Genetically engineered strains producing L-alanine and methods for their construction and 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] The present invention relates to the field of biotechnology, in particular to genetically engineered strains that produce L-alanine, and methods for their construction and use. [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, medicine, 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 medicine and health care, L-alanine can not only be used in amino acid-based dietary 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 to provide high biocompatibility.

[0003] The methods for producing L-alanine mainly include chemical synthesis, enzyme catalysis and fermentation. The current main chemical synthesis approach is the propionic acid chlorination process, which uses liquid chlorine and propionic acid as the main raw materials. Due to the long synthesis route, high cost and low yield, the chemical synthesis approach has been practically abandoned. Enzyme catalysis of L-alanine essentially involves the decarboxylation of aspartate catalyzed immobilized L-aspartate-β-decarboxylase. However, the enzyme catalysis approach is not suitable for large-scale production due to low carbon utilization of the 4-carbon substrate to the 3-carbon product and the relatively high price of aspartic acid. L-alanine can be produced by fermentation using strains constructed by genetic engineering, mainly using glucose as a substrate. This method has attracted wide attention due to its advantages such as cheap and easily available raw materials, high product yield and low cost.

[0004] Compared with fermentation at common temperatures below 42°C, high-temperature fermentation can significantly reduce the risk of contamination, improve the conversion efficiency of raw materials, and reduce heat exchange costs, making it valuable for large-scale production by fermentation. In the production of alanine by fermentation, genetically engineered E. coli strains are often used as production strains. However, the optimal growth temperature of these strains is 37°C, making them unsuitable for use in high-temperature fermentation.

[0005] Therefore, those skilled in the art are striving to develop 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 a method for constructing and using the same.

[0007] In order to achieve the above object, a first aspect of the present invention provides a method for constructing a genetically engineered strain for producing L-alanine, comprising the steps of: providing a source strain having a pyruvate synthesis pathway; manipulating the genome of the original strain through any one, any two or three of the following steps S200, S300 and S400; S200:6 - Inserting a copy of the phosphofructokinase gene pfk and a copy of the pyruvate kinase gene pyk; S300: inserting the gene GSald of alanine dehydrogenase, which 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 A method is provided that includes:

[0008] The genetically engineered L-alanine producing strain 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 invention, the original strain further comprises a lactate synthesis pathway, and the genome of the original strain contains a lactate dehydrogenase gene. In these cases, the method comprises: S100: A step of inactivating or deleting the lactate dehydrogenase gene in the genome of the original strain Further includes:

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

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

[0012] In a fourth aspect of the present invention, there is provided a method for producing L-alanine, comprising 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 between 42°C and 55°C.

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

[0014] Advantages provided by the techniques disclosed in this invention include:

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

[0016] 2. Using a thermophilic strain as the starting strain, a genetically engineered strain capable of producing L-alanine by fermentation at high temperatures of 42°C to 55°C (particularly, for example, 50°C) can be constructed. Furthermore, a significantly increased alanine yield can be obtained by enhancing the glycolytic pathway or / and introducing a thermostable alanine dehydrogenase. The production of optically pure L-alanine is possible by knocking out the alanine racemase gene. These strategies can be synergistically combined to enable high-yield production of optically pure L-alanine by fermentation at high temperatures of 42°C to 55°C, and the resulting L-alanine typically exhibits an optical purity of 98% or more, or even 99% or more. In some examples, the yield of optically pure L-alanine can be as high as 95 g / L or more, or even 100 g / L or more. 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 by fermentation at high temperatures, which has high industrial utility.

[0017] 3. In the case of an original strain having a lactic acid synthesis pathway, blocking the lactic acid synthesis pathway can increase the proportion of the pyruvate synthesis pathway and increase the yield of L-alanine. In the case of an original strain producing both lactic acid and pyruvate, blocking the lactic acid synthesis pathway in the original strain can increase the proportion of the alanine synthesis pathway. Furthermore, by strengthening the glycolysis pathway and introducing a thermostable alanine dehydrogenase, a genetically engineered strain capable of producing alanine at a high yield can be constructed. Knocking out the alanine racemase gene allows 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. The Bacillus licheniformis BN11 strain, which can produce D-lactic acid at high yields, can be used as the starting strain, and the D-lactic acid synthesis pathway therein can be eliminated to enhance the flow of the glycolysis pathway. In addition, a thermostable heterologous alanine dehydrogenase can be introduced, and the D-alanine synthesis pathway can be eliminated. In this way, high-yield production of optically pure L-alanine can be achieved at high temperatures of 42°C to 55°C. The obtained L-alanine typically exhibits an optical purity of 98% or more, or even 99% or more. In some instances, the yield of optically pure L-alanine can be as high as 95 g / L or more, or even 100 g / L or more.

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

[0020] Other characteristics, objects and advantages of the present invention will become more apparent from the following detailed description of non-limiting examples, taken in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0021] [Figure 1] 1 illustrates a schematic diagram of alanine synthesis and metabolic pathways in an exemplary engineered Bacillus licheniformis strain according to one embodiment of the present invention. [Diagram 2] 1 is a plot of L-alanine production by fed-batch fermentation in a 5L fermentor at 50° C. using the L-alanine producing genetically engineered strain BLA-1 according to an embodiment of the present invention (▲: OD600; ■: glucose; ●: alanine). [Diagram 3]1 shows the structure of the pKVM vector used in some embodiments of the present invention, which contains two antibiotic resistance genes: ampR (ampicillin resistance) and ermC (erythromycin resistance), and the bgaB gene encoding galactosidase. Either of these genes can be used as a selection marker. The plasmid has a thermosensitive origin of replication (oriT pE194ts) as well as an origin of replication in E. coli (ori) and in Bacillus licheniformis (oriT). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0023] In the following, some preferred embodiments and examples of the present invention will be described with reference to the drawings attached hereto, so that the technology thereof will be more clear and easily understood. The present invention can be embodied and exemplified 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 those detailed in the following description (including, but not limited to, the embodiments and examples) can be combined in any suitable manner to create new embodiments so long as the invention can be practiced.

[0025] As used herein, terms such as "preferred," "particular," "specific," "furthermore," and "furthermore" are used merely to describe embodiments or examples having favorable effects or a certain degree of specificity. It will be understood that they are not intended to limit the scope of the present invention in any way.

