Bacillus licheniformis strain according to one embodiment of the present invention are schematically shown. [Figure 2] This is a plot diagram (▲: OD600; ■: glucose; ●: alanine) of L-alanine production by feeding-boil fermentation at 50°C in a 5L fermenter using a genetically modified bacterial strain BLA-1 that produces L-alanine according to an embodiment of the present invention. [Figure 3]The structure of a pKVM vector used in several embodiments of the present invention is shown, containing two antibiotic resistance genes: ampR (ampicillin resistance) and ermC (erythromycin resistance), as well as the bgaB gene encoding galactosidase. Any of these genes can be used as selection markers. This plasmid has a heat-sensitive origin of replication (oriT pE194ts), as well as an origin of replication in Escherichia coli (ori) and an origin of replication in Bacillus licheniformis (oriT). [Modes for carrying out the invention]

[0022] Hereinafter, specific embodiments of the present invention will be described in detail with reference to the attached drawings.

[0023] Hereinafter, several preferred embodiments and examples of the present invention will be described with reference to the drawings accompanying this specification, so that the art may become clearer and easier to understand. The present invention may be embodied and illustrated in many different forms, and its scope is not limited to the embodiments and examples disclosed herein.

[0024] It will be understood that the features described above and in detail in the following description (including, but not limited to, embodiments and examples) can be combined in any suitable way to create new embodiments, as long as the present invention can be carried out.

[0025] Where used herein, terms such as “preferred,” “specific,” “concrete,” “furthermore,” and “also” are used simply to describe embodiments or examples that have a good effect or a certain degree of specificity. It will be understood that they are not in any way intended to limit the scope of the invention.

[0026] As used herein, both "and / or" and "or / and" mean that the listed items are substitutes, but the substitutes also include any combination of the listed items.

[0027] As used herein, “their combination,” “any combination,” “any combination,” and “in any combination” all mean that the enumerated items can be combined in any suitable way, insofar as the present invention can be carried out. The number of items that can be combined is two or more.

[0028] Where used herein, references to concentrations in percentages should be understood to refer to the final concentration in the system unless otherwise defined or specified.

[0029] When used herein, any numerical range listed is intended to include both a lower and upper limit unless otherwise specified.

[0030] Where used herein, “greater than or equal to” or “less than or equal to” following a number is intended to refer to a range that includes that specific number, unless otherwise defined.

[0031] The features of the present invention described above and below can be combined in any suitable manner, as long as they are consistent and the resulting embodiments can be carried out to solve the problems that the present invention seeks to solve. These features can be combined in any preferred manner, as long as the resulting embodiments can solve the problems that the present invention seeks to solve and produce the expected effects.

[0032] As used herein, "starting strain" and "original strain" have the same meaning and can be used interchangeably.

[0033] Where used herein, references to temperature control should be understood to mean that the temperature is kept constant or controlled to vary within a certain range.

[0034] As used herein, “high temperature” is relative to a general temperature (below 42°C, typically below 37°C) when used to describe the production of L-alanine. As used herein, unless otherwise defined, “high temperature production” is intended to refer to production by fermentation at 42°C to 55°C, for example, but 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, or 55°C.

[0035] As used herein, unless otherwise specified, “pyruvate synthesis pathway” refers to the pathway by which pyruvate is synthesized from a carbon source via glycolysis. As used herein, “having a pyruvate synthesis pathway” means “having the ability to synthesize pyruvate endogenously.”

[0036] As used herein, unless otherwise specified, “lactic acid synthesis pathway” refers to the synthesis pathway that converts pyruvate to lactate. As used herein, unless otherwise specified, “having a lactic acid synthesis pathway” means “having the ability to produce lactate via the lactic acid synthesis pathway.” In other words, “having a lactic acid synthesis pathway” means “having the ability to synthesize lactate endogenously.”

[0037] As used herein, unless otherwise specified, “alanine synthesis pathway” refers to the synthesis pathway that converts pyruvate to alanine. As used herein, unless otherwise specified, “having an alanine synthesis pathway” means “having the ability to produce alanine via the alanine synthesis pathway,” that is, “having the ability to synthesize alanine endogenously.” As used herein, when it is stated that an organism has an alanine synthesis pathway, unless otherwise specified, it is intended to mean that it has both the pathway for synthesizing L-alanine from pyruvate and the pathway for synthesizing D-alanine from L-alanine.

[0038] As used herein, unless otherwise specified, “D-alanine synthesis pathway” refers to the pathway for synthesizing D-alanine from L-alanine, and “having a D-alanine synthesis pathway” means “having the ability to synthesize D-alanine endogenously.”

[0039] As used herein, a gene cluster fragment refers to a fragment containing at least two target genes, which may be identical or different. When describing a gene cluster fragment composed of identical genes, the term “repetition” shall be interpreted broadly to mean that the genes may be directly adjacent to each other or that a spacer sequence may be present between any two of the genes.

[0040] According to the present invention, one typical approach to deleting a target gene is to knock it out.

[0041] As used herein, unless otherwise specified, “inactivation” and “deletion” may refer to partial or complete inactivation and partial or complete deletion, respectively.

[0042] [Table 1]

[0043] [Table 2]

[0044] Table 3 summarizes the amino acid and nucleotide sequences referred to herein.

[0045] [Table 3-1]

[0046] [Table 3-2]

[0047] Sequence ID 1 (GSald)

[0048] Sequence ID 2 (Pals promoter) AAGGTGACGCCTATTTCACTTTCTAGCTGTTTAATCTGCTGGCTGAGCGGAGGCTGAGTCATGTTCAGCCGAAGAGCTGCTTTTCCGAAATGCAGTTCTTCGGCAACAACCATAAAATAACGAAGATGGCGCAGCTCCATTAATCACTCA TTCCTTTCTGAATGCGATTTCAGTCGTTTTACATATTAATTGTAAGACAAAGAAGTATTGGAAAACAATTTCCACAAGATGTATATTTAATAATACAATAATTTTATTAAAAATTCATTGTAAATGAATGAAAATGGAGGAGTGAGGGCT

[0049] Sequence ID 3: CCTCGCGTCGGGCGATATCGGATCCGAAGGGGAAAGTCTTCGATTTCT Sequence ID 4: AGAGGGCTTTTTCATGCTGAAGAGGTCAAAAAGAGCC Sequence ID 5: TTTGACCTCTTCAGCATGAAAAAGCCCTCTTTGAAAAG Sequence ID 6: CCATGGTACCCGGGAGCTCGAATTCCATAAGACCGCTGATGACAAGC Sequence ID 7: TCCAGCCTCGCGTCGGGCGATATCGTCCCCATAACAACGGAATCATC Sequence ID 8: AATAGGCGTCACCTTGACTCATCATTCCTTTGCCGTT Sequence ID 9: AAGGAATGATGAGTCAAGGTGACGCCTATTTCACTTTC Sequence ID 10: TCCAATTCGCTTCATAGCCCTCACTCCTCCATTTTC Sequence ID 11: GGAGGAGTGAGGGCTATGAAGCGAATTGGAGTATTGACA Sequence ID 12: CTTCATGGTGTTCAGTTACAATACAGTCGCATGGCC Sequence ID 13: GCGACTGTATTGTAACTGAACACCATGAAGATACTAACATCA Sequence ID 14: ACTAGACAGATCTATCGATGCATGCTTTCCCTTATTCCTTTAAACCCG Sequence ID 15: AATAGGCGTCACCTTGCTGAAGAGGTCAAAAAGAGCC Sequence ID 16: TTTGACCTCTTCAGCAAGGTGACGCCTATTTCACTTTCT Sequence ID 17: AGAGGGCTTTTTCATTTAGCCATGCAGCAGGCTATG Sequence ID 18: CTGCTGCATGGCTAAATGAAAAAGCCCTCTTTGAAAAG Sequence ID 19: CCTCGCGTCGGGCGATATCGGATCCAAAATATGACGCTGTCTCAAATTGA Sequence ID 20: CTAATTCATCAATTTGACACTTCCTGTTCCTTGTTTCACT Sequence ID 21: GGAACAGGAAGTGTCAAATTGATGAATTAGCGGAAAAAC Sequence ID 22: CCATGGTACCCGGGAGCTCGAATTCCGGAGTCTCTTTCAAAACCGTAG Sequence ID 23: CCTCGCGTCGGGCGATATCGGATCCAAAATCATGTAAGCCCATTCCG Sequence ID 24: GTGAGTATGGGAAAACAACGCTCCCTTCTTTCTTGTC Sequence ID 25: AAGAAGGGAGCGTTGTTTTCCCATACTCACAGGCCG Sequence ID 26: CCATGGTACCCGGGAGCTCGAATTCTAAAATGAAGGTGGTCCGGGAT

