De novo synthesis type corynebacterium glutamicum for producing L-hydroxyproline and construction method of corynebacterium glutamicum
By genetically modifying Corynebacterium glutamicum, a de novo synthetic strain was constructed, solving the problem of synthesizing L-hydroxyproline from inexpensive carbon sources, reducing production costs, and improving conversion efficiency and yield.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-17
- Publication Date
- 2026-04-07
AI Technical Summary
In existing technologies, when producing L-hydroxyproline using microbial fermentation, it is impossible to achieve de novo synthesis starting from inexpensive carbon sources. It heavily relies on expensive precursors, and Corynebacterium glutamicum has a weak endogenous L-proline synthesis capacity and lacks a hydroxylation system, thus failing to drive the complete synthetic pathway.
By genetically modifying Corynebacterium glutamicum, a de novo synthetic strain was constructed, which included overexpressing endogenous proline biosynthesis pathway genes and introducing exogenous proline-4-hydroxylase, knocking out competitive amino acid synthesis pathway genes, forming recombinant plasmids, and performing resistance screening and PCR verification, and optimizing fermentation conditions.
This method enables the direct synthesis of L-hydroxyproline from inexpensive carbon source glucose, reducing production costs, improving conversion efficiency and yield, reducing by-product generation, and increasing raw material utilization.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of microbial engineering, in particular to a de novo synthesis type of Corynebacterium glutamicum for producing L-hydroxyproline and a construction method thereof. BACKGROUND
[0002] Microbial engineering, also known as fermentation engineering, is a technology system for using modern engineering techniques to produce products on a large scale by utilizing the metabolic function of microorganisms. The core links include strain breeding, medium preparation, fermentation process control and product purification.
[0003] At present, in the process of producing L-hydroxyproline by microbial fermentation method, it mainly depends on the modification of traditional hosts such as Escherichia coli and yeast, and the conversion by adding L-proline as a precursor. This method has limitations: it cannot realize the de novo synthesis of L-hydroxyproline from cheap carbon sources, it is seriously dependent on high-priced precursor materials, resulting in high production cost, and it is difficult to achieve high yield due to the inhibition of precursor materials on the growth of bacterial cells; at the same time, in the commonly used host Corynebacterium glutamicum, the endogenous L-proline synthesis ability is weak, the metabolic flux is insufficient, and there is no efficient hydroxylation system to drive the complete synthesis path from central metabolites to L-hydroxyproline.
[0004] Therefore, the present application provides a de novo synthesis type of Corynebacterium glutamicum for producing L-hydroxyproline and a construction method thereof to solve the above problems. SUMMARY
[0005] In view of the deficiencies of the prior art, the present application provides a de novo synthesis type of Corynebacterium glutamicum for producing L-hydroxyproline and a construction method thereof, which solves the problems of being unable to realize the de novo synthesis of L-hydroxyproline from cheap carbon sources, being seriously dependent on high-priced precursor materials, and being unable to drive the complete synthesis path from central metabolites to L-hydroxyproline.
[0006] To achieve the above purpose, the present application provides the following technical scheme: a de novo synthesis type of Corynebacterium glutamicum for producing L-hydroxyproline and a construction method thereof, the construction method comprising the following steps: Step 1: Construction of a gene expression vector, cloning an exogenous proline-4-hydroxylase gene into an expression vector containing a strong promoter to form a recombinant plasmid; Step 2: Preparation of host bacteria, selecting wild-type Corynebacterium glutamicum as host bacteria and pretreating the host bacteria, including activation culture and preparation of competent cells; Step 3: Genetic modification, overexpressing endogenous proline biosynthesis pathway genes and knocking out competitive amino acid synthesis pathway genes in the host bacteria genome by homologous recombination and CRISPR-Cas9 technology; Step four: transformation, transforming the recombinant plasmid constructed in step one into the modified host bacteria obtained in step three; Step five: screening and verification, obtaining positive transformants, i.e. the L-hydroxyproline-producing de novo synthesis Corynebacterium glutamicum, through resistance screening and PCR verification. Step six: fermentation optimization, optimizing the culture conditions of the positive transformants to improve the yield of L-hydroxyproline.