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

[0027] As used herein, "combinations thereof," "any combination," "any combination," and "in any combination" all mean that the listed items can be combined in any suitable manner so long as the invention can be practiced. The number of items that can be combined is two or more.

[0028] As 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] As used herein, any numerical range recited is intended to be inclusive of both the lower and upper limits, unless otherwise stated.

[0030] As used herein, the words "greater than or equal to" or "less than or equal to" following a number are intended to refer to a range that includes that particular number, unless otherwise defined.

[0031] The features of the invention described above and below can be combined in any suitable manner, as long as they are not inconsistent and allow the resulting embodiment to be implemented to solve the problem that the invention is intended to solve. These features can be combined in any suitable manner, as long as the resulting embodiment can solve the problem that the invention is intended to solve and achieve the expected effects.

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

[0033] As used herein, references to temperature control should be understood to mean that the temperature remains at a constant value or is controlled to vary within a range.

[0034] As used herein, "high temperature", when used to describe the production of L-alanine, is relative to common temperatures (below 42°C, typically below 37°C). As used herein, unless otherwise defined, "high temperature production" is intended to refer to production by fermentation at temperatures between 42°C and 55°C, such as, 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, the term "pyruvate synthesis pathway" refers to a pathway for synthesizing pyruvate by glycolysis of a carbon source. As used herein, "having a pyruvate synthesis pathway" means "having the ability to endogenously synthesize pyruvate."

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

[0037] As used herein, unless otherwise specified, "alanine synthesis pathway" refers to a synthetic pathway that converts pyruvate to alanine. As used herein, unless otherwise specified, "having an alanine synthesis pathway" means "having the ability to produce alanine through an alanine synthesis pathway", i.e., "having the ability to endogenously synthesize alanine". As used herein, when it is described as having an alanine synthesis pathway, unless otherwise specified, it is intended to mean having both a pathway for synthesizing L-alanine from pyruvate and a pathway for synthesizing D-alanine from L-alanine.

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

[0039] As used herein, gene cluster fragment refers to a fragment that contains at least two target genes, which may be the same or different.When describing gene cluster fragments that are composed of the same genes, the term "repeat" shall be broadly interpreted to mean that the genes may be directly adjacent to each other, or there may be a spacer sequence between any two of the genes.

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

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

[0042] [Table 1]

[0043] [Table 2]

[0044] The amino acid and nucleotide sequences referred to herein are summarized in Table 3.

[0045] [Table 3-1]

[0046] [Table 3-2]

[0047] SEQ ID NO:1 (GSald)

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

[0049] 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: CTGCTGCATGGCTAAATGAAAAAAGCCCTCTTTGAAAAG 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

[0050] SEQ ID NO:27 (Pfk) ATGAAGCGAATTGGAGTATTGACAAGCGGCGGCGATGCACCGGGGATGAATGCGGCGGTCCGCGCGATTGCCCGTAAAGGGATTTATCACGGCCTGGAAGTTTACGGCATTCGCCAAGGTTATAACGGATTGATTCAAGGAAACATCCAAAAGCTCGAAGCAGGATCTGTTGGCGATATTCTCCAGCGGGGCGGCACGGTTTTGCAGTCGGCAAGAAGCGAAGAATTCAAAACGCCGGAAGGGCAGCAAAAAGCGATCAGGCAGCTGAAAGACCATGGCATTGAAGCGCTCGTTGTGATCGGCGGCGACGGTTCCTACCAAGGGGCCAAAAAGTTGACGGAACAGGGCTTTAACTGCATTGGTGTGCCAGGGACAATCGATAACGACATCCCGGGGACGGATTTTACAATCGGTTTTGATACGGCATTGAACACAGTGCTTGATGCGATTGATAAAATTCGCGACACCGCTTCTTCCCACGAACGCACCTTTATTATTGAAGTCATGGGCAGAAATGCCGGGGATATCGCGCTCTGGTCCGGCCTGGCCGGCGGAGCCGAATCGATTATTATTCCGGAAGAAAAATATGACTTAAAAGATGTCGTGGAGCGTCTTGAACAGGGGAGAAAACGCGGCAAACGCCACAGCATCATCATTGTCGCGGAAGGCGTGATGAGCGGCAACGAGTTTGCTGAACAATTGAAAAAAACCGGTGTGATCGGCGATACCCGCGTTTCTGTTCTCGGCCATATCCAGCGCGGCGGTTCTCCGACGGCATTTGACCGCGTGCTTGCAAGCCGCCTCGGCGCAAGGGCTGTTGAACTGCTGCTTGAAGGAAAAGGGGGCCGCGCTGTCGGCATTCAAAATAACCAGCTGGTTGACCACGATATCCTTGAGATTCTCGGAAAACCGCACGCCGTTAATAAAAACATGTACAAGCTGTCGAAAGAATTGTCGATCTAA

[0051] SEQ ID NO:28 (Pyk)

[0052] SEQ ID NO:29 (Alr1)

[0053] SEQ ID NO:30 (Alr2)

