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A kind of genetic recombination engineering bacteria and its application in the catalytic synthesis of d-pantolactone

A technology of gene recombination and pantothenolactone, which is applied in the field of biocatalysis research, can solve the problems of low catalytic efficiency, low expression level, and lack of recombinant cells, and achieve high catalytic activity, promotion of upgrading, and good catalytic stability.

Active Publication Date: 2021-02-09
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the gene resource of conjugated polyketide reductase is relatively scarce. At present, there are only CPR-C1 and CPR-2 derived from Candida parapsilosis IFO 0708 (Appl.Microbiol.Biotechnol.2004,64:359-366), derived from Candida orthopsilosis Gene reports such as CorCPR (J.Biotechnol.2019,291:26-34) and CduCPR (Enzyme Microb.Tech.2019,126:77-85) derived from Candida dubliniensis
Moreover, genes such as CPR-C1 and CPR-C2 are expressed at low levels in E. coli, which directly leads to low catalytic efficiency of recombinant cells.

Method used

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  • A kind of genetic recombination engineering bacteria and its application in the catalytic synthesis of d-pantolactone
  • A kind of genetic recombination engineering bacteria and its application in the catalytic synthesis of d-pantolactone
  • A kind of genetic recombination engineering bacteria and its application in the catalytic synthesis of d-pantolactone

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1 Construction of recombinant expression plasmid pACYC-CPR-GDH

[0043] The GDH gene of glucose dehydrogenase from Bacillus subtilis 168 was cloned with primers F_GDH / R_GDH. The nucleotide sequences of primers F_GDH and R_GDH are respectively:

[0044] 5'-GGAATTCCATATGTACCCGGACCTGAAAGG-3';

[0045] 5'-CCGCTCGAGTTAACCACGACCAGCCTGGA-3'.

[0046] The GDH gene was double-digested with Nde I and Xho I, and the gene fragment after digestion was recovered; at the same time, the expression plasmid pACYCDuet-1 was double-digested with Nde I and XhoI, and the plasmid fragment after digestion was recovered. T4 ligase was used for ligation, and the ligated product was transformed into the cloning host E.coli DH5α. Screen with chloramphenicol-resistant LB solid plates to select positive transformants, and the sequence results show that the recombinant plasmid after the gene sequence is correct is the recombinant expression plasmid pACYC-GDH, which is stored at -20°C for f...

Embodiment 2

[0051] Construction and catalyst preparation of embodiment 2 genetically engineered bacteria E.coli PL01

[0052] The recombinant expression plasmid pACYC-CPR-GDH constructed in Example 1 was transformed into the expression host E.coli BL21(DE3) to obtain the genetically engineered strain E.coli PL01. Inoculate E.coli PL01 in liquid LB medium (38 μg / mL chloramphenicol) in a 10 mL test tube, and shake and culture at 37° C. and 200 rpm for 12 to 16 hours. The culture solution was inoculated into 50 mL liquid LB medium (38 μg / mL chloramphenicol) in a 250 mL triangular conical flask according to the inoculum amount of 1-5%, and cultured with shaking at 37°C and 200 rpm for 2-3 hours. species density OD 600 When the value reaches 0.6-0.8, add 0.1-0.5 mM isopropylthiogalactopyranoside (IPTG), and continue culturing for 12 hours at 18° C. and 200 rpm.

[0053] The cells are collected by centrifugation for 5-10 minutes under the condition of 5000-10000×g, which is used as a catalyst...

Embodiment 3

[0054] Example 3 Optimization of reaction conditions for the catalytic synthesis of D-pantolactone by genetically engineered bacteria E.coli PL01

[0055] In a 5mL 0.1M potassium phosphate buffer (pH 6.5) reaction system containing 0.5g genetically engineered bacteria E.coliPL01 resting cells, 100mM KPL, 125mM glucose was reacted at 35°C and 200rpm for 20min.

[0056] The optimum pH value for the synthesis of D-PL catalyzed by E.coli PL01 was 6.5 ( image 3 ), the optimum reaction temperature is 35°C ( Figure 4 ), the mol ratio of adding glucose and substrate KPL in the reaction solution is 1.25:1 ( Figure 5 ), the intracellular coenzymes of genetically engineered bacteria can be self-sufficient, and there is no need to add NADP to the reaction system + ( Figure 6 ).

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Abstract

The invention discloses a gene recombination engineering bacterium and its application in catalyzing the synthesis of D-pantolactone, which belongs to the field of biocatalysis research. The genetically recombined engineering bacterium uses E. coli as a host bacterium, and the host bacterium contains a recombinant expression plasmid comprising a conjugated polyketide reductase gene and a glucose dehydrogenase gene, and the conjugated polyketide reductase gene The nucleotide sequence is shown in SEQ ID NO.1. The engineering bacterium is induced to express polyketide reductase and glucose dehydrogenase at the same time, and it is applied to the asymmetric catalytic synthesis of D-pantolactone. Through the synergistic effect of the above two enzymes, the in situ regeneration of the coenzyme NADPH can be realized The purpose of high-efficiency production from ketopantoolactone to D-pantoolactone is achieved. The method simplifies the production process, avoids the addition of extra coenzymes and reduces the production cost.

Description

technical field [0001] The invention relates to the field of biocatalysis research, in particular to a genetically engineered bacterium and a method for asymmetrically catalytically synthesizing D-pantolactone using the engineered bacterium. Background technique [0002] Pantothenic acid, also known as vitamin B5, is a water-soluble vitamin B family. It is widely found in animals and plants in nature, and beneficial bacteria in the human intestine can also synthesize it by itself. Pantothenic acid is mainly involved in the metabolism of sugar, lipid and protein in the form of coenzyme A (Coenzyme A), and is an essential nutrient for the brain and nerves. In addition, pantothenic acid has anti-lipid peroxidation effect. There are two configurations of pantothenic acid in nature, D- and L-, but only the D-type has physiological activity. Due to the poor stability of D-pantothenic acid, calcium D-pantothenate is usually used in the form of calcium D-pantothenate, which is wid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12P17/04C12R1/19
CPCC12N9/0006C12N15/70C12P17/04C12Y101/01214C12Y101/9901
Inventor 裴晓林王加跑吴益锋
Owner HANGZHOU NORMAL UNIVERSITY
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