[0007] Further, in step one, the expression vector is pEC-XK99E and pCGL plasmid, and the vector contains kanamycin resistance gene as a screening marker.
[0008] Further, in step two, the pretreatment of the host bacteria includes: inoculating the wild-type Corynebacterium glutamicum into LB culture medium, activating it at 30-37°C for 12-16 hours, and then preparing competent cells by calcium ion method.
[0009] Further, in step three, genetic modification uses CRISPR-Cas9 system, wherein the sgRNA target sequence is directed to the coding region of dihydropyridine dicarboxylic acid synthase gene, and the length of the homologous arm is 500-800 bp.
[0010] Further, in step six, the fermentation culture conditions include: using culture medium containing glucose, ammonium sulfate, phosphate and trace elements, the culture temperature is 30-35°C, the pH is controlled at 6.5-7.5, the dissolved oxygen is maintained at 20%-40%, and the fermentation time is 48-72 hours.
[0011] Further, the Corynebacterium glutamicum is Corynebacterium glutamicum ATCC 13032 and its derivative strains, and the yield of L-hydroxyproline is detected by HPLC, which can reach 8.0-12.0 g / L under optimized conditions.
[0012] Further, the Corynebacterium glutamicum is genetically modified, including: overexpressing endogenous glutamate kinase gene, glutamate-5-semialdehyde dehydrogenase gene and pyrroline-5-carboxylate reductase gene in the proline biosynthesis pathway, introducing exogenous proline-4-hydroxylase gene, and knocking out dihydropyridine dicarboxylic acid synthase gene and homoserine dehydrogenase gene in the competitive amino acid synthesis pathway, wherein the exogenous proline-4-hydroxylase gene is derived from mammals and plants, and the yield of L-hydroxyproline of the Corynebacterium glutamicum in the fermentation medium with glucose as carbon source is greater than 5.0 g / L.
[0013] Further, the exogenous proline-4-hydroxylase gene is derived from humans, mice and Arabidopsis thaliana.
[0014] Further, the overexpression of the endogenous proline biosynthesis pathway gene is achieved by integrating a strong promoter, and the strong promoter is selected from the group consisting of a Ptac promoter, a Pgap promoter and a Psod promoter, and the promoter strength is 3-5 times that of the endogenous promoter.
[0015] Further, the knockout of the competitive amino acid synthesis pathway gene includes complete knockout of dihydrodipicolinic acid synthase gene and homoserine dehydrogenase gene to reduce the synthesis competition of lysine and threonine.
[0016] Compared with the prior art, the present application provides a de novo synthesis type corynebacterium glutamicum for producing L-hydroxyproline and a construction method thereof, which has the following beneficial effects: 1. In the present application, the corynebacterium glutamicum is genetically modified, and a complete biosynthesis path for directly synthesizing L-hydroxyproline from cheap carbon source glucose is constructed in vivo, so that the de novo synthesis of L-hydroxyproline is realized, thereby eliminating the dependence on the addition of expensive L-proline precursor in the traditional method, reducing the production cost, and laying an economically feasible foundation for the industrialized biological manufacturing of L-hydroxyproline.
[0017] 2. In the present application, the endogenous glutamate kinase gene, glutamate-5-semialdehyde dehydrogenase gene and pyrroline-5-carboxylate reductase gene in the proline biosynthesis pathway are overexpressed, and the exogenous proline-4-hydroxylase gene is introduced, which solves the problems of weak L-proline synthesis ability and lack of hydroxylation activity of the host itself, so that the cell can convert central metabolites into L-proline and further hydroxylate into the target product L-hydroxyproline, thereby improving the overall conversion efficiency from carbon source to end product.