[0054] SEQ ID NO:31 (No) ATGAAAGTAATTTTTTTTTCTATGCACCCGTATGAAGAGGAATTTCTGGGTCCGATTCTGCCGTCTGACTGGGACGTAGAAATGACCCCGGACTTTCTGGACGAAACCACCGTGGAAAAGGCTAAAGGTGCCCAGGTAGTAAGCCTGTTTGTTTCTGACAAAGCTGATGGTCCGGTACTGGAAGCGCTGCATTCTTACGGTGTGGGCCTGCTGGCCCTGCGTTCTGCTGGCTATGATCACATCGATATTGAGACCGCAAAACGCCTGGGTATCAAAGTAGTTAACGTGCCAGCCTATTCTCCGCACGCTATCGCTGACCATACTCTGGCTATCATGCTGGCTCTGATTCGTCGTCTGCACCGTGCCCATGATAAAGTGCGCCTGGGTGATTTTGATCTGGATGGTCTGATGGGCTTTGATCTGAACGGCAAAGTTGCTGGTGTAATTGGTCTGGGTAAAATCGGTCGCCTGGTAGCTACCCGCCTGAAAGCGTTTGGTTGCAAAGTTCTGGGCTATGATCCATACATTCAGCCGGAAATCGTAGAAAACGTTGATCTGGATACCCTGATCACTCAGGCTGATATCATTTCTATTCATTGTCCGCTGACCCGTGAAAACTTTCATATGTTTAACGAAGAGACTTTTAAGCGTATGAAACCGGGTGCTATTCTGGTTAACACCGCGCGTGGTGGTCTGATCGATACCAAGGCCCTGCTGGAGGCCCTGAAGTCTGGTAAACTGGGCGGCGCAGCCCTGGATGTGTATGAATATGAACGTGGCCTGTTTTTTAAAAACCACCAAAAAGAAGGTATCAAAGACCCGTATCTGGCCCAGCTGCTGGGTCTGGCCAACGTAGTGCTGACCGGTCATCAGGCCTTTCTGACCCGTGAGGCTGTAAAAAACATCGAAGAAACTACCGTAGAAAACATTCTGGAATGGCAAAAGAACCCGCAGGCAAAGCTGAAAAACGAAATCTAA

[0055] SEQ ID NO:32 (GSald) MKIGIPKEIKNNENRVAITPAGVMTLVKAGHDVYVETEAGAGSGFSDSEYEKAGAVIVPNAEDAWTAEMVLKVKEPLAEEFRYFRPGLILFTYLHLAAAEALTKALVEQKVVGIAYETVQLANGSLPLLTPMSEVAGRMSVQVGAQFLEKPHGGKGILLGGVPGVRRGKVTIIGGGTAGTNAAKIA VGLGSDVTILDINAERLRELDDLFGDHVTTLMSNSYHIAECVRESDLVVGAVLIPGAKAPKLVTEEMVRSMTPGSVLVDIAIDQGGIFETTDRVTTHDDPTYVKHGVVHYAVANMPGAVRTSTFALTNVTIPYALQIANKGYRAACLDNPALLKGINTLDGHIVYEAVAAAHNMPYTDVHSLLHG

[0056] In a first aspect of the present invention, there is provided a method for constructing an engineered strain for producing L-alanine, comprising the steps of: providing a source strain having a pyruvate synthesis pathway; manipulating the genome of the original strain by one, two or three of the following steps S200, S300 and S400; S200: introducing overexpressed copies of the pfk gene encoding 6-phosphofructokinase and the pyk gene encoding pyruvate kinase; S300: A step of introducing an overexpressed gene encoding a thermostable alanine dehydrogenase (preferably, an overexpressed gene GSald encoding a thermostable alanine dehydrogenase); S400: Inactivating or deleting the alanine racemase gene if the genome of the original strain contains the alanine racemase gene. A method is provided that includes:

[0057] In some embodiments of the present invention, S200, S300, and S400 include: S200:6 - inserting a copy of the phosphofructokinase gene pfk and a copy of the pyruvate kinase gene pyk; S300: inserting the overexpressed gene GSald encoding alanine dehydrogenase, which is thermostable at 42℃~55℃; S400: Partially or completely inactivating the alanine racemase gene, for example by knocking out the alanine racemase gene, or partially or completely deleting the alanine racemase gene. This can be achieved separately by

[0058] The genetically engineered 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 can enhance the glycolysis pathway by enabling overexpression of 6-phosphofructokinase and pyruvate kinase involved in the pathway, thereby increasing the supply of pyruvate and promoting the production of L-alanine. In step S300, a gene encoding a thermostable alanine dehydrogenase is introduced to enhance the pathway for synthesizing L-alanine from pyruvate, effectively increasing the yield of L-alanine. In step S400, the alanine racemase gene can be inactivated or deleted to promote the production of optically pure L-alanine. By using any one of steps S200, S300, and S400, the yield of L-alanine can be increased, and by using two or three of them in combination, a synergistic effect can be obtained.

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

[0060] FIG. 1 shows a schematic diagram of alanine synthesis and metabolic pathways in genetically engineered strains producing L-alanine according to some embodiments of the present invention. A carbon source is converted to pyruvate via the glycolysis pathway, which then reacts with NADH and ammonium ions under the catalytic action of alanine dehydrogenase to consume them and form L-alanine. A part of the resulting L-alanine is directly secreted outside the cells, and the rest is converted to D-alanine under the catalytic action of alanine racemase and then secreted outside the cells. If the carbon flow downstream of pyruvate is dominated by the lactate synthesis pathway of the original strain, the lactate synthesis pathway can be blocked to direct the carbon flow to the alanine synthesis pathway as much as possible. In addition, overexpressing 6-phosphofructokinase and pyruvate kinase involved in the glycolysis pathway can enhance the glycolysis pathway and increase the supply of pyruvate, thereby promoting the production of alanine. In addition, knocking out the alanine racemase gene can block the natural D-alanine synthesis pathway, resulting in optically pure L-alanine.

[0061] In some embodiments of the present invention, the original strain further comprises a D-lactate synthesis pathway, and the genome contains an ldh gene encoding D-lactate dehydrogenase. Ti In these cases, the method comprises the step of extracting the ldh gene encoding D-lactate dehydrogenase in the genome of the original strain. Ti In some preferred embodiments of the present invention, the step S500 further comprises the step of inactivating or deleting a gene, ldh, which encodes D-lactate dehydrogenase, in the genome of the original strain. Ti This involves knocking out a gene.

[0062] In some embodiments of the present invention, the ldh gene encoding D-lactate dehydrogenase Ti The gene has the sequence shown in SEQ ID NO:31.

[0063] In some embodiments of the 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 invention, the alanine dehydrogenase gene has the sequence shown in SEQ ID NO:1.

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

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

[0067] In some embodiments of the invention, step S400 comprises inactivating or deleting the alr1 or alr2 genes encoding alanine racemase. In some embodiments of the invention, step S400 comprises inactivating or deleting the alr1 and alr2 genes encoding alanine racemase. In some preferred embodiments of the invention, step S400 comprises knocking out the alr1 and alr2 genes encoding alanine racemase.