[0050] Sequence ID 27 (Pfk) ATGAAGCGAATTGGAGTATTGACAAGCGGCGGCGATGCACCGGGGATGAATGCGGCGGTCCGCGCGATTGCCCGTAAAGGGATTTATCACGGCCTGGAAGTTTACGGCATTCGCCAAGGTTATAACGGATTGATTCAAGGAAACATCCAAAAGCTCGAAGCAGGATCTGTTGGCGATATTCTCCAGCGGGGCGGCACGGTTTTGCAGTCGGCAAGAAGCGAAGAATTCAAAACGCCGGAAGGGCAGCAAAAAGCGATCAGGCAGCTGAAAGACCATGGCATTGAAGCGCTCGTTGTGATCGGCGGCGACGGTTCCTACCAAGGGGCCAAAAAGTTGACGGAACAGGGCTTTAACTGCATTGGTGTGCCAGGGACAATCGATAACGACATCCCGGGGACGGATTTTACAATCGGTTTTGATACGGCATTGAACACAGTGCTTGATGCGATTGATAAAATTCGCGACACCGCTTCTTCCCACGAACGCACCTTTATTATTGAAGTCATGGGCAGAAATGCCGGGGATATCGCGCTCTGGTCCGGCCTGGCCGGCGGAGCCGAATCGATTATTATTCCGGAAGAAAAATATGACTTAAAAGATGTCGTGGAGCGTCTTGAACAGGGGAGAAAACGCGGCAAACGCCACAGCATCATCATTGTCGCGGAAGGCGTGATGAGCGGCAACGAGTTTGCTGAACAATTGAAAAAAACCGGTGTGATCGGCGATACCCGCGTTTCTGTTCTCGGCCATATCCAGCGCGGCGGTTCTCCGACGGCATTTGACCGCGTGCTTGCAAGCCGCCTCGGCGCAAGGGCTGTTGAACTGCTGCTTGAAGGAAAAGGGGGCCGCGCTGTCGGCATTCAAAATAACCAGCTGGTTGACCACGATATCCTTGAGATTCTCGGAAAACCGCACGCCGTTAATAAAAACATGTACAAGCTGTCGAAAGAATTGTCGATCTAA

[0051] Sequence ID 28 (Pyk)

[0052] Sequence ID 29 (Alr1)

[0053] Sequence ID 30 (Alr2)

[0054] Sequence ID 31 (No.) ATGAAAGTAATTTTTTTTTCTATGCACCCGTATGAAGAGGAATTTCTGGGTCCGATTCTGCCGTCTGACTGGGACGTAGAAATGACCCCGGACTTTCTGGACGAAACCACCGTGGAAAAGGCTAAAGGTGCCCAGGTAGTAAGCCTGTTTGTTTCTGACAAAGCTGATGGTCCGGTACTGGAAGCGCTGCATTCTTACGGTGTGGGCCTGCTGGCCCTGCGTTCTGCTGGCTATGATCACATCGATATTGAGACCGCAAAACGCCTGGGTATCAAAGTAGTTAACGTGCCAGCCTATTCTCCGCACGCTATCGCTGACCATACTCTGGCTATCATGCTGGCTCTGATTCGTCGTCTGCACCGTGCCCATGATAAAGTGCGCCTGGGTGATTTTGATCTGGATGGTCTGATGGGCTTTGATCTGAACGGCAAAGTTGCTGGTGTAATTGGTCTGGGTAAAATCGGTCGCCTGGTAGCTACCCGCCTGAAAGCGTTTGGTTGCAAAGTTCTGGGCTATGATCCATACATTCAGCCGGAAATCGTAGAAAACGTTGATCTGGATACCCTGATCACTCAGGCTGATATCATTTCTATTCATTGTCCGCTGACCCGTGAAAACTTTCATATGTTTAACGAAGAGACTTTTAAGCGTATGAAACCGGGTGCTATTCTGGTTAACACCGCGCGTGGTGGTCTGATCGATACCAAGGCCCTGCTGGAGGCCCTGAAGTCTGGTAAACTGGGCGGCGCAGCCCTGGATGTGTATGAATATGAACGTGGCCTGTTTTTTAAAAACCACCAAAAAGAAGGTATCAAAGACCCGTATCTGGCCCAGCTGCTGGGTCTGGCCAACGTAGTGCTGACCGGTCATCAGGCCTTTCTGACCCGTGAGGCTGTAAAAAACATCGAAGAAACTACCGTAGAAAACATTCTGGAATGGCAAAAGAACCCGCAGGCAAAGCTGAAAAACGAAATCTAA

[0055] Sequence ID 32 (GSald) MKIGIPKEIKNNENRVAITPAGVMTLVKAGHDVYVETEAGAGSGFSDSEYEKAGAVIVPNAEDAWTAEMVLKVKEPLAEEFRYFRPGLILFTYLHLAAAEALTKALVEQKVVGIAYETVQLANGSLPLLTPMSEVAGRMSVQVGAQFLEKPHGGKGILLGGVPGVRRGKVTIIGGGTAGTNAAKIA VGLGSDVTILDINAERLRELDDLFGDHVTTLMSNSYHIAECVRESDLVVGAVLIPGAKAPKLVTEEMVRSMTPGSVLVDIAIDQGGIFETTDRVTTHDDPTYVKHGVVHYAVANMPGAVRTSTFALTNVTIPYALQIANKGYRAACLDNPALLKGINTLDGHIVYEAVAAAHNMPYTDVHSLLHG

[0056] A first aspect of the present invention relates to a method for constructing a genetically modified bacterial strain that produces L-alanine, A step of preparing a progenitor strain that possesses the pyruvate synthesis pathway; A step of manipulating the genome of the progenitor strain by one, two, or three of the following steps S200, S300, and S400; A step of introducing overexpression copies of the pfk gene encoding S200:6-phosphofructokinase and the pyk gene encoding pyruvate kinase; S300: Step of introducing an overexpression gene encoding a thermostable alanine dehydrogenase (preferably the overexpression gene GSald encoding a thermostable alanine dehydrogenase); S400: If the genome of the bacterial strain contains the alanine racemase gene, a step to inactivate or delete the alanine racemase gene. A method including this is provided.

[0057] In some embodiments of the present invention, S200, S300, and S400 are S200: Insertion of a copy of the phosphofructokinase gene pfk and a copy of the pyruvate kinase gene pyk; S300: Insert the overexpression gene GSald, which encodes alanine dehydrogenase that is heat-resistant at temperatures between 42°C and 55°C. S400: For example, partially or completely inactivating the alanine racemase gene by knocking it out, or partially or completely deleting the alanine racemase gene. These can be achieved separately.

[0058] A genetically modified strain for producing L-alanine, constructed according to the first aspect of the present invention, can produce L-alanine by fermentation under high-temperature conditions of 42°C or higher (e.g., 42°C to 55°C). Step S200 enhances the glycolysis pathway by enabling the overexpression of 6-phosphofructokinase and pyruvate kinase involved in the pathway, thereby increasing the supply of pyruvate and promoting L-alanine production. In step S300, a gene encoding a thermostable alanine dehydrogenase is introduced to strengthen the pathway for synthesizing L-alanine from pyruvate, effectively increasing the L-alanine yield. In step S400, the alanine racemase gene can be inactivated or deleted to promote the production of optically pure L-alanine. The L-alanine yield can be increased by using any one of steps S200, S300, or S400, and a synergistic effect can be obtained by using two or three of them in combination.

[0059] In some embodiments of the present invention, the original strain further possesses a lactate synthesis pathway, and the genome of the original strain contains a lactate dehydrogenase gene. In these cases, the present method is S100: Further comprises the step of inactivating or deleting the lactate dehydrogenase gene in the genome of the intrinsic bacterial strain.

[0060] Figure 1 schematically illustrates the alanine synthesis and metabolic pathways in genetically modified bacterial strains producing L-alanine according to several embodiments of the present invention. The carbon source is converted to pyruvate via the glycolysis pathway, and pyruvate then reacts with NADH and ammonium ions under the catalytic action of alanine dehydrogenase, consuming them to form L-alanine. A portion of the resulting L-alanine is secreted directly extracellularly, while the remainder is converted to D-alanine under the catalytic action of alanine racemase and then secreted extracellularly. If the carbon flow downstream of pyruvate is dominated by the lactate synthesis pathway in the original strain, blocking the lactate synthesis pathway can direct the carbon flow to the alanine synthesis pathway as much as possible. Furthermore, overexpression of 6-phosphofructokinase and pyruvate kinase, which are involved in the glycolysis pathway, can enhance the glycolysis pathway, increasing the supply of pyruvate and thereby promoting alanine production. In addition, knocking out the alanine racemase gene can block the innate D-alanine synthesis pathway, yielding optically pure L-alanine.

[0061] In some embodiments of the present invention, the original strain further possesses a D-lactate synthesis pathway, and its genome encodes ldh Ti It contains genes. In these cases, this method involves the ldh encoding D-lactate dehydrogenase in the genome of the inoculum strain. Ti The process further includes a step (S500) to inactivate or delete a gene. In some preferred embodiments of the present invention, step S500 involves deactivating the ldh encoding D-lactate dehydrogenase in the genome of the intrinsic strain. Ti This includes knocking out genes.