[0018] 3. In the present application, the dihydrodipicolinic acid synthase gene and homoserine dehydrogenase gene in the competitive amino acid synthesis pathway are specifically knocked out, which solves the competition of the internal metabolic network of the host strain for the common precursor, can guide the carbon metabolic flow to the synthesis of the target product L-hydroxyproline, thereby reducing the generation of by-products such as lysine and threonine, and improving the utilization rate of raw materials and the yield of L-hydroxyproline. DETAILED DESCRIPTION
[0019] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.
[0020] Embodiment 1: A de novo synthesis type corynebacterium glutamicum for producing L-hydroxyproline and a construction method thereof, the construction method comprising the following steps: Step one: construction of gene expression vector, cloning exogenous proline-4-hydroxylase gene into expression vector containing strong promoter to form recombinant plasmid; Step two: preparation of host bacteria, selecting wild-type Corynebacterium glutamicum as host bacteria and pretreating host bacteria, including activation culture and preparation of competent cells; Step three: genetic modification, overexpressing endogenous proline biosynthesis pathway genes and knocking out competitive amino acid synthesis pathway genes in host bacteria genome through homologous recombination and CRISPR-Cas9 technology; Step four: transformation, transforming recombinant plasmid constructed in step one into modified host bacteria obtained in step three; Step five: screening and verification, obtaining positive transformants, i.e. de novo synthesis of L-hydroxyproline Corynebacterium glutamicum, through resistance screening and PCR verification; Step six: fermentation optimization, optimizing culture conditions to improve L-hydroxyproline yield by fermentation culture of positive transformants.
[0021] In step one, the expression vector is pEC-XK99E and pCGL plasmid, and the vector contains kanamycin resistance gene as a selection marker.
[0022] In step two, the pretreatment of host bacteria includes: inoculating wild-type Corynebacterium glutamicum in LB medium, activating for 12 hours at 30°C, and then preparing competent cells by calcium ion method.
[0023] In step three, the genetic modification uses CRISPR-Cas9 system, wherein the sgRNA target sequence is directed to the coding region of dihydropyridine dicarboxylic acid synthase gene, and the homologous arm length is 500 bp.
[0024] In step six, the fermentation culture conditions include: using medium containing glucose, ammonium sulfate, phosphate and trace elements, culture temperature is 30°C, pH is controlled at 6.5, dissolved oxygen is maintained at 20%, and fermentation time is 48 hours.
[0025] Corynebacterium glutamicum is Corynebacterium glutamicum ATCC 13032 and its derivative strains, and the L-hydroxyproline yield is detected by HPLC, which can reach 8.0 g / L under optimized conditions.
[0026] The coryneform bacterium is genetically modified, including overexpressing endogenous glutamate kinase gene, glutamate-5-semialdehyde dehydrogenase gene and pyrroline-5-carboxylate reductase gene in the proline biosynthesis pathway, introducing exogenous proline-4-hydroxylase gene, and knocking out dihydrodipicolinate synthase gene and homoserine dehydrogenase gene in the competitive amino acid synthesis pathway, wherein the exogenous proline-4-hydroxylase gene is derived from mammals and plants, and the coryneform bacterium has an L-hydroxyproline yield of greater than 5.0 g / L in a fermentation medium with glucose as the carbon source.
[0027] The exogenous proline-4-hydroxylase gene is derived from humans, mice and Arabidopsis thaliana.
[0028] The overexpression of the endogenous proline biosynthesis pathway gene is achieved by integrating a strong promoter, and the strong promoter is selected from the group consisting of Ptac promoter, Pgap promoter and Psod promoter, and the strength of the promoter is 3 times that of the endogenous promoter.
[0029] The knockout of the competitive amino acid synthesis pathway gene includes complete knockout of the dihydrodipicolinate synthase gene and the homoserine dehydrogenase gene to reduce the synthesis competition of lysine and threonine.