[0068] In some embodiments of the invention, the alr1 gene encoding an alanine racemase has the sequence set forth in SEQ ID NO:29, and the alr2 gene encoding an alanine racemase has the sequence set forth in SEQ ID NO:30.

[0069] In some embodiments of the present invention, the manipulation of the genome of the original strain comprises 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 overexpressing 6-phosphofructokinase by the pfk gene and overexpressing pyruvate kinase by the pyk gene. Preferably, the manipulation of the genome of the original strain further comprises step S300. In this way, the yield of L-alanine can be further increased by inserting a gene encoding a thermostable alanine dehydrogenase.

[0070] In some embodiments of the present invention, the manipulation of the genome of the original strain includes steps S100, S200, S300 and S400. In this way, in addition to blocking the lactate synthesis pathway by inactivating or deleting the lactate dehydrogenase gene, enhancing the glycolysis pathway, and thereby increasing the supply of pyruvate by introducing genes that overexpress 6-phosphofructokinase and pyruvate kinase into the original strain, the introduced overexpressed alanine dehydrogenase gene can enhance the synthesis pathway of L-alanine from pyruvate, and the alanine racemase gene of the original 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 original strain comprises steps S500 and S200. Thus, the lactate synthesis pathway is modified with the ldh gene encoding D-lactate dehydrogenase. Ti The glycolytic pathway is blocked by inactivating or deleting genes, and the glycolytic pathway is enhanced by overexpressing the pfk and pyk genes, which code for 6-phosphofructokinase and pyruvate kinase, respectively.

[0072] In a preferred embodiment of the invention, the method comprises the steps of:

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

[0074] S200: overexpressing a copy of the pfk gene encoding 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: overexpressing a heterologous GSald gene encoding alanine dehydrogenase (to enhance the synthesis pathway from pyruvate to L-alanine); and

[0076] S400: Complete inactivation or complete deletion of the alr1 and alr2 genes encoding alanine racemase (thereby blocking the conversion of L-alanine to D-alanine and facilitating high yield production of optically pure L-alanine).

[0077] In a preferred embodiment of the invention, the method comprises the steps of:

[0078] S500: a lactate dehydrogenase gene in the genome of the original strain, preferably ldh encoding D-lactate dehydrogenase Ti A step of knocking out a gene (and thereby blocking the lactate synthesis pathway), preferably LDH Ti The gene has the sequence shown in SEQ ID NO:31;

[0079] S200: inserting a copy of the pfk gene encoding 6-phosphofructokinase and a copy of the pyk gene encoding pyruvate kinase (to allow overexpression of the pfk gene encoding 6-phosphofructokinase and the pyk gene encoding pyruvate kinase in the original strain, 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: Inserting a heterologous GSald gene encoding alanine dehydrogenase (to enable overexpression of the heterologous GSald gene encoding alanine dehydrogenase and enhance the synthesis pathway from pyruvate to L-alanine), wherein more preferably, the GSald gene encoding alanine dehydrogenase has the sequence shown in SEQ ID NO:1; and

[0081] S400: knocking out the alr1 gene and the alr2 gene encoding alanine racemase (thereby blocking the conversion of L-alanine to D-alanine and facilitating the production of optically pure L-alanine), wherein more preferably, the alr1 gene encoding alanine racemase has the sequence shown in SEQ ID NO:29 and the alr2 gene encoding alanine racemase has the sequence shown in SEQ ID NO:30.

[0082] It will be understood that, unless otherwise stated, features of the embodiments of the invention are preferably independent but can be combined as appropriate.

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

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

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

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

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

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

[0089] In some preferred embodiments of the present invention, the source strain is Bacillus licheniformis, which is a facultative anaerobic Gram-positive endospore-forming bacterium with a fast growth rate, a broad substrate spectrum, and the ability of thermophilic fermentation under 50°C conditions. Furthermore, it allows stable genetic manipulation and is recognized as a "Generally Regarded 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 source 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 thereof. Examples of derivatives include, but are not limited to, Bacillus licheniformis MW3, Bacillus licheniformis BN11, etc.

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

[0093] In some preferred embodiments of the invention, the original 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 invention, the source strain is Bacillus licheniformis BN11 and the GSald gene encoding alanine dehydrogenase has the sequence shown in SEQ ID NO:1.

[0095] In some preferred embodiments of the invention, the source strain is Bacillus licheniformis BN11 and the endogenous alanine racemase genes comprise the alr1 and alr2 genes having the sequences shown in SEQ ID NO:29 and SEQ ID NO:30, respectively.

[0096] In a second aspect of the present invention, there is provided a genetically engineered strain constructed according to the first aspect of the present invention. The genetically engineered strain is capable of producing L-alanine in significantly increased yields by fermentation at 42°C or higher (e.g., 42°C to 55°C). It is also capable of producing optically pure L-alanine in high yields.

[0097] In a third aspect of the present invention, there is provided the use of the genetically engineered 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 significantly increased yields under high temperature conditions. It can also be used for the high yield production of optically pure L-alanine under high temperature conditions.

[0098] In a fourth aspect of the present invention, there is provided a method for producing L-alanine, comprising subjecting the genetically engineered strain according to the second aspect of the present invention to fermentation culture at a temperature between 42° C. and 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 inoculating an activated seed 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 an agitation speed of 50 rpm to 350 rpm until a predetermined fermentation density is achieved. Further, the inoculum volume of the activated seed can be 3% to 5%, for example 3%, 4% or 5%, by volume percentage.

[0100] In some embodiments of the present invention, the method further comprises, prior to the fermentation culture, culturing a seed culture to obtain an activated seed culture, which is then inoculated into a fermentation culture medium and cultured for fermentation.

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

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

[0103] In some embodiments of the invention, the seed culture medium is composed of the following components: peptone, yeast powder and sodium chloride.

[0104] In some embodiments of the invention, the method comprises the steps of:

[0105] Seed culture: a step of inoculating the genetically engineered strain into a seed culture medium and culturing it under a seed culture temperature condition of 42°C to 55°C to obtain an activated seed; and

[0106] Fermentation culture: a process of inoculating an activated seed culture medium containing a carbon source with fed-batch fermentation at a fermentation culture temperature of 42°C to 55°C until a predetermined fermentation density is reached, where the inoculum volume can be 3% to 5% by volume percentage.