[0062] In some embodiments of the present invention, ldh encodes D-lactate dehydrogenase. Ti The gene has the sequence shown in Sequence ID No. 31.

[0063] In some embodiments of the present invention, the pfk gene encoding 6-phosphofructokinase has the sequence shown in SEQ ID NO: 27, and the pyk gene encoding pyruvate kinase has the sequence shown in SEQ ID NO: 28.

[0064] In some embodiments of the present invention, the alanine dehydrogenase gene has the sequence shown in Sequence ID No. 1.

[0065] In some embodiments of the present invention, one, two, or more types of alanine racemase genes are inactivated or deleted in step S400.

[0066] In some embodiments of the present invention, the target gene is deleted by knockout.

[0067] In some embodiments of the present invention, step S400 includes inactivating or deleting the alfr1 or alfr2 gene encoding alanine racemase. In some embodiments of the present invention, step S400 includes inactivating or deleting the alfr1 and alfr2 genes encoding alanine racemase. In some preferred embodiments of the present invention, step S400 includes knocking out the alfr1 and alfr2 genes encoding alanine racemase.

[0068] In some embodiments of the present invention, the alur1 gene encoding alanine racemase has the sequence shown in SEQ ID NO: 29, and the alur2 gene encoding alanine racemase has the sequence shown in SEQ ID NO: 30.

[0069] In some embodiments of the present invention, the manipulation of the genome of the intrinsic strain includes steps S100 and S200. In this way, the lactate synthesis pathway can be blocked and eliminated by inactivating or deleting the lactate dehydrogenase gene, and the glycolysis pathway can be enhanced by overexpression of 6-phosphofructokinase by the pfk gene and overexpression of pyruvate kinase by the pyk gene. Preferably, the manipulation of the genome of the intrinsic strain further includes step S300. In this way, the yield of L-alanine can be further increased by inserting a gene encoding thermostable alanine dehydrogenase.

[0070] In some embodiments of the present invention, the manipulation of the genome of the intrinsic strain comprises steps S100, S200, S300, and S400. In this manner, the lactate synthesis pathway is blocked by inactivating or deleting the lactate dehydrogenase gene, the glycolysis pathway is enhanced, and pyruvate supply is increased by introducing genes that overexpress 6-phosphofructokinase and pyruvate kinase into the intrinsic strain. In addition, the introduced overexpressed alanine dehydrogenase gene can enhance the L-alanine synthesis pathway from pyruvate, and the alanine racemase gene of the intrinsic strain is inactivated or deleted. As a result of these synergistic strategies, high yield production of optically pure L-alanine can be achieved.

[0071] In some embodiments of the present invention, the manipulation of the genome of the progenitor strain includes steps S500 and S200. Thus, the lactate synthesis pathway is modified to encode D-lactate dehydrogenase ldh Ti The glycolytic pathway is blocked by inactivating or deleting genes, and is enhanced by overexpressing the pfk and pyk genes, which encode 6-phosphofructokinase and pyruvate kinase, respectively.

[0072] In a preferred example of the present invention, the method includes the following steps:

[0073] S500: Lactate dehydrogenase gene in the genome of the progenitor strain, preferably ldh encoding D-lactate dehydrogenase Ti A process of completely inactivating or deleting a gene (the thereby blocking the lactate synthesis pathway);

[0074] A process of overexpressing a copy of the pfk gene encoding S200:6-phosphofructokinase and a copy of the pyk gene encoding pyruvate kinase (thereby enhancing the glycolytic pathway and increasing the supply of pyruvate);

[0075] S300: A step of overexpressing a heterologous GSald gene encoding alanine dehydrogenase (thereby enhancing the synthesis pathway from pyruvate to L-alanine); and

[0076] S400: A step to completely inactivate or delete the alur1 and alur2 genes that encode alanine racemase (thereby blocking the conversion of L-alanine to D-alanine and promoting high-yield production of optically pure L-alanine).

[0077] In a preferred example of the present invention, the method includes the following steps:

[0078] S500: Lactate dehydrogenase gene in the genome of the progenitor strain, preferably ldh encoding D-lactate dehydrogenase Ti A process of knocking out a gene (thereby blocking the lactate synthesis pathway), more preferably LDH Ti The gene has the sequence shown in Sequence ID No. 31;

[0079] S200: A step of inserting a copy of the pfk gene encoding 6-phosphofructokinase and a copy of the pyk gene encoding pyruvate kinase (thereby enabling overexpression of the pfk gene encoding 6-phosphofructokinase and the pyk gene encoding pyruvate kinase in the intrinsic strain, thereby enhancing the glycolysis pathway and increasing the supply of pyruvate), wherein more preferably, the pyk gene encoding pyruvate kinase has the sequence shown in SEQ ID NO: 27, and the pyk gene encoding pyruvate kinase has the sequence shown in SEQ ID NO: 28;

[0080] S300: A step of inserting a heterologous GSald gene encoding alanine dehydrogenase (thereby enabling overexpression of the heterologous GSald gene encoding alanine dehydrogenase and enhancing the synthesis pathway from pyruvate to L-alanine), wherein the GSald gene encoding alanine dehydrogenase has the sequence shown in SEQ ID NO: 1; and

[0081] S400: A step of knocking out the alur1 and alur2 genes encoding alanine racemase (thereby blocking the conversion of L-alanine to D-alanine and promoting the production of optically pure L-alanine), wherein the alur1 gene encoding alanine racemase has the sequence shown in SEQ ID NO: 29, and the alur2 gene encoding alanine racemase has the sequence shown in SEQ ID NO: 30.

[0082] Unless otherwise specified, the features of the embodiments of the present invention are preferably independent, but it should be understood that they can be combined as needed.

[0083] In some embodiments of the present invention, in steps S200 and S300, each of the target genes is inserted by adding a copy (one or more) of the gene (i.e., one or more copies) to a chromosome and ligating a promoter in series upstream thereof.

[0084] In some embodiments of the present invention, the promoter is P als , any one of Plac, Ptrc, Ptac and Pc P43.

[0085] In some embodiments of the present invention, the promoter is P als .

[0086] In some embodiments of the present invention, the promoter has the sequence set forth in SEQ ID NO: 2.

[0087] In some embodiments of the present invention, the original strain is a thermophilic strain.

[0088] In some preferred embodiments of the present invention, the original strain is a strain of the genus Bacillus.

[0089] In some preferred embodiments of the present invention, the original strain is Bacillus licheniformis, which is a facultative anaerobic Gram-positive endospore-forming bacterium with fast growth rate, wide substrate spectrum, and high-temperature fermentation capacity under 50°C conditions. Furthermore, it enables stable genetic manipulation and is recognized as a "Generally Recognized As Safe" (GRAS) bacterium by the U.S. Food and Drug Administration. Therefore, it is promising to provide an ideal platform strain.

[0090] In some preferred embodiments of the present invention, the original strain is Bacillus licheniformis, Bacillus coagulans, Bacillus methylotrophicus, thermophilic Bacillus inulinus or Geobacillus stearothermophilus.

[0091] In some preferred embodiments of the present invention, the original strain is Bacillus licheniformis ATCC14580 or a derivative strain thereof. Examples of the derivative strain include, but are not limited to, Bacillus licheniformis MW3, Bacillus licheniformis BN11, and the like.

[0092] In some preferred embodiments of the present invention, the original strain is Bacillus licheniformis BN11, deposited with the China Center for Typical Cultures on January 8, 2016, under CCTCC number M2016026.

[0093] In some preferred embodiments of the present invention, the progenitor strain is Bacillus licheniformis BN11, the pfk gene encoding 6-phosphofructokinase has the sequence shown in SEQ ID NO: 27, and the pyk gene encoding pyruvate kinase has the sequence shown in SEQ ID NO: 28.

[0094] In some preferred embodiments of the present invention, the original strain is Bacillus licheniformis BN11, and the GSald gene encoding alanine dehydrogenase has the sequence shown in Sequence ID No. 1.

[0095] In some preferred embodiments of the present invention, the progenitor strain is Bacillus licheniformis BN11, and the endogenous alanine racemase genes include the alfr1 gene and the alfr2 gene, having sequences shown in SEQ ID NO: 29 and SEQ ID NO: 30, respectively.

[0096] In a second aspect of the present invention, a genetically modified strain constructed according to the first aspect of the present invention is provided. The genetically modified strain can be produced by fermentation at 42°C or higher (e.g., 42°C to 55°C) with a significantly increased yield of L-alanine. High yield production of optically pure L-alanine is also possible.

[0097] A third aspect of the present invention provides the use of a genetically modified strain according to the second aspect of the present invention for producing L-alanine. It can be used for the production of L-alanine by fermentation with a significantly increased yield under high-temperature conditions. It can also be used for the high-yield production of optically pure L-alanine under high-temperature conditions.