[0030] Embodiment 2: A de novo synthesis type coryneform bacterium producing L-hydroxyproline and a construction method thereof, the construction method comprising the following steps: Step one: construction of a gene expression vector, cloning the exogenous proline-4-hydroxylase gene into an expression vector containing a strong promoter to form a recombinant plasmid; Step two: preparation of a host bacterium, selecting wild-type coryneform bacterium as the host bacterium and pretreating the host bacterium, including activation culture and preparation of competent cells; Step three: genetic modification, overexpressing the endogenous proline biosynthesis pathway gene in the host bacterium genome and knocking out the competitive amino acid synthesis pathway gene by homologous recombination and CRISPR-Cas9 technology; Step four: transformation, transforming the recombinant plasmid constructed in step one into the modified host bacterium obtained in step three; Step five: screening and verification, obtaining positive transformants, i.e. the de novo synthesis type coryneform bacterium producing L-hydroxyproline, by resistance screening and PCR verification; Step six: fermentation optimization, optimizing the culture conditions of the positive transformants to improve the yield of L-hydroxyproline.
[0031] In step one, the expression vector is pEC-XK99E and pCGL plasmid, and the vector contains kanamycin resistance gene as a screening marker.
[0032] In step two, the pre-treatment of the host bacteria includes: inoculating the wild-type Corynebacterium glutamicum into LB medium, activating for 14 hours at 33.5℃, and then preparing competent cells by calcium ion method.
[0033] In step three, the genetic modification uses the CRISPR-Cas9 system, in which the sgRNA target sequence is directed to the coding region of the dihydropyridine dicarboxylic acid synthase gene, and the length of the homologous arm is 650bp.
[0034] In step six, the fermentation culture conditions include: using a medium containing glucose, ammonium sulfate, phosphate and trace elements, the culture temperature is 32.5℃, the pH is controlled at 7.0, the dissolved oxygen is maintained at 30%, and the fermentation time is 60 hours.
[0035] The Corynebacterium glutamicum is Corynebacterium glutamicum ATCC 13032 and its derivative strains, and the L-hydroxyproline yield can reach 10.0g / L under optimized conditions.
[0036] The Corynebacterium glutamicum is genetically modified, including: overexpressing the endogenous glutamate kinase gene, glutamate-5-semialdehyde dehydrogenase gene and pyrroline-5-carboxylate reductase gene in the proline biosynthesis pathway, introducing the exogenous proline-4-hydroxylase gene, and knocking out the dihydropyridine dicarboxylic acid synthase gene and the homoserine dehydrogenase gene in the competitive amino acid synthesis pathway, wherein the exogenous proline-4-hydroxylase gene is derived from mammals and plants, and the L-hydroxyproline yield of the Corynebacterium glutamicum in the fermentation medium with glucose as the carbon source is greater than 5.0g / L.
[0037] The exogenous proline-4-hydroxylase gene is derived from humans, mice and Arabidopsis thaliana.
[0038] The overexpression of the endogenous proline biosynthesis pathway gene is achieved by integrating a strong promoter, and the strong promoter is selected from the group consisting of Ptac promoter, Pgap promoter and Psod promoter, and the strength of the promoter is 4 times that of the endogenous promoter.
[0039] Knocking out the competitive amino acid synthesis pathway genes includes completely knocking out the dihydropyridine dicarboxylic acid synthase gene and the homoserine dehydrogenase gene to reduce the synthesis competition of lysine and threonine.