[0107] In some embodiments of the invention, the method comprises the steps of:

[0108] Seed culture: a step of inoculating the genetically engineered strain into a seed culture medium and culturing it for 12 to 16 hours under a seed culture temperature condition of 42°C to 55°C to obtain an activated seed; and

[0109] Fermentation culture: a process of inoculating an activated seed culture medium containing a carbon source with fed-batch fermentation at a fermentation culture temperature of 42°C to 55°C and an agitation speed of 50 rpm to 350 rpm until a predetermined fermentation density is reached, where the inoculum volume can be 3% to 5% by volume percentage.

[0110] In some embodiments of the present invention, in the fermentation culture process, the carbon source 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 invention, achievement of a predetermined fermentation density is determined by a detected OD 600 For example, an OD of 6.5 to 8.0 600 Fermentation is stopped when

[0113] In some embodiments of the present invention, the fermentation culture process includes adjusting the pH to about 7.0 with aqueous ammonia; 600 and continuing the fermentation culture with a reduced agitation speed until a predetermined fermentation density is reached (second fermentation culture stage), after which the fermentation is stopped. During the fermentation process, the carbon source can be replenished when its concentration drops to a certain value.

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

[0115] Preferably, the ventilation is accomplished with air.

[0116] The ventilation may be performed at an airflow rate of 0.8 vvm to 1.2 vvm, for example, 0.9 vvm, 1.0 vvm, 1.1 vvm, 1.2 vvm, and the like.

[0117] The fermentation culture is divided into multiple 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 allows rapid growth of the strain, and the second fermentation culture stage allows efficient L-alanine production. In the first fermentation culture stage, the stirring is preferably performed at a speed of 280 rpm to 320 rpm, for example, 280 rpm, 290 rpm, 300 rpm, 310 rpm, 320 rpm, etc. The first fermentation culture stage is performed at an OD 600 The second fermentation culture stage lasts for a period of time depending on the OD, for example 6 hours to 8 hours, for example 6 hours, 6.5 hours, 7 hours, 7.5 hours or 8 hours. The second fermentation culture stage is preferably stirred 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 lasts for a period of time depending on the OD 600 It lasts for a period that also depends on the value (eg 1.2), for example 50 hours to 70 hours, for example 50 hours, 55 hours, 60 hours, 65 hours or 70 hours.

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

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

[0120] In some embodiments of the 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 invention, the carbon source is replenished twice: the first replenishment may be performed when the concentration drops to 30g / L-50g / L (e.g., 30g / L, 35g / L, 40g / L, 45g / L or 50g / L), resulting in a new concentration of 80g / L-120g / L (e.g., 80g / L, 90g / L, 100g / L, 110g / L or 120g / L); the second replenishment may be performed when the concentration drops to 20g / L-40g / L (e.g., 20g / L, 25g / L, 30g / L, 35g / L or 40g / L), returning the concentration to 60g / L-80g / L (e.g., 60g / L, 65g / L, 70g / L, 75g / L or 80g / L). Preferably, the carbon source is glucose.

[0122] In some embodiments of the present invention, the fermentation culture process includes adjusting the pH to 7.0±0.2 with 20% to 30% (v / v) aqueous ammonia; aeration at 0.8 vvm to 1.2 vvm and stirring at an initial speed of 280 rpm to 320 rpm for 6 to 8 hours; and culturing the culture to an OD of 6.5 to 8.0. 600and continuing the fermentation culture for 50 to 70 hours (e.g., 60 hours) at a modified agitation speed of 60 rpm to 100 rpm, followed by stopping the aeration. Furthermore, during the fermentation process, the carbon source may be replenished once, twice, or more times when its concentration drops to a certain value. In some embodiments, 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, and the concentration may be increased 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 only when its concentration drops to 30 g / L to 50 g / L, resulting in the concentration returning to 80 g / L to 120 g / L. Subsequently, when the concentration further drops to 20 g / L to 40 g / L, the carbon source is replenished a second time to bring the concentration back 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 process is glucose, and the fermentation culture process includes adjusting the pH to pH 7.0 with 25% aqueous ammonia and maintaining the pH at 7.0; aerating with an aeration rate of 1 vvm and stirring at an initial speed of 300 rpm for 6 to 8 hours; and increasing the OD of the Bacillus licheniformis strain to 1. 600 the value is 6.5-8.0, followed by stopping the aeration; and continuing the fermentation culture for 60 hours at a modified agitation speed of 80 rpm, followed by stopping the fermentation. During the fermentation process, glucose is replenished when its concentration drops to 30 g / L-50 g / L, thereby increasing the glucose concentration to 100 g / L. If the glucose concentration subsequently drops to 20 g / L-40 g / L, glucose is replenished again to increase the glucose concentration to 70 g / L.

[0124] The present invention will be described in more detail below by some examples that are intended to illustrate, but not limit the scope of the present invention in any way. These examples are provided in accordance with the teachings of the present invention. Although specific implementation and operation details are described, the scope of the present invention is in no way limited to the following examples. For any experimental procedure (e.g., PCR amplification, transformation, gene knockout, gene insertion, etc.) used in the following examples, if no specific conditions are specified, it is preferably performed in the manner and conditions described above. Otherwise, it is generally performed in the conventional manner and conditions, for example, as taught in Molecular Cloning: A Laboratory Manual by Sambrook et al. (New York: Cold Spring Harbor Laboratory Press, 1989), or under the conditions suggested by the manufacturer. EXAMPLES

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

[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 then the pH was adjusted to 2.1 with phosphoric acid. Then, 100 mL of methanol and ultrapure water were added successively to obtain a volume of 1 L as the mobile phase. The sample was injected in a volume of 10 μL into a ZORBAX Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 μm) at a temperature of 30 °C, run 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 min and centrifuged at 8000 rpm for 5 min. The supernatant was then collected and diluted at an appropriate ratio so that alanine was present in it 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 2 mM copper sulfate aqueous solution as the mobile phase. The samples were injected in a volume of 10 μL into a SUMICHIRAL OA-5000 (150 mm × 4.6 mm, 5 μm) at a temperature of 30 °C, run through the column at a flow rate of 0.5 mL / min, and measured with a DAD detector at a wavelength of 254 nm. Before the measurement, the samples were boiled for 10 min and centrifuged at 8000 rpm for 5 min. The supernatant was then collected and diluted by an appropriate factor so that alanine was present in it at a final concentration of 0.2 g / L to 1 g / L.