[0098] A fourth aspect of the present invention provides a method for producing L-alanine by subjecting a genetically modified bacterial strain according to the second aspect of the present invention to fermentation culture at a temperature of 42°C to 55°C. Examples of 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, and 55°C.

[0099] In some embodiments of the present invention, the fermentation culture step includes sowing activated inoculum into a fermentation culture medium containing a carbon source; and carrying out fed-batch fermentation under conditions of a fermentation culture temperature of 42°C to 55°C and a stirring speed of 50 rpm to 350 rpm until a predetermined fermentation density is achieved. Furthermore, the volume of the sowing material of activated inoculum may be 3% to 5% by volume, for example, 3%, 4%, or 5%.

[0100] In some embodiments of the present invention, the method further comprises, prior to fermentation culture, a seed culture to obtain activated seed cells, which are then seeded into a fermentation culture medium and fermented.

[0101] In some embodiments of the present invention, the inoculum culture step includes seeding a genetically modified strain into an inoculum culture medium and culturing it for 12 to 16 hours at an inoculum culture temperature of 42°C to 55°C to obtain an inoculum culture solution, i.e., activated inoculum.

[0102] In some embodiments of the present invention, the culture medium contains the following components: peptone, yeast powder, and sodium chloride.

[0103] In some embodiments of the present invention, the culture medium for the inoculum consists of the following components: peptone, yeast powder, and sodium chloride.

[0104] In some embodiments of the present invention, the method includes the following steps:

[0105] Spawn culture: A process of obtaining activated spawn by seeding a genetically modified strain onto a spawn culture medium and culturing it under spawn culture temperature conditions of 42°C to 55°C; and

[0106] Fermentation culture: A process in which activated inoculum is seeded into a fermentation culture medium containing a carbon source, and fed-batch fermentation is carried out under fermentation culture temperature conditions of 42°C to 55°C until a predetermined fermentation density is achieved, where the volume of seeded material may be 3% to 5% by volume percentage.

[0107] In some embodiments of the present invention, the method includes the following steps:

[0108] Spawn culture: A process of obtaining activated spawn by seeding a genetically modified strain onto a spawn culture medium and culturing it for 12 to 16 hours under spawn culture temperature conditions of 42°C to 55°C; and

[0109] Fermentation culture: This process involves inoculating a fermentation culture medium containing a carbon source with activated inoculum, and carrying out fed-boil fermentation under conditions of a fermentation culture temperature of 42°C to 55°C and a stirring speed of 50 rpm to 350 rpm until a predetermined fermentation density is achieved. Here, the volume of the inoculated material may be 3% to 5% by volume.

[0110] In some embodiments of the present invention, the carbon source in the fermentation culture step is glucose, glycerol, xylose, or arabinose.

[0111] In some embodiments of the present invention, in the fermentation culture step, the fermentation culture medium contains the following components: glucose, yeast powder, ammonium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, ferrous sulfate, and manganese sulfate. In some embodiments of the present invention, the fermentation culture medium consists of the following components: glucose, yeast powder, ammonium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, ferrous sulfate, and manganese sulfate.

[0112] In some embodiments of the present invention, the achievement of a predetermined fermentation density is determined by the detected OD. 600 It is determined based on the value. For example, OD 6.5 to 8.0 600 When detected, fermentation is stopped.

[0113] In some embodiments of the present invention, the fermentation culture step involves adjusting the pH to about 7.0 using aqueous ammonia; OD 600 The process includes fermenting and culturing under aeration and stirring until the carbon content reaches a value of 6.5 to 8.0 (first fermentation and culturing stage), after which aeration is stopped; and continuing the fermentation and culturing at a reduced stirring rate until a predetermined fermentation density is achieved (second fermentation and culturing stage), after which fermentation is stopped. During the fermentation process, the carbon source may be replenished when its concentration falls to a specific value.

[0114] Ammonia solution for pH adjustment may have a concentration of 20% to 30% by volume (v / v), such as 20%, 25%, or 30%.

[0115] Preferably, ventilation is achieved with air.

[0116] Aeration may be performed at a rate of 0.8vvm to 1.2vvm, for example, 0.9vvm, 1.0vvm, 1.1vvm, 1.2vvm, etc.

[0117] Fermentation culture can be divided into several stages. For example, it may be divided into two stages: a first fermentation culture stage with aeration and a second fermentation culture stage without aeration. The first fermentation culture stage enables rapid proliferation of the strain, and the second fermentation culture stage enables efficient L-alanine production. In the first fermentation culture stage, it is preferable to stir at a speed of 280 rpm to 320 rpm, for example, 280 rpm, 290 rpm, 300 rpm, 310 rpm, 320 rpm. The first fermentation culture stage is OD 600 The period of time depends on the value, for example, 6 to 8 hours, for example, 6 hours, 6.5 hours, 7 hours, 7.5 hours, or 8 hours. In the second fermentation culture stage, it is preferable to stir at a speed of 60 rpm to 100 rpm, for example, 60 rpm, 70 rpm, 80 rpm, 90 rpm, 100 rpm. The second fermentation culture stage is OD 600 The duration also depends on the value (e.g., 1.2), for example, 50 to 70 hours, or lasting 50, 55, 60, 65, or 70 hours.

[0118] During the fermentation process, the carbon source may be replenished once, twice, or more times.

[0119] Regarding the timing of carbon source replenishment during the fermentation process, for example, it may be replenished when the concentration drops to 30 g / L to 50 g / L, and then again when the concentration drops to 20 g / L to 40 g / L. Preferably, the carbon source is glucose.

[0120] In some embodiments of the present invention, the carbon source is replenished only once. In these cases, the carbon source may be replenished when the concentration drops to 30 g / L to 50 g / L (e.g., 30 g / L, 35 g / L, 40 g / L, 45 g / L, or 50 g / L), thereby increasing the concentration to 80 g / L to 120 g / L (e.g., 80 g / L, 90 g / L, 100 g / L, 110 g / L, or 120 g / L). Preferably, the carbon source is glucose.

[0121] In some embodiments of the present invention, the carbon source is replenished twice: the first replenishment may be performed when the concentration has decreased to 30 g / L to 50 g / L (e.g., 30 g / L, 35 g / L, 40 g / L, 45 g / L, or 50 g / L), resulting in a new concentration of 80 g / L to 120 g / L (e.g., 80 g / L, 90 g / L, 100 g / L, 110 g / L, or 120 g / L); the second replenishment may be performed when the concentration has decreased to 20 g / L to 40 g / L (e.g., 20 g / L, 25 g / L, 30 g / L, 35 g / L, or 40 g / L), returning the concentration to 60 g / L to 80 g / L (e.g., 60 g / L, 65 g / L, 70 g / L, 75 g / L, or 80 g / L). Preferably, the carbon source is glucose.

[0122] In some embodiments of the present invention, the fermentation culture step involves adjusting the pH to 7.0 ± 0.2 with 20% to 30% (v / v) aqueous ammonia; stirring for 6 to 8 hours with an aeration rate of 0.8 vvm to 1.2 vvm and an initial speed of 280 rpm to 320 rpm to an OD of 6.5 to 8.0. 600The process includes fermenting and culturing until a value is achieved, followed by stopping aeration; and continuing the fermentation and culturing for 50 to 70 hours (e.g., 60 hours) at a modified stirring speed of 60 to 100 rpm, followed by stopping the fermentation. Furthermore, during the fermentation process, the carbon source may be replenished once, twice, or more times when its concentration falls to a specific value. In some embodiments, the carbon source is replenished only once. In these cases, the carbon source may be replenished when the concentration falls to 30 g / L to 50 g / L, increasing the concentration to 80 g / L to 120 g / L. In some other embodiments, the carbon source is replenished twice. In these cases, the carbon source is replenished for the first time when its concentration falls to 30 g / L to 50 g / L, resulting in a concentration returning to 80 g / L to 120 g / L. Next, when the concentration further decreases to 20 g / L to 40 g / L, the carbon source is replenished a second time to return the concentration to 60 g / L to 80 g / L. Preferably, the carbon source is glucose.

[0123] In some embodiments of the present invention, the carbon source in the fermentation culture step is glucose, and the fermentation culture step involves adjusting the pH to 7.0 and maintaining the pH at 7.0 using 25% aqueous ammonia; aeration at 1 vvm and stirring at an initial speed of 300 rpm for 6 to 8 hours to OD of Bacillus licheniformis strain. 600 This involves fermenting the culture until the value reaches 6.5-8.0, followed by stopping aeration; and continuing the fermentation culture for 60 hours at a modified stirring speed of 80 rpm, followed by stopping fermentation. During the fermentation process, glucose is replenished when its concentration falls to 30 g / L-50 g / L, thereby increasing the glucose concentration to 100 g / L. If the glucose concentration subsequently falls to 20 g / L-40 g / L, glucose is replenished again to increase the glucose concentration to 70 g / L.