[0040] Example 3: A de novo synthesis type Corynebacterium glutamicum for producing L-hydroxyproline and a construction method thereof, the construction method comprising the following steps: Step one: construction of a gene expression vector, cloning the exogenous proline-4-hydroxylase gene into an expression vector containing a strong promoter to form a recombinant plasmid; Step two: preparation of host bacteria, wild-type Corynebacterium glutamicum was selected as the host bacteria, and the host bacteria was pretreated, including activation culture and preparation of competent cells; Step three: genetic modification, overexpression of endogenous proline biosynthesis pathway genes and knockout of competitive amino acid synthesis pathway genes in the host bacteria genome by homologous recombination and CRISPR-Cas9 technology; Step four: transformation, the recombinant plasmid constructed in step one was transformed into the modified host bacteria obtained in step three; Step five: screening and verification, positive transformants were obtained through resistance screening and PCR verification, which were de novo synthesis of L-hydroxyproline Corynebacterium glutamicum; Step six: fermentation optimization, the positive transformants were cultured by fermentation, and the culture conditions were optimized to improve the yield of L-hydroxyproline.
[0041] In step one, the expression vector is pEC-XK99E and pCGL plasmid, and the vector contains kanamycin resistance gene as a selection marker.
[0042] In step two, the pretreatment of host bacteria includes: inoculating wild-type Corynebacterium glutamicum in LB medium, activating at 37℃ for 16 hours, and then preparing competent cells by calcium ion method.
[0043] In step three, the genetic modification uses CRISPR-Cas9 system, wherein the sgRNA target sequence is directed to the coding region of dihydropyridine dicarboxylic acid synthase gene, and the homologous arm length is 800bp.
[0044] In step six, the fermentation culture conditions include: using medium containing glucose, ammonium sulfate, phosphate and trace elements, culture temperature is 35℃, pH is controlled at 7.5, dissolved oxygen is maintained at 40%, and fermentation time is 72 hours.
[0045] Corynebacterium glutamicum is Corynebacterium glutamicum ATCC 13032 and its derivative strains, and the yield of L-hydroxyproline is detected by HPLC, which can reach 12.0g / L under optimized conditions.
[0046] Corynebacterium glutamicum is genetically modified, including: overexpression of glutamate kinase gene, glutamate-5-semialdehyde dehydrogenase gene and pyrroline-5-carboxylate reductase gene in endogenous proline biosynthesis pathway, introduction of exogenous proline-4-hydroxylase gene, and knockout of dihydropyridine dicarboxylic acid synthase gene and homoserine dehydrogenase gene in competitive amino acid synthesis pathway, wherein the exogenous proline-4-hydroxylase gene is derived from mammals and plants, and the yield of L-hydroxyproline of Corynebacterium glutamicum in fermentation medium with glucose as carbon source is greater than 5.0g / L.
[0047] The exogenous proline-4-hydroxylase gene is derived from human, mouse and Arabidopsis.
[0048] The overexpression of the endogenous proline biosynthesis pathway genes is achieved by integrating a strong promoter, which is selected from the group consisting of a Ptac promoter, a Pgap promoter and a Psod promoter, and the strength of the promoter is 5 times that of the endogenous promoter.
[0049] The knockout of the competitive amino acid synthesis pathway genes includes complete knockout of dihydrodipicolinate synthase genes and homoserine dehydrogenase genes to reduce the synthesis competition of lysine and threonine.
[0050] Comparative Example 1, the difference between the comparative example and example 1 is that the exogenous proline-4-hydroxylase gene is not introduced in the construction of the engineering strain.
[0051] Comparative Example 2, the difference between the comparative example and example 2 is that the glutamate kinase gene, glutamate-5-semialdehyde dehydrogenase gene and pyrroline-5-carboxylate reductase gene in the endogenous proline biosynthesis pathway are not overexpressed in the genetic modification process.
[0052] Comparative Example 3, the difference between the comparative example and example 3 is that the dihydrodipicolinate synthase gene and homoserine dehydrogenase gene in the competitive amino acid synthesis pathway are not knocked out in the genetic modification process.
[0053] Comparative Example 4, the difference between the comparative example and example 3 is that lactic acid is used instead of glucose as the main carbon source in the fermentation process.
