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A single-cell factory for synthesizing l-phenylglycine and its construction and application

A technology of phenylglycine and amino acid, applied in the field of microorganisms, can solve the problems of high cost, affect the stability of transformation, complicated process, etc., and achieve the effects of improving transformation efficiency, low cultivation cost and reducing cost.

Active Publication Date: 2021-03-02
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Although the production of L-phenylglycine by this multi-enzyme cascade conversion method has increased compared with previous reports, the conversion rate will decrease significantly if the substrate concentration is further increased, making it difficult to achieve large-scale industrial production.
At the same time, using this transformation system to produce L-phenylosine requires cell disruption of the three enzyme-producing recombinant bacteria. The process is cumbersome and expensive, and the transformation process is affected by the inactivation of the enzyme. Stability, in addition, it is necessary to add exogenous cofactors, which further increases the production cost of L-phenylosine

Method used

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  • A single-cell factory for synthesizing l-phenylglycine and its construction and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Preparation of Escherichia coli Competent and Transformation of Plasmid

[0035] (1) Preparation of competent Escherichia coli. Activate the monoclonal Escherichia coli in 10ml LB medium, then transfer to 37°C shaking culture to OD 600 0.35 to prepare the competent state; put the cultured bacterial solution in ice water, shake gently to cool the bacterial solution for about 10 minutes; prepare several 1.5mL centrifuge tubes that have been sterilized, and divide the bacterial solution into the tubes. The amount of bacteria in the tube is 1.2mL, put the centrifuge tube in ice; centrifuge the bacteria liquid at 8000r / min for 10-20s, let it stand in ice water for 2min, discard the supernatant, add pre-cooled 0.1M CaCl 2 400μL, gently blow the suspension, put it in ice for 15min (repeat this step 2-3 times); finally, add pre-cooled 0.1M CaCl 2 80 μL, gently pipette the suspension and place it on ice.

[0036] (2) Transformation of plasmids: Take the competen...

Embodiment 2

[0037] Example 2: Expression and enzyme activity determination of LeuDH mutant N71S in Escherichia coli

[0038] (1) The acquisition of mutant N71S, using the nucleotide sequence shown in SEQ ID NO.4 as a template, Fprimer (sequence shown in SEQ ID NO.5) and Rprimer (sequence shown in SEQ ID NO.6) as follows: primers, PCR is performed to obtain the recombinant gene shown in SEQ ID NO.3.

[0039] (2) The recombinant gene and pET-28a were digested with EcoR I and Xho I, respectively, and after purification, they were ligated with T4 DNA ligase overnight at 16°C. The ligation product was chemically transformed into E.coli BL21 competent cells. Incubate at 37°C for 1-2 hours, apply the transformation solution to an LB plate containing kanamycin (50mg / L), extract the plasmid, and double-enzyme digest to verify the constructed recombinant plasmid, which is named pET-28a-N71S, and the sequencing work was provided by Shanghai Sangong Finish. The obtained bacterial strain containing...

Embodiment 3

[0048] Example 3: Construction of the promoter of the FDH mutant A10C that provides the cofactor NADH and the optimized recombinant Escherichia coli of the RBS sequence

[0049] (1) The tac promoter was selected, and according to the tac promoter on the pXMJ-19 plasmid and the gene sequence of the mutant A10C, RBS sequences containing different strengths were designed (indicated in bold underline, such as SEQ ID NO: 7~SEQ ID NO: 13) PCR primers r1FDH, r2FDH, r3FDH, r4FDH, r5FDH, r6FDH and r7FDH (SEQ ID NO: 14~SEQ ID NO: 20), and the end primer pFDHRBamHI of the formate dehydrogenase gene (SEQ ID NO :twenty one).

[0050] (2) Using the existing vector pET28a-A10C carrying the FDH mutant A10C as a template, primers containing different intensities of the RBS sequence and pFDHRBamHI were used to form a primer pair, and PCR was performed to obtain multiple sequences containing RBS sequences and formate dehydrogenase The gene fragment was connected with the pXMJ-19 plasmid (the nu...

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Abstract

The invention discloses a single-cell factory for efficiently synthesizing L-phenylglycine and its construction and application, belonging to the technical field of microorganisms. The present invention first realizes the high-efficiency expression of leucine dehydrogenase derived from Bacillus cereus in Escherichia coli, and obtains mutant N71S with significantly improved reduction properties through site-directed mutation, and combines the mutant enzyme with formate dehydrogenase mutant Co-expressed in Escherichia coli to form an intracellular in situ cofactor NADH cycle system, optimized the expression of formate dehydrogenase mutants through promoter and RBS sequence optimization, successfully constructed a recombinant Escherichia coli single-cell factory, and used the single-cell factory Carry out whole-cell transformation to prepare L-phenylglycine. The conversion process of this method is simple and fast, with low cost and no by-products, which is beneficial to separation and purification. After 4 hours of conversion in a 5L fermenter, the output of L-phenylglycine can reach 105.7g / l. The conversion rate was 93.3%, and the space-time yield of L-phenylosine was 26.3 g / L·h, which provided a practical and effective strategy for the industrial production of L-phenylosine.

Description

technical field [0001] The invention relates to a single-cell factory for synthesizing L-phenylglycine and its construction and application, belonging to the technical field of microorganisms. Background technique [0002] Phenylglycine and its derivatives are important pharmaceutical intermediates, which can be used in the synthesis of ampicillin, cephalexin, cefaclor, amoxicillin, benzamicillin and other lactam antibiotics. O-chlorophenylglycine is an important intermediate in the synthesis of the antiplatelet inhibitor clopidogrel. In addition, phenylglycine is also an important intermediate for the synthesis of polypeptide hormones and various chiral pesticides. With the rapid development of my country's pharmaceutical and chemical industry, it is believed that the demand for phenylglycine and its derivatives will continue to increase, which has a wide range of applications. market. [0003] The synthesis methods of L-phenylglycine mainly include chemical synthesis meth...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/70C12P13/04C12R1/19
CPCC12N9/0008C12N9/0016C12P13/04C12Y102/01002C12Y104/01009
Inventor 饶志明刘巧利杨套伟周俊平张显徐美娟
Owner JIANGNAN UNIV
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