[0124] The present invention is described in more detail below by several examples intended to illustrate, rather than limit, the scope of the invention in any sense. These examples are provided in accordance with the teachings of the invention. While specific details of implementation and operation are described, the scope of the invention is by no means limited to the following examples. Any experimental procedure used in the following examples (e.g., PCR amplification, transformation, gene knockout, gene insertion, etc.) is preferably carried out in the manner and conditions described above unless specific conditions are specified. Otherwise, it is generally carried out in the conventional manner and conditions, such as taught, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions suggested by the manufacturer. [Examples]

[0125] All materials and reagents used in the following examples are available from commercial sources unless otherwise specified.

[0126] The alanine concentration in each fermentation broth sample was measured by high-performance liquid chromatography (HPLC). 0.54 g of sodium 1-octanesulfonate was weighed and added to 800 mL of ultrapure water, and the pH was adjusted to 2.1 with phosphoric acid. Then, 100 mL of methanol and ultrapure water were continuously added as the mobile phase to obtain a volume of 1 L. The sample was injected at a temperature of 30°C into a ZORBAX Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 μm) in a volume of 10 μL, flowed through the column at a flow rate of 0.8 mL / min, and measured with a DAD detector at a wavelength of 210 nm. Before measurement, the sample was boiled for 10 minutes and centrifuged at 8000 rpm for 5 minutes. Then, the supernatant was collected and diluted to an appropriate level so that alanine was present at a final concentration of 0.2 g / L to 1 g / L.

[0127] The optical purity of alanine in each fermentation broth sample was measured by HPLC using a 2 mM copper sulfate aqueous solution as the mobile phase. The sample was injected at 30°C in a SUMICHIRAL OA-5000 column (150 mm × 4.6 mm, 5 μm) in a volume of 10 μL, flowed through the column at a flow rate of 0.5 mL / min, and measured at a wavelength of 254 nm using a DAD detector. Prior to measurement, the sample was boiled for 10 minutes and centrifuged at 8000 rpm for 5 minutes. The supernatant was then collected and diluted to an appropriate concentration so that alanine was present at a final concentration of 0.2 g / L to 1 g / L.

[0128] Glucose concentration was measured using an SBA-40D biosensor analyzer (Biology Institute of Shandong Academy of Sciences).

[0129] In the following examples, Bacillus licheniformis strains were used as the starting strain to construct genetically modified bacterial strains capable of producing L-alanine under high-temperature conditions. In the following examples, it will be understood that other bacterial strains (e.g., Bacillus coagulans, Bacillus methylotropicus, thermophilic Bacillus inulinus, Geobacillus stearothermophilus, etc., but not limited to these) can also be used as starting strains to construct genetically modified bacterial strains capable of producing L-alanine under high-temperature conditions, without departing from the scope of the present invention.

[0130] Figure 3 shows the structure of a pKVM vector containing two antibiotic resistance genes: ampR (ampicillin resistance) and ermC (erythromycin gene), as well as the bgaB gene encoding galactosidase. All of these genes can be used as selection markers. This plasmid possesses a heat-sensitive origin of replication (oriT pE194ts), as well as origins of replication in *Escherichia coli* (ori) and *Bacillus licheniformis* (oriT).

[0131] Bacillus licheniformis BN11 is a lactic acid-producing strain constructed from ATCC 14580. It was deposited with the China Center for Typical Cultures on January 8, 2016, under the CCTCC number M2016026.

[0132] The volume of seeding material is measured as a volume percentage.

[0133] Unless otherwise specified, one copy of each newly inserted gene was used.

[0134] Example 1: Construction of a Bacillus licheniformis strain in which the D-lactic acid synthesis pathway is knocked out. 1.1 Primers were designed according to the sequence of the Bacillus licheniformis ATCC14580 genome (GenBank number NC_006270.3). Separately, PCR amplification was performed using pKVM-XPDdldh-UpF (SEQ ID NO: 3) and XPDDldh-UpR (SEQ ID NO: 4), and XPDDldh-DownF (SEQ ID NO: 5) and pKVM-XPDdldh-DownR (SEQ ID NO: 6) as upstream and downstream primers, respectively, to obtain the ldh gene. Ti A fragment containing homologous arms upstream and downstream of the promoter was obtained.

[0135] 1.2 The PCR product from step 1.1 was purified, and recombinant PCR amplification was performed using pKVM-XPDdldh-UpF and pKVM-XPDdldh-DownR as primers. After purification, the PCR product was cloned into a pKVM vector by seamless cloning, and then transformed into E. coli S17 to produce S17-pKVMΔldh Ti I obtained it.

[0136] 1.3 S17-pKVMΔldh obtained in step 1.2 Ti The (E. coli) strain and the Bacillus licheniformis BN11 strain (original strain) were separately administered via OD. 600 The culture was fermented until the value reached 1.2. Next, the resulting strain was centrifuged at 6000 rpm for 5 minutes and washed twice with PBS. Then, the same OD (Oral-Density Spectroscopy) was performed. 600Genetically modified E. coli S17-pKVMΔldh (therefore, having the same concentration) Ti The Bacillus licheniformis BN11 strain was mixed in a 7:1 volume ratio, and the mixture was dropped onto LB plates. After incubation overnight at 30°C, the strain was diluted and spread onto LB plates containing erythromycin and polymyxin, and then incubated at 30°C. The screened transformants were incubated in LB medium containing erythromycin at 37°C, and then transferred to LB medium at 50°C for incubation. Subsequently, to screen for single cross-transformers, they were diluted, spread onto LB plates containing erythromycin, and incubated at 50°C. The single cross-transformers were cultured for two generations in LB medium at 30°C, diluted, spread onto LB plates, and incubated overnight at 37°C. Double cross-transformers were then selected by reverse screening based on their resistance to erythromycin. As confirmed by PCR and sequencing using pKVM-XPDdldh-UpF and pKVM-XPDdldh-DownR as primers, the D-lactate synthesis pathway (i.e., the gene ldh encoding D-lactate dehydrogenase (SEQ ID NO: 31)) is involved. Ti We have successfully constructed a genetically modified strain in which ) is knocked out (BN11Δldh Ti (This is how it is expressed.)

[0137] Example 2: Construction of a Bacillus licheniformis strain overexpressing 6-phosphofructokinase and pyruvate kinase. 2.1 The primers were designed according to the sequence of the Bacillus licheniformis ATCC14580 genome (GenBank number NC_006270.3). Separately, pKVM-XPDldh-UpF (SEQ ID NO: 7) and P were used as upstream and downstream primers, respectively. als -XPDldh-UpR (Sequence ID 8), XPDldh-P als -F (Sequence ID 9) and PFYAK-P als -R (Sequence ID 10), P alsPCR amplification was performed using -PFYAK-F (SEQ ID NO: 11) and XPDldh-PFYAK-R (SEQ ID NO: 12), as well as PFYAK-XPDldh-DownF (SEQ ID NO: 13) and pKVM-XPDldh-DownR (SEQ ID NO: 14), and homologous arms were added upstream of the ldh gene. als We obtained a fragment containing a promoter (SEQ ID NO: 2), a pfk (SEQ ID NO: 27), and a pyk (SEQ ID NO: 28) gene cluster, as well as homologous arms downstream of the ldh gene.

[0138] 2.2 The PCR product from step 2.1 was purified, and recombinant PCR amplification was performed using pKVM-XPDldh-UpF and pKVM-XPDldh-DownR as primers. After purification, the PCR product was cloned into a pKVM vector by seamless cloning, and then transformed into E. coli S17 to obtain S17-pKVM-PFYAK.

[0139] 2.3 Escherichia coli S17-pKVM-PFYAK strain and Bacillus licheniformis BN11Δldh obtained in step 2.2 Ti The strain was subjected to conjugation and homologous recombination in the same manner as in Example 1, and a strain overexpressing the pfk gene and pyk gene was obtained (BN11Δldh Ti (Represented as -PFYAK).

[0140] Example 3: Construction of a strain that overexpresses heterologous alanine dehydrogenase 3.1 Based on the amino acid sequence of alanine dehydrogenase, a potentially thermostable alanine dehydrogenase (with the amino acid sequence shown in SEQ ID NO: 32 and corresponding protein SEQ ID NO: WP_033014465.1) in the genome of Geobacillus stearothermophilus was identified using the BLAST method. This alanine dehydrogenase gene from Geobacillus stearothermophilus (named GSald) was subjected to codon optimization based on the Bacillus licheniformis genome (the optimized version of the sequence is shown in SEQ ID NO: 1), and the P from 5' to 3' was modified. alsIt was synthesized together with the promoter (sequence number 2). The merged sequence is P als - Represented as GSald

[0141] 3.2 Primers were designed according to the sequence of the Bacillus licheniformis ATCC14580 genome (GenBank number NC_006270.3). Separately, pKVM-XPDdldh-UpF (SEQ ID NO: 3) and P als -XPDdldh-UpR (Sequence ID 15), XPDDLDH-P als PCR amplification was performed using -F (SEQ ID NO: 16) and XPDDldh-GSald-R (SEQ ID NO: 17), and GSald-XPDDDldh-DownF (SEQ ID NO: 18) and pKVM-XPDDDldh-DownR (SEQ ID NO: 6) as upstream and downstream primers, respectively, and ldh Ti Homologous arm upstream of gene promoter, P als -GSald gene and ldh Ti We obtained a fragment containing homologous arms downstream of the gene.