[0128] Glucose concentrations were measured with an SBA-40D biosensor analyzer (Biology Institute of Shandong Academy of Sciences).

[0129] In the following examples, Bacillus licheniformis strains were used as the original strains to construct genetically engineered strains capable of producing L-alanine under high temperature conditions. It will be understood that in the following examples, other bacterial strains (such as, but not limited to, Bacillus coagulans, Bacillus methylotrophicus, thermophilic Bacillus inulinus, Geobacillus stearothermophilus, etc.) can also be used as the original strains to construct genetically engineered 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 the pKVM vector, which contains two antibiotic resistance genes: ampR (ampicillin resistance) and ermC (erythromycin gene), and the bgaB gene encoding galactosidase. Both of these genes can be used as selection markers. The plasmid has a thermosensitive origin of replication (oriT pE194ts) as well as an origin of replication in E. coli (ori) and B. licheniformis (oriT).

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

[0132] Inoculum volume is measured as a volume percentage.

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

[0134] Example 1: Construction of a Bacillus licheniformis strain with a knockout D-lactate synthesis pathway 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 gene ldh Ti A fragment containing homology arms upstream and downstream of the promoter of was obtained.

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

[0136] 1.3 S17-pKVMΔldh obtained in step 1.2 Ti (Escherichia coli) strain and Bacillus licheniformis BN11 strain (original strain) were separately measured using OD 600 The fermentation culture was performed until the OD value reached 1.2. The resulting strain was then centrifuged at 6000 rpm for 5 min and washed twice with PBS. 600(hence, the same concentration) of engineered E. coli S17-pKVMΔldh Ti and Bacillus licheniformis BN11 strains were mixed in a volume ratio of 7:1, and the mixture was dropped onto LB plates. After overnight cultivation at 30 °C, the strains were diluted and spread on LB plates containing erythromycin and polymyxin, and subsequently cultivated at 30 °C. The screened transformants were cultivated in LB medium containing erythromycin at 37 °C, and then transferred to LB medium at 50 °C for cultivation. Then, to screen for single-crossover transformants, they were diluted, spread on LB plates containing erythromycin, and cultivated at 50 °C. The single-crossover transformants were serially cultivated for two generations in LB medium at 30 °C, diluted, spread on LB plates, and cultivated overnight at 37 °C. Then, double-crossover transformants were selected by reverse screening based on their resistance to erythromycin. The D-lactate synthesis pathway (i.e., the gene ldh encoding D-lactate dehydrogenase (SEQ ID NO: 31)) was identified as a marker for the D-lactate synthesis pathway (i.e., the gene ldh encoding D-lactate dehydrogenase (SEQ ID NO: 31)) as confirmed by PCR and sequencing using pKVM-XPDdldh-UpF and pKVM-XPDdldh-DownR as primers. Ti We successfully constructed a genetically engineered strain in which the Δldh Ti (expressed as:

[0137] Example 2: Construction of Bacillus licheniformis strains overexpressing 6-phosphofructokinase and pyruvate kinase 2.1 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 als - XPDldh-UpR (SEQ ID NO: 8), XPDldh-P als -F (SEQ ID NO: 9) and PFYAK-P als -R (SEQ ID NO: 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), to insert a homology arm, P als A fragment was obtained containing the promoter (SEQ ID NO:2), the pfk (SEQ ID NO:27) and pyk (SEQ ID NO:28) gene clusters, and a homology arm 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 pKVM vector by seamless cloning, which was then transformed into E. coli S17 to obtain S17-pKVM-PFYAK.

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

[0140] Example 3: Construction of strains overexpressing heterologous alanine dehydrogenase 3.1 Based on the amino acid sequence of alanine dehydrogenase, a potentially thermostable alanine dehydrogenase (having the amino acid sequence shown in SEQ ID NO: 32 and protein sequence corresponding to WP_033014465.1) was identified in the genome of Geobacillus stearothermophilus 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 sequence of the optimized version is as shown in SEQ ID NO: 1), and the 5' to 3' P alsThe merged sequence was synthesized with the promoter (SEQ ID NO: 2). als -It is represented as GSald.

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

[0142] 3.3 The PCR product from step 3.2 was purified and subjected to recombinant PCR amplification using pKVM-XPDdldh-UpF and pKVM-XPDdldh-DownR as primers. After purification, the PCR product was cloned into pKVM vector by seamless cloning, which was 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 conjugative transfer and homologous recombination as in Example 1 to obtain a strain expressing heterologous GSald (designated BA-1).

[0143] Example 4: Knockout of the alanine racemase gene in Bacillus licheniformis 4.1 Primers were designed according to the sequence of 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 a fragment containing homologous arms upstream and downstream of gene alr1.

[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 pKVM vector by seamless cloning, which was then transformed into E. coli S17 to obtain S17-pKVMΔalr1.

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

[0146] 4.4. Primers were designed according to the sequence of 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 a fragment containing homologous arms upstream and downstream of gene alr2.

[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 pKVM vector by seamless cloning, which was then transformed into E. coli S17 to obtain S17-pKVMΔalr2.

[0148] 4.6 The E. coli S17-pKVMΔalr2 strain and the Bacillus licheniformis BA-1Δalr1 strain were subjected to conjugation and homologous recombination in the same manner as in Example 1 to obtain a strain in which both the alr1 gene (SEQ ID NO: 29) and the alr2 gene (SEQ ID NO: 30) were knocked out (designated as BLA-1).

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

[0150] 5.2 Seed culture BLA-1 was transferred from the glycerol tube to a shaker tube containing 5 mL of LB medium and incubated at 50° C. for 12 hours with shaking at 200 rpm. It was then inoculated at 3%-5% of the inoculum volume 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 to obtain a seed solution (i.e., activated seed). In this step, seed activation was achieved.