[0054] The performance of one L-hydroxyproline-producing de novo synthesis type C. glutamicum and the construction method thereof in examples 1-3 and comparative examples 1-4 are tested, and the test items and test methods are as follows: L-hydroxyproline yield test: after fermentation, the fermentation broth is centrifuged, the concentration of L-hydroxyproline in the supernatant is detected by high performance liquid chromatography, and the yield per unit volume is calculated.
[0055] Glucose conversion rate test: the total amount of glucose consumed in the fermentation process is measured, and the number of grams of L-hydroxyproline converted from each gram of glucose is calculated.
[0056] By-product analysis: the accumulation amount of key by-products lysine and threonine in the fermentation broth is detected by high performance liquid chromatography.
[0057] Genetic stability test: the engineering strain is continuously subcultured on a non-resistant plate, and the retention of the exogenous gene and the L-hydroxyproline yield are verified periodically.
[0058] The test data of one L-hydroxyproline-producing de novo synthesis Corynebacterium glutamicum and its construction method in Examples 1-3 and Comparative Examples 1-4 are recorded in the following table:
[0059] By comparing and analyzing the data in the table, it can be seen that the one L-hydroxyproline-producing de novo synthesis Corynebacterium glutamicum and its construction method in Examples 1-3 have more excellent performance than the one L-hydroxyproline-producing de novo synthesis Corynebacterium glutamicum and its construction method in Comparative Examples 1-4, which indicates that by genetically modifying Corynebacterium glutamicum, a complete biosynthetic pathway for directly synthesizing L-hydroxyproline from cheap carbon source glucose is constructed in the body of the Corynebacterium glutamicum, realizing the de novo synthesis of L-hydroxyproline, thereby getting rid of the dependence on the addition of expensive L-proline precursor in the traditional method, reducing the production cost, laying an economically feasible foundation for the industrialized biological manufacturing of L-hydroxyproline, and solving the problems of weak L-proline synthesis ability and lack of hydroxylation activity of the host by synergistically overexpressing the glutamate kinase gene, glutamate-5-semialdehyde dehydrogenase gene and pyrroline-5-carboxylate reductase gene in the endogenous proline biosynthesis pathway and introducing the exogenous proline-4-hydroxylase gene, so that the bacteria can convert central metabolites into L-proline and further hydroxylate into the target product L-hydroxyproline, thereby improving the overall conversion efficiency from carbon source to end product, solving the competition of the host's internal metabolic network for common precursors by specifically knocking out the dihydrodipicolinate synthase gene and homoserine dehydrogenase gene in the competitive amino acid synthesis pathway, guiding the carbon metabolic flow to the synthesis of the target product L-hydroxyproline, thereby reducing the generation of by-products such as lysine and threonine, and improving the utilization rate of raw materials and the yield of L-hydroxyproline.
[0060] By comparing and analyzing the relevant data in the table, it can be seen that the one L-hydroxyproline-producing de novo synthesis Corynebacterium glutamicum and its construction method has more excellent comprehensive performance.
[0061] In the description of the present specification, the description referring to the terms "one embodiment", "an example", "a specific example" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.
[0062] The preferred embodiments of the application disclosed above are only to facilitate the elucidation of the application. The preferred embodiments do not describe all the details of the application and limit the application to the specific embodiments described. Obviously, many modifications and variations can be made in light of the teachings above. The description is chosen and described in order to best explain the principles of the application and its practical application to thereby enable others skilled in the art to best utilize the application and get the best results from the application. The application is only limited by the claims and their full scope and equivalents.