[0142] 3.3 The PCR product from step 3.2 was purified, and recombinant PCR amplification was performed using pKVM-XPDdldh-UpF and pKVM-XPDdldh-DownR as primers. After purification, the PCR product was cloned into a pKVM vector by seamless cloning, and then transformed into E. coli S17 to obtain S17-pKVM-GSald. E. coli S17-pKVM-GSald strain and Bacillus licheniformis BN11Δldh Ti -The PFYAK strain was subjected to conjugation and homologous recombination in the same manner as in Example 1, and a strain expressing heterologous GSald was obtained (represented as BA-1).

[0143] Example 4: Knockout of the alanine racemase gene in Bacillus licheniformis 4.1 Primers were designed according to the sequence of the Bacillus licheniformis ATCC14580 genome (GenBank number NC_006270.3). Separately, PCR amplification was performed using pKVM-XPDalr1-UpF (SEQ ID NO: 19) and XPDalr1-UpR (SEQ ID NO: 20), and XPDalr1-DownF (SEQ ID NO: 21) and pKVM-XPDalr1-DownR (SEQ ID NO: 22) as upstream and downstream primers, respectively, to obtain fragments containing homologous arms upstream and downstream of the alr1 gene.

[0144] 4.2 The PCR product from step 4.1 was purified, and recombinant PCR amplification was performed using pKVM-XPDalr1-UpF and pKVM-XPDalr1-DownR as primers. After purification, the PCR product was cloned into a pKVM vector by seamless cloning, and then transformed into E. coli S17 to obtain S17-pKVMΔalr1.

[0145] 4.3 Escherichia coli S17-pKVMΔalr1 strain and Bacillus licheniformis BA-1 strain were subjected to conjugation and homologous recombination in the same manner as in Example 1, and a strain in which the alr1 gene (SEQ ID NO: 29) was knocked out was obtained (represented as BA-1Δalr1).

[0146] 4.4. Primers were designed according to the sequence of the Bacillus licheniformis ATCC14580 genome (GenBank number NC_006270.3). Separately, PCR amplification was performed using pKVM-XPDalr2-UpF (SEQ ID NO: 23) and XPDalr2-UpR (SEQ ID NO: 24), and XPDalr2-DownF (SEQ ID NO: 25) and pKVM-XPDalr2-DownR (SEQ ID NO: 26) as upstream and downstream primers, respectively, to obtain fragments containing homologous arms upstream and downstream of the alr2 gene.

[0147] 4.5 The PCR product from step 4.4 was purified, and recombinant PCR amplification was performed using pKVM-XPDalr2-UpF and pKVM-XPDalr2-DownR as primers. After purification, the PCR product was cloned into a pKVM vector by seamless cloning, and then transformed into E. coli S17 to obtain S17-pKVMΔalr2.

[0148] 4.6 Escherichia coli S17-pKVMΔalr2 strain and Bacillus licheniformis BA-1Δalr1 strain were subjected to conjugation and homologous recombination in the same manner as in Example 1, and a strain was obtained in which both the arl1 gene (SEQ ID NO: 29) and the arl2 gene (SEQ ID NO: 30) were knocked out (represented as BLA-1).

[0149] Example 5: Recombinant Bacillus licheniformis BLA-1 fed-batch fermentation at 42°C (in a 5L fermenter) 5.1 Incubator culture medium and fermentation culture medium Composition of the culture medium for inoculum: LB medium, yeast powder 5g / L, tryptone 10g / L, sodium chloride 10g / L. Composition of fermentation culture medium: 100 g / L glucose, 5 g / L yeast powder, 5 g / L ammonium sulfate, 1.3 g / L dipotassium hydrogen phosphate trihydrate, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 20 mg / L ferrous sulfate heptahydrate, 20 mg / L manganese sulfate tetrahydrate. Glucose was sterilized separately at 115°C for 20 minutes.

[0150] 5.2 Spawn culture BLA-1 was transferred from a glycerol tube to a shaking tube containing 5 mL of LB medium and incubated at 50°C for 12 hours while shaking at 200 rpm. Subsequently, 150 mL of LB medium contained in a 500 mL Erlenmeyer flask was seeded with a seeding material volume of 3% to 5%, and incubated at 50°C for 12 hours while shaking at 200 rpm to obtain a spawn solution (i.e., activated spawn). Spawn activation was achieved in this process.

[0151] 5.3 Fermentation Culture The inoculum solution obtained in step 5.2 was inoculated into the fermentation culture medium contained in a 5L fermentation tank at a volume of 5% of the seeding material, bringing the total volume to 3L. After controlling the fermentation pH to 7.0 with 25% (v / v) ammonia water, fermentation culture was started at a temperature of 42°C with aeration rate of 1.0 vvm and stirring speed of 300 rpm while aerating (air). After 6 to 8 hours, Bacillus licheniformis was cultured to an OD of 6.5 to 8.0. 600 The mixture was allowed to ferment until [amount missing]. At this point, aeration was stopped and the stirring speed was reduced to 80 rpm. Fermentation was then restarted and completed after 60 hours. During the fermentation process, when the glucose (carbon source) concentration decreased to 30 g / L to 50 g / L, glucose was replenished to increase the concentration to 100 g / L. After 60 hours of fermentation, the L-alanine concentration in the fermentation broth was detected at 93.7 g / L, which corresponds to a glucose-to-alanine conversion rate of 74.4%, and the optical purity of L-alanine was 98.9%.

[0152] Example 6: Flow-bottle fermentation of recombinant Bacillus licheniformis BLA-1 at 50°C (in a 5L fermenter) 6.1 Incubator culture medium and fermentation culture medium See 5.1 of Example 5. 6.2 Spawn culture See 5.2 of Example 5. 6.3 Fermentation Culture The inoculum solution obtained in step 6.2 was inoculated into the fermentation culture medium contained in a 5L fermentation tank at a volume of 5% of the seeding material, bringing the total volume to 3L. After controlling the fermentation pH to 7.0 with 25% (v / v) ammonia water, fermentation culture was started at a temperature of 50°C with aeration rate of 1.0 vvm and stirring speed of 300 rpm. After 6 to 8 hours, Bacillus licheniformis was cultured to an OD of 6.5 to 8.0. 600The mixture was allowed to ferment until [amount missing]. At this point, aeration was stopped and the stirring speed was reduced to 80 rpm. Fermentation was then restarted and completed after 60 hours. During the fermentation process, when the glucose concentration fell to 30 g / L to 50 g / L, glucose was replenished to increase the concentration to 100 g / L. Figure 2 is a plot of this fermentation process. After 60 hours of fermentation, the L-alanine concentration in the fermentation broth was detected to be 96.8 g / L, which corresponds to a glucose-to-alanine conversion rate of 76.8%, and the optical purity of L-alanine was 98.9%.

[0153] Example 7: Flow-bottle fermentation of recombinant Bacillus licheniformis BLA-1 at 55°C (in a 5L fermenter) 7.1 Incubator culture medium and fermentation culture medium See 5.1 of Example 5. 7.2 Spawn culture See 5.2 of Example 5. 7.3 Fermentation Culture The inoculum solution obtained in step 7.2 was inoculated into the fermentation culture medium contained in a 5L fermentation tank at a volume of 5% of the seeding material, bringing the total volume to 3L. After controlling the fermentation pH to 7.0 with 25% (v / v) ammonia water, fermentation culture was started at a temperature of 55°C with aeration rate of 1.0 vvm and stirring speed of 300 rpm. After 6 to 8 hours, Bacillus licheniformis was cultured to an OD of 3.0 to 4.5. 600 The mixture was allowed to ferment until [amount missing]. At this point, aeration was stopped and the stirring speed was reduced to 80 rpm. Fermentation was then restarted and completed after 60 hours. After 60 hours of fermentation, the L-alanine concentration in the fermentation broth was detected at 21.4 g / L, which corresponds to a glucose-to-alanine conversion rate of 63.8%, and the optical purity of L-alanine was 97.2%.