[0151] 5.3 Fermentation culture The seed solution obtained in step 5.2 was inoculated into the fermentation culture medium contained in a 5 L fermenter at 5% of the inoculum volume to make the total volume 3 L. 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 (air) at 1.0 vvm and agitation speed of 300 rpm. After 6 to 8 hours, Bacillus licheniformis was cultured at an OD of 6.5 to 8.0. 600 The fermentation was continued for 60 h until the aeration was stopped and the stirring speed was reduced to 80 rpm. The fermentation was then resumed and terminated after 60 h. During the fermentation process, when the concentration of glucose (carbon source) decreased to 30 g / L-50 g / L, glucose was supplemented to increase the concentration to 100 g / L. After 60 h of fermentation, the L-alanine concentration in the fermentation broth was detected to be 93.7 g / L, which corresponds to a conversion rate of glucose to alanine of 74.4%, and the optical purity of L-alanine was 98.9%.

[0152] Example 6: Fed-batch fermentation of recombinant Bacillus licheniformis BLA-1 at 50 °C (in a 5 L fermenter) 6.1 Seed and fermentation culture media See Example 5, 5.1 6.2 Seed culture See 5.2 of Example 5 6.3 Fermentation Culture The seed solution obtained in step 6.2 was inoculated into the fermentation culture medium contained in a 5 L fermenter at 5% of the inoculum volume to make the total volume 3 L. 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 an aeration volume of 1.0 vvm and an agitation speed of 300 rpm. After 6 to 8 hours, Bacillus licheniformis was cultured at an OD of 6.5 to 8.0. 600The fermentation was continued until 60 h. At this point, aeration was stopped and the agitation speed was reduced to 80 rpm. Fermentation was then resumed and terminated after 60 h. During the fermentation process, when the concentration of glucose decreased to 30 g / L-50 g / L, glucose was supplemented to increase the concentration to 100 g / L. Figure 2 shows a plot of this fermentation process. After 60 h of fermentation, the L-alanine concentration in the fermentation broth was detected to be 96.8 g / L, which corresponds to a conversion rate of glucose to alanine of 76.8%, and the optical purity of L-alanine was 98.9%.

[0153] Example 7: Fed-batch fermentation of recombinant Bacillus licheniformis BLA-1 at 55 °C (in a 5 L fermentor) 7.1 Seed and fermentation culture media See Example 5, 5.1 7.2 Seed culture See 5.2 of Example 5 7.3 Fermentation Culture The seed solution obtained in step 7.2 was inoculated into the fermentation culture medium contained in a 5 L fermenter at 5% of the inoculum volume to make the total volume 3 L. 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 an aeration volume of 1.0 vvm and an agitation speed of 300 rpm. After 6 to 8 hours, Bacillus licheniformis was cultured at an OD of 3.0 to 4.5. 600 The fermentation was continued for 60 h until the end of the incubation period. At this point, aeration was stopped and the agitation speed was reduced to 80 rpm. The fermentation was then resumed and terminated after 60 h. After 60 h of fermentation, the L-alanine concentration in the fermentation broth was detected to be 21.4 g / L, which corresponds to a conversion of glucose to alanine of 63.8%, and the optical purity of L-alanine was 97.2%.

[0154] Example 8: Fed-batch fermentation of recombinant Bacillus licheniformis BLA-1 at 42 °C (in a 50 L fermentor) 8.1 Seed and fermentation culture media See Example 5, 5.1 8.2 Seed culture BLA-1 was transferred from the glycerol tube to a shaker tube containing 5 mL of LB medium and incubated at 50°C for 12 hours with shaking at 200 rpm. It was then inoculated with 3%-5% of the inoculum volume 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, it was further inoculated with 3%-5% of the inoculum volume 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 seed solution obtained in step 8.2 was inoculated into the fermentation culture medium contained in a 50 L fermenter at 5% of the inoculum volume to make the total volume 30 L. 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 an aeration volume of 1.0 vvm and an agitation speed of 300 rpm. After 6 to 8 hours, Bacillus licheniformis was cultured at an OD of 6.5 to 8.0. 600 The fermentation was continued for 60 h until the end of the incubation period. At this point, aeration was stopped and the stirring speed was reduced to 80 rpm. Fermentation was then resumed and terminated after 60 h. During the fermentation process, when the concentration of glucose decreased to 30 g / L-50 g / L, glucose was supplemented to increase the concentration to 100 g / L. After 60 h of fermentation, the L-alanine concentration in the fermentation broth was detected to be 40.5 g / L, which corresponds to a conversion rate of glucose to alanine of 70.7%, and the optical purity of L-alanine was 96.6%.

[0155] Example 9: Fed-batch fermentation of recombinant Bacillus licheniformis BLA-1 at 50 °C (in a 50 L fermentor) 9.1 Seed and fermentation culture media See Example 5, 5.1 9.2 Seed culture See Example 8, 8.2 9.3 Fermentation Culture The seed solution obtained in step 9.2 was inoculated into the fermentation culture medium contained in a 50 L fermenter at 5% of the inoculum volume to make the total volume 30 L. 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 an aeration volume of 1.0 vvm and an agitation speed of 300 rpm. After 6 to 8 hours, Bacillus licheniformis was cultured at an OD of 6.5 to 8.0. 600 The fermentation was continued for 60 h until the end of the incubation period. At this point, aeration was stopped and the stirring speed was reduced to 80 rpm. Fermentation was then resumed and terminated after 60 h. During the fermentation process, when the concentration of glucose decreased to 30 g / L-50 g / L, glucose was supplemented to increase the concentration to 100 g / L. After 60 h of fermentation, the L-alanine concentration in the fermentation broth was detected to be 108.2 g / L, which corresponds to a conversion rate of glucose to alanine of 73.3%, and the optical purity of L-alanine was 99.1%.

