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Single cell factory capable of efficiently synthesizing L-phenylglycine as well as construction and application of single cell factory

A single-cell, factory technology, applied in the field of microorganisms, can solve the problems of affecting the transformation stability, high cost, cumbersome process, etc., and achieve the effects of low culture cost, improved transformation efficiency, and simple operation

Active Publication Date: 2018-06-01
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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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  • Single cell factory capable of efficiently synthesizing L-phenylglycine as well as construction and application of single cell factory

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 competent c...

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 capable of efficiently synthesizing L-phenylglycine as well as construction and application of the single cell factory and belongs to the technical fieldof microorganisms. Firstly, efficient expression of leucine dehydrogenase obtained from Bacillus cereus in escherichia coli is realized, and site-directed mutation is carried out to obtain a mutant N71S with a remarkably improved reduction property; a mutant enzyme and a formate dehydrogenase mutant are co-expressed in the escherichia coli to form an intracellular in-situ co-factor NADH (Nicotinamide Adenine Dinucleotide) circulating system; the expression amount of the formate dehydrogenase mutant is optimized and controlled through a promoter and an RBS (Ribosomal Binding Site) sequence to successfully construct a recombinant escherichia coli single cell factory; the single cell factory is subjected to whole-cell conversion to prepare the L-phenylglycine. The method disclosed by the invention has the advantages of simple and rapid conversion process, low cost, no byproduct and easiness for separation and purification; when conversion is carried out in a 5L fermentation tank for 4h, the yield of the L-phenylglycine can reach 105.7g / l, the conversion rate is 93.3 percent and the space-time yield of the L-phenylglycine is 26.3g / L; an actually practical and effective strategy is provided for industrial production of the L-phenylglycine.

Description

technical field [0001] The invention relates to a single-cell factory for efficiently 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 sy...

Claims

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Application Information

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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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