[0154] Example 8: Recombinant Bacillus licheniformis BLA-1 fed-batch fermentation at 42°C (in a 50L fermenter) 8.1 Incubator culture medium and fermentation culture medium See 5.1 of Example 5. 8.2 Spawn culture BLA-1 was transferred from a glycerol tube to a shaking tube containing 5 mL of LB medium and incubated at 50°C for 12 hours with shaking at 200 rpm. Then, 3% to 5% of the seed material volume was seeded into 150 mL of LB medium contained in a 500 mL Erlenmeyer flask and incubated at 50°C for 12 hours with shaking at 200 rpm. Finally, 3% to 5% of the seed material volume was seeded into 1.5 L of LB medium contained in a 5 L Erlenmeyer flask and incubated at 50°C for 12 hours with shaking at 200 rpm. 8.3 Fermentation Culture The inoculum solution obtained in step 8.2 was inoculated into the fermentation culture medium contained in a 50L fermentation tank at a volume of 5% of the seeding material, bringing the total volume to 30L. After controlling the fermentation pH to 7.0 with 25% (v / v) ammonia water, fermentation culture was started at a temperature of 42°C with aeration rate of 1.0 vvm and stirring speed of 300 rpm. After 6 to 8 hours, Bacillus licheniformis was cultured to an OD of 6.5 to 8.0. 600 The mixture was allowed to ferment until [amount missing]. At this point, aeration was stopped and the stirring speed was reduced to 80 rpm. Fermentation was then restarted and completed after 60 hours. During the fermentation process, when the glucose concentration decreased to 30 g / L to 50 g / L, glucose was replenished to increase the concentration to 100 g / L. After 60 hours of fermentation, the L-alanine concentration in the fermentation broth was detected to be 40.5 g / L, which corresponds to a glucose-to-alanine conversion rate of 70.7%, and the optical purity of L-alanine was 96.6%.

[0155] Example 9: Recombinant Bacillus licheniformis BLA-1 fed-batch fermentation at 50°C (in a 50L fermenter) 9.1 Incubator culture medium and fermentation culture medium See 5.1 of Example 5. 9.2 Spawn culture See 8.2 of Example 8. 9.3 Fermentation Culture The inoculum solution obtained in step 9.2 was inoculated into the fermentation culture medium contained in a 50L fermentation tank at a volume of 5% of the seeding material, bringing the total volume to 30L. After controlling the fermentation pH to 7.0 with 25% (v / v) ammonia water, fermentation culture was started at a temperature of 50°C with aeration rate of 1.0 vvm and stirring speed of 300 rpm. After 6 to 8 hours, Bacillus licheniformis was cultured to an OD of 6.5 to 8.0. 600 The mixture was allowed to ferment until [amount missing]. At this point, aeration was stopped and the stirring speed was reduced to 80 rpm. Fermentation was then restarted and completed after 60 hours. During the fermentation process, when the glucose concentration decreased to 30 g / L to 50 g / L, glucose was replenished to increase the concentration to 100 g / L. After 60 hours of fermentation, the L-alanine concentration in the fermentation broth was detected to be 108.2 g / L, which corresponds to a glucose-to-alanine conversion rate of 73.3%, and the optical purity of L-alanine was 99.1%.

[0156] Example 10: Recombinant Bacillus licheniformis BLA-1 fed-batch fermentation at 55°C (in a 50L fermenter) 10.1 Incubator culture medium and fermentation culture medium See 5.1 of Example 5. 10.2 Spawn culture See 8.2 of Example 8. 10.3 Fermentation Culture The inoculum solution obtained in step 10.2 was inoculated into the fermentation culture medium contained in a 50L fermentation tank at a volume of 5% of the seeding material, bringing the total volume to 30L. After controlling the fermentation pH to 7.0 with 25% (v / v) ammonia water, fermentation culture was started at a temperature of 55°C with aeration rate of 1.0 vvm and stirring speed of 300 rpm. After 6 to 8 hours, Bacillus licheniformis was cultured to an OD of 4.0 to 6.0. 600 The mixture was allowed to ferment until [amount missing]. At this point, aeration was stopped and the stirring speed was reduced to 80 rpm. Fermentation was then restarted and completed after 60 hours. After 60 hours of fermentation, the L-alanine concentration in the fermentation broth was detected at 26.6 g / L, which corresponds to a glucose-to-alanine conversion rate of 59.0%, and the optical purity of L-alanine was 98.7%.

[0157] Example 11: BN11Δldh constructed in Example 2Ti - Results of fermentation culture of the PFYAK strain BN11Δldh obtained in Example 2 Ti - Fermentation culture of the PFYAK strain was carried out according to the method and experimental parameters of Example 6. As a result, a mixture of L-alanine and D-alanine was produced in a yield of 64 g / L, and the optical purity of L-alanine was only 67.7%, which was therefore unsatisfactory (normally, optical purity should be 98% or higher).

[0158] Example 12: Fermentation culture results of the BA-1 strain constructed in Example 3 The BA-1 strain obtained in Example 3 was fermented and cultured according to the method and experimental parameters of Example 6. As a result, a mixture of L-alanine and D-alanine was produced in a yield of 129 g / L, and the optical purity of L-alanine was only 70.8%, which was therefore unsatisfactory.

[0159] The results from Examples 11 and 12 indicate that knockout of the alanine racemase gene in Bacillus licheniformis is necessary to achieve high optical purity of L-alanine.

[0160] Example 13: Knockout of only one alanine racemase gene Fermentation culture of strains in which either the alur1 (sequence number 29) or alur2 (sequence number 30) gene encoding alanine racemase was knocked out using the method of Example 4 was performed according to the method of Example 6. As a result, it was found to be unsatisfactory in terms of optical purity.