[0156] Example 10: Fed-batch fermentation of recombinant Bacillus licheniformis BLA-1 at 55 °C (in a 50 L fermenter) 10.1 Seed and fermentation culture media See Example 5, 5.1 10.2 Seed culture See Example 8, 8.2 10.3 Fermentation Culture The seed solution obtained in step 10.2 was inoculated into the fermentation culture medium contained in a 50 L fermenter at 5% of the inoculum volume to make the total volume 30 L. 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 an aeration volume of 1.0 vvm and an agitation speed of 300 rpm. After 6 to 8 hours, Bacillus licheniformis was cultured at an OD of 4.0 to 6.0. 600 The fermentation was continued for 60 hours until the end of the incubation period. At this point, aeration was stopped and the agitation speed was reduced to 80 rpm. The fermentation was then resumed and terminated after 60 hours. After 60 hours of fermentation, the L-alanine concentration in the fermentation broth was detected to be 26.6 g / L, which corresponds to a conversion of glucose to alanine of 59.0%, and the optical purity of L-alanine was 98.7%.

[0157] Example 11: BN11Δldh constructed in Example 2Ti -Fermentation culture results of PFYAK strain BN11Δldh obtained in Example 2 Ti Fermentation 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 with a yield of 64 g / L, and the optical purity of L-alanine was only 67.7%, which was therefore unsatisfactory (normally, the optical purity should be 98% or more).

[0158] Example 12: Fermentation culture results of the BA-1 strain constructed in Example 3 Fermentation of the BA-1 strain obtained in Example 3 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 with a yield of 129 g / L, and the optical purity of L-alanine was only 70.8%, which was therefore unsatisfactory.

[0159] The results of Examples 11 and 12 indicate that knockout of the alanine racemase gene of 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 a strain in which either the alr1 (SEQ ID NO: 29) or alr2 (SEQ ID NO: 30) gene encoding alanine racemase was knocked out using the method of Example 4 was carried out according to the method of Example 6. As a result, it was found that the optical purity was unsatisfactory.

[0161] Above, the preferred specific embodiments of the present invention have been described in detail. It should be understood that those skilled in the art can make various modifications and changes based on the concept of the present invention without any creative efforts. Therefore, all technical solutions that those skilled in the art can obtain according to the concept of the present invention based on the prior art through logical analysis, reasoning, or limited experiments are intended to fall within the protection scope defined by the claims.

Claims

1. 1. A method for constructing an L-alanine producing genetically engineered strain, comprising: Providing a source strain having a pyruvate synthesis pathway; manipulating the genome of said original strain through any one, any two or three of the following steps S200, S300 and S400; S200: inserting a copy of the 6-phosphofructokinase gene pfk and a copy of the pyruvate kinase gene pyk; S300: Inserting the gene GSald of alanine dehydrogenase, which 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 The method according to claim 1, further comprising:

2. The original strain further has a lactic acid synthesis pathway, and the genome of the original strain contains a lactate dehydrogenase gene; The method of constructing S100: A step of inactivating or deleting the lactate dehydrogenase gene in the genome of the original strain The method for constructing a genetically engineered strain for producing L-alanine according to claim 1, further comprising:

3. Inactivating or deleting one, two or more types of alanine racemase genes in step S400; and / or The step S400 includes inactivating or deleting the alanine racemase gene alr1 and the alanine racemase gene alr2. It is characterized by:

2. The method for constructing a genetically engineered strain for producing L-alanine according to claim 1, wherein 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.

4. 2. The method for constructing a genetically engineered strain for producing L-alanine according to claim 1, wherein in steps S200 and S300, the gene of interest is inserted by adding a copy of the gene of interest to a chromosome and ligating a promoter upstream of the gene in tandem.

5. The promoter is P ａｌｓ 5. The method for constructing a genetically engineered strain for producing L-alanine according to claim 4, wherein the sequence of the promoter is as shown in SEQ ID NO:

2.

6. The method for constructing the genetically engineered strain producing L-alanine according to any one of claims 1 to 5, wherein the genetically engineered strain producing L-alanine is capable of producing L-alanine by fermentation at 42°C to 55°C.

7. The method for constructing a genetically engineered strain for producing L-alanine according to any one of claims 1 to 5, wherein the original strain is a thermophilic strain.

8. 8. The method for constructing a genetically engineered strain for producing L-alanine according to claim 7, wherein the original strain is Bacillus licheniformis, Bacillus coagulans, Bacillus methylotrophicus, thermophilic Bacillus inulinus or Geobacillus stearothermophilus.

9. 8. The method for constructing a genetically engineered strain for producing L-alanine according to claim 7, wherein the original strain is Bacillus licheniformis BN11, deposited in the China Center for Typical Culture Collection on January 8, 2016 under CCTCC No. M2016026.

10. A genetically engineered strain constructed according to the method of any one of claims 1 to 9.

11. Use of the genetically engineered strain according to claim 10 for producing L-alanine.

12. The genetically engineered strain of claim 10 is subjected to a fermentation culture, wherein the fermentation culture is carried out at a temperature between 42° C. and 55° C.; The method for producing L-alanine, wherein the fermentation culture step comprises inoculating an activated seed culture medium containing a carbon source.

13. The method according to claim 12, wherein the carbon source in the fermentation culture step is glucose, glycerol, xylose or arabinose.

14. The fermentation culture process comprises adjusting the pH to 7.0±0.2 with 20%-30% (w / v) aqueous ammonia; culturing the culture at an OD of 6.5-8.0 for 6-8 hours with an aeration rate of 0.8 vvm-1.2 vvm and an initial agitation rate of 280 rpm-320 rpm; ６００ 13. The method according to claim 12, further comprising: cultivating the carbon source at a modified agitation speed of 60-100 rpm for 50-70 hours and then stopping the aeration; and continuing the fermentation culture at a modified agitation speed of 60-100 rpm for 50-70 hours and then stopping the fermentation, wherein during the fermentation process, the carbon source is replenished when its concentration drops to a predetermined value, Preferably, during the fermentation process, if the concentration of the carbon source falls to 30 g / L to 50 g / L, the carbon source is replenished once to make the concentration of the carbon source 80 g / L to 120 g / L; Preferably, during the fermentation process, if the concentration of the carbon source drops to between 30 g / L and 50 g / L, a first supplement of the carbon source is made to increase the concentration of the carbon source to between 80 g / L and 120 g / L, and thereafter, if the concentration of the carbon source drops to between 20 g / L and 40 g / L, a second supplement of the carbon source is made to increase the concentration of the carbon source to between 60 g / L and 80 g / L.

15. The method according to claim 14, wherein the carbon source in the fermentation culture step is glucose.

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