Claims
1. A method for constructing a de novo synthetic glutamate-producing Corynebacterium, characterized in that: Includes the following steps: Step 1: Construction of gene expression vector. The exogenous proline-4-hydroxylase gene is cloned into an expression vector containing a strong promoter to form a recombinant plasmid. Step 2: Preparation of host bacteria. Wild-type Corynebacterium glutamicum is selected as the host bacteria, and the host bacteria are pretreated, including activation culture and preparation of competent cells. Step 3: Genetic modification. Through homologous recombination and CRISPR-Cas9 technology, endogenous proline biosynthesis pathway genes are overexpressed in the host bacterial genome, and competitive amino acid synthesis pathway genes are knocked out. Step 4: Transformation, transforming the recombinant plasmid constructed in Step 1 into the modified host bacteria obtained in Step 3; Step 5: Screening and verification. Positive transformants were obtained through resistance screening and PCR verification, namely the de novo synthetic glutamate-producing Corynebacterium glutamicum. Step 6: Fermentation optimization. The positive transformants are fermented and cultured, and the culture conditions are optimized to increase the yield of L-hydroxyproline.
2. The method for constructing a de novo synthetic glutamate-producing Corynebacterium according to claim 1, characterized in that: In step one, the expression vectors are pEC-XK99E and pCGL plasmids, and the vectors contain a kanamycin resistance gene as a selection marker.
3. The method for constructing a de novo synthetic glutamate-producing Corynebacterium according to claim 1, characterized in that: In step two, the pretreatment of the host bacteria includes: inoculating wild-type Corynebacterium glutamicum into LB medium, activating it at 30-37℃ for 12-16 hours, and then preparing competent cells by calcium ion method.
4. The method for constructing a de novo synthetic glutamate-producing Corynebacterium according to claim 1, characterized in that: In step three, the genetic modification uses the CRISPR-Cas9 system, where the sgRNA target sequence targets the coding region of the dihydropyridine dicarboxylic acid synthase gene, and the homologous arm length is 500-800 bp.
5. The method for constructing a de novo synthetic glutamate-producing Corynebacterium according to claim 1, characterized in that: In step six, the fermentation culture conditions include: using a culture medium containing glucose, ammonium sulfate, phosphate and trace elements, a culture temperature of 30-35℃, a pH controlled at 6.5-7.5, dissolved oxygen maintained at 20%-40%, and a fermentation time of 48-72 hours.
6. The method for constructing a de novo synthetic glutamate-producing Corynebacterium according to claim 1, characterized in that: The *Corynebacterium glutamicum* strain is *Corynebacterium glutamicum* ATCC 13032 and its derivative strains, and the L-hydroxyproline yield was detected by HPLC, which showed that it could reach 8.0-12.0 g / L under optimized conditions.
7. A de novo synthetic glutamate-producing Corynebacterium that produces L-hydroxyproline, characterized in that: The *Corynebacterium glutamicum* has undergone genetic engineering modification, including: overexpression of the glutamate kinase gene, glutamate-5-semialdehyde dehydrogenase gene, and pyrrolinoline-5-carboxylic acid reductase gene in the endogenous proline biosynthesis pathway; introduction of the exogenous proline-4-hydroxylase gene; and knockout of the dihydropyridine dicarboxylic acid synthase gene and homoserine dehydrogenase gene in the competitive amino acid synthesis pathway. The exogenous proline-4-hydroxylase gene is derived from mammals and plants, and the *Corynebacterium glutamicum* exhibits an L-hydroxyproline yield greater than 5.0 g / L in a glucose-based fermentation medium.
8. The de novo synthetic glutamate-producing Corynebacterium as described in claim 7, characterized in that: The exogenous proline-4-hydroxylase gene was derived from humans, mice, and Arabidopsis thaliana.
9. The de novo synthetic glutamate-producing Corynebacterium as described in claim 7, characterized in that: The overexpression of the endogenous proline biosynthesis pathway gene is achieved by integrating a strong promoter, which is selected from the Ptac promoter, Pgap promoter and Psod promoter, and the promoter strength is 3-5 times that of the endogenous promoter.
10. A de novo synthetic glutamate-producing Corynebacterium that produces L-hydroxyproline according to claim 7, characterized in that: The knockout of competitive amino acid synthesis pathway genes includes the complete knockout of the dihydropyridine dicarboxylic acid synthase gene and the homoserine dehydrogenase gene to reduce the competition for synthesis between lysine and threonine.