[0161] Preferred specific embodiments of the present invention have been described in detail above. Those skilled in the art should understand that various modifications and changes can be made based on the concept of the present invention without any creative effort. Accordingly, all technical solutions that those skilled in the art can obtain by logical analysis, reasoning, or limited experimentation based on the prior art and in accordance with the concept of the present invention are intended to fall within the scope of protection as defined by the claims. Exemplary embodiments of the present invention are described below. <1> A method for constructing a genetically modified bacterial strain that produces L-alanine, A step of preparing a progenitor strain that possesses the pyruvate synthesis pathway; A step of manipulating the genome of the progenitor strain through one, two, or three of the following steps S200, S300, and S400; A step of inserting a copy of the S200:6-phosphofructokinase gene pfk and a copy of the pyruvate kinase gene pyk; S300: A process to insert the GSald gene, which is heat-resistant to alanine dehydrogenase at temperatures between 42°C and 55°C; S400: A step of inactivating or deleting the alanine racemase gene contained in the genome of the aforementioned bacterial strain. A method characterized by including the following. <2> The aforementioned strain further possesses a lactate synthesis pathway, and the genome of the aforementioned strain contains a lactate dehydrogenase gene. The aforementioned construction method S100: A step of inactivating or deleting the lactate dehydrogenase gene in the genome of the aforementioned bacterial strain. A further comprising, <1> A method for constructing a genetically modified bacterial strain that produces L-alanine as described above. <3> The aforementioned strain further possesses a D-lactate synthesis pathway, and the genome of the aforementioned strain contains the D-lactate dehydrogenase gene ldh Ti Includes, The aforementioned construction method is, S500: The D-lactate dehydrogenase gene ldh contained in the genome of the aforementioned bacterial strain. Ti A process of inactivating or deactivating It further includes, Preferably, the D-lactate dehydrogenase gene ldh Ti The sequence is as shown in sequence number 31. <2> A method for constructing a genetically modified bacterial strain that produces L-alanine as described above. <4> The sequence of the 6-phosphofructokinase gene pfk is as shown in SEQ ID NO: 27, and the sequence of the pyruvate kinase gene pyk is as shown in SEQ ID NO: 28; and / or The sequence of the GSald gene of the alanine dehydrogenase is as shown in Sequence ID No. 1. <1> A method for constructing a genetically modified bacterial strain that produces L-alanine as described above. <5> In step S400, inactivating or deleting one, two or more types of alanine racemase genes; and / or The aforementioned step S400 includes inactivating or deleting the alanine racemase gene alfr1 and the alanine racemase gene alfr2. It is characterized by, Preferably, the sequence of the alanine racemase gene alur1 is as shown in SEQ ID NO: 29, and the sequence of the alanine racemase gene alur2 is as shown in SEQ ID NO: 30. <1> A method for constructing a genetically modified bacterial strain that produces L-alanine as described above. <6> The manipulation of the genome of the aforementioned bacterial strain is characterized by including steps S100 and S200. Preferably, the manipulation of the genome of the progenitor strain includes steps S100, S200, and S300. <2> A method for constructing a genetically modified bacterial strain that produces L-alanine as described above. <7> The manipulation of the genome of the aforementioned bacterial strain is characterized by including steps S100, S200, S300, and S400. <2> A method for constructing a genetically modified bacterial strain that produces L-alanine as described above. <8> The following steps: S500: The D-lactate dehydrogenase gene ldh contained in the genome of the aforementioned bacterial strain. Ti A process to knock out; A step of inserting a copy of the S200:6-phosphofructokinase gene pfk and a copy of the pyruvate kinase gene pyk; S300: Step of inserting a heterologine dehydrogenase gene GSald; and S400: A process to knock out the alanine racemase genes arl1 and alanine racemase genes arl2. Features including, <3> A method for constructing a genetically modified bacterial strain that produces L-alanine as described above. <9> In steps S200 and S300, a copy of the target gene is added to the chromosome, and a promoter is ligated in series upstream thereof, thereby inserting the target gene. <1> A method for constructing a genetically modified bacterial strain that produces L-alanine as described above. <10> The aforementioned promoter is P als Characterized by, <9> A method for constructing a genetically modified bacterial strain that produces L-alanine as described above. <11> The sequence of the promoter is as shown in Sequence ID No. 2, characterized in that <10> A method for constructing a genetically modified bacterial strain that produces L-alanine as described above. <12> The genetically modified bacterial strain that produces L-alanine is characterized in that it can produce L-alanine by fermentation at 42°C to 55°C. <1> ~ <11> A method for constructing a genetically modified bacterial strain that produces L-alanine as described in any one of the following. <13> The aforementioned bacterial strain is characterized by being a thermophilic strain. <1> ~ <11> A method for constructing a genetically modified bacterial strain that produces L-alanine as described in any one of the following. <14> The aforementioned strain is characterized by being a Bacillus. <1> ~ <11> A method for constructing a genetically modified bacterial strain that produces L-alanine as described in any one of the following. <15> The aforementioned bacterial strain is characterized by being Bacillus licheniformis, Bacillus coagulans, Bacillus methylotrophicus, thermophilic Bacillus inulinus, or Geobacillus stearothermophilus. <14> A method for constructing a genetically modified bacterial strain that produces L-alanine as described above. <16> The aforementioned strain is characterized in that it is Bacillus licheniformis ATCC14580 or a derivative thereof. <14> A method for constructing a genetically modified bacterial strain that produces L-alanine as described above. <17> The aforementioned strain is characterized by being Bacillus licheniformis BN11, deposited with the China Center for Typical Cultures on January 8, 2016, under the CCTCC number M2016026. <14> A method for constructing a genetically modified bacterial strain that produces L-alanine as described above. <18> <1> ~ <17> A genetically modified bacterial strain constructed according to one of the following methods. <19> To produce L-alanine, <18> Use of genetically modified bacterial strains as described above. <20> <18> A method for producing L-alanine, wherein the genetically modified bacterial strain described is subjected to fermentation culture, and the fermentation culture is carried out at a temperature of 42°C to 55°C. <21> The fermentation culture step is characterized by including inoculating activated inoculum into a fermentation culture medium containing a carbon source, and carrying out feed-boil fermentation at a stirring speed of 50 rpm to 350 rpm until a predetermined fermentation density is reached. <20> The production method described above. <22> The fermentation culture step is characterized in that the carbon source is glucose, glycerol, xylose, or arabinose. <21> The production method described above. <23> In the fermentation culture step, the fermentation culture medium is characterized by containing the following components: glucose, yeast powder, ammonium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, ferrous sulfate, and manganese sulfate. <21> The production method described above. <24> The aforementioned fermentation culture process involves adjusting the pH to 7.0±0.2 using 20%~30% (w / v) aqueous ammonia; aeration rate of 0.8vvm~1.2vvm and initial stirring speed of 280rpm~320rpm for 6~8 hours, with an OD of 6.5~8.0. 600 The process includes fermenting and culturing until a certain value is reached, then stopping aeration; and continuing the fermentation and culturing for 50 to 70 hours at a modified stirring speed of 60 rpm to 100 rpm, and then stopping the fermentation, characterized in that during the fermentation process, the carbon source is replenished when its concentration falls to a predetermined value. <21> The production method described above, Preferably, if the concentration of the carbon source decreases to 30 g / L to 50 g / L during the fermentation process, the carbon source is replenished once so that the concentration of the carbon source becomes 80 g / L to 120 g / L. Preferably, the method wherein, during the fermentation process, if the concentration of the carbon source decreases to 30 g / L to 50 g / L, a first replenishment of the carbon source is performed to increase the concentration of the carbon source to 80 g / L to 120 g / L, and thereafter, if the concentration of the carbon source decreases to 20 g / L to 40 g / L, a second replenishment of the carbon source is performed to increase the concentration of the carbon source to 60 g / L to 80 g / L. <25> The fermentation culture step is characterized in that the carbon source is glucose. <24> The production method described above. <26> The method further includes obtaining activated starter cultures by starter culture before the fermentation culture step, and sowing the activated starter cultures into a fermentation culture medium for the fermentation culture. <20> ~ <25> The production method described in any one of the following. <27> The aforementioned inoculum culture step is characterized by including sowing the genetically modified strain onto an inoculum culture medium, culturing it at an inoculum culture temperature of 42°C to 55°C for 12 to 16 hours, thereby obtaining the activated inoculum. <26> The production method described above. <28> In the above-mentioned inoculum culture step, the inoculum culture medium is characterized by containing the following components: peptone, yeast powder, and sodium chloride. <26> The production method described above.

Claims

1. A method for constructing a genetically modified bacterial strain that produces L-alanine, which can produce L-alanine by fermentation at 42°C to 55°C, A step of preparing a progenitor strain having a pyruvate synthesis pathway, wherein the progenitor strain is Bacillus licheniformis; A process of manipulating the genome of the progenitor strain through the following three steps S200, S300, and S400, or two steps S300 and S400; S200: A step of inserting a copy of the 6-phosphofructokinase gene pfk and a copy of the pyruvate kinase gene pyk; S300: A step of inserting the GSald gene, which is alanine dehydrogenase heat-resistant at 42°C to 55°C; S400: A step of inactivating or deleting the alanine racemase gene contained in the genome of the aforementioned bacterial strain. A method characterized by including the following.

2. The aforementioned strain further possesses a lactate synthesis pathway, and the genome of the aforementioned strain contains a lactate dehydrogenase gene. The aforementioned construction method S100: A step of inactivating or deleting the lactate dehydrogenase gene in the genome of the aforementioned bacterial strain. A method for constructing a genetically modified bacterial strain that produces L-alanine according to claim 1, further comprising the above.

3. A method for constructing a genetically modified bacterial strain that produces L-alanine according to claim 1, comprising inactivating or deleting one, two, or more types of alanine racemase genes in step S400.

4. A method for constructing a genetically modified bacterial strain that produces L-alanine according to claim 1, wherein step S400 includes inactivating or deleting the alanine racemase gene alfr1 and the alanine racemase gene alfr2.

5. A method for constructing a genetically modified bacterial strain that produces L-alanine according to claim 4, wherein the sequence of the alanine racemase gene arl1 is as shown in SEQ ID NO: 29, and the sequence of the alanine racemase gene arl2 is as shown in SEQ ID NO:

30.

6. A method for constructing a genetically modified bacterial strain that produces L-alanine according to claim 1, characterized in that in steps S200 and S300, a copy of the target gene is added to a chromosome and a promoter is ligated in series upstream thereof, thereby inserting the target gene.

7. The promoter is P ａｌｓ A method for constructing a genetically modified bacterial strain that produces L-alanine according to claim 6, characterized in that the sequence of the promoter is as shown in Sequence ID No.

2.

8. A method for constructing a genetically modified bacterial strain that produces L-alanine, as described in claim 1, characterized in that the original strain is Bacillus licheniformis BN11, deposited with the China Center for Typical Cultures on January 8, 2016, under CCTCC number M2016026.

9. A genetically modified bacterial strain constructed according to the method described in any one of claims 1 to 8.

10. Use of the genetically modified bacterial strain according to claim 9 for producing L-alanine.

11. The genetically modified bacterial strain described in claim 9 is subjected to fermentation culture, where the fermentation culture is carried out at a temperature of 42°C to 55°C. A method for producing L-alanine, wherein the fermentation culture step includes sowing activated inoculum into a fermentation culture medium containing a carbon source.

12. The production method according to claim 11, characterized in that the carbon source in the fermentation culture step is glucose, glycerol, xylose, or arabinose.

13. The fermentation culture process involves adjusting the pH to 7.0 ± 0.2 using 20% ​​to 30% (w / v) aqueous ammonia; and culturing for 6 to 8 hours with an aeration rate of 0.8 vvm to 1.2 vvm and an initial stirring speed of 280 rpm to 320 rpm, until the OD reaches 6.5 to 8.

0. ６００ A production method according to claim 11, comprising: fermenting and culturing until a certain value is reached, then stopping aeration; and continuing fermenting and culturing for 50 to 70 hours at a modified stirring speed of 60 rpm to 100 rpm, then stopping fermentation, wherein during the fermentation process, the carbon source is replenished when its concentration falls to a predetermined value, If the concentration of the carbon source decreases to 30 g / L to 50 g / L during the fermentation process, the carbon source will be replenished once so that its concentration becomes 80 g / L to 120 g / L, or, The method comprising: if the concentration of the carbon source decreases to 30 g / L to 50 g / L during the fermentation process, a first replenishment of the carbon source is performed to increase the concentration of the carbon source to 80 g / L to 120 g / L; and thereafter, if the concentration of the carbon source decreases to 20 g / L to 40 g / L, a second replenishment of the carbon source is performed to increase the concentration of the carbon source to 60 g / L to 80 g / L.

14. The production method according to claim 13, characterized in that the carbon source in the fermentation culture step is glucose.

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