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Genetic recombinant engineering bacterium and application thereof in catalytic synthesis of D-pantolactone

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

Active Publication Date: 2019-11-15
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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  • Genetic recombinant engineering bacterium and application thereof in catalytic synthesis of D-pantolactone
  • Genetic recombinant engineering bacterium and application thereof in catalytic synthesis of D-pantolactone
  • Genetic recombinant engineering bacterium and application thereof in catalytic synthesis of D-pantolactone

Examples

Experimental program
Comparison scheme
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 + ( Image 6 ).

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Abstract

The invention discloses a genetic recombinant engineering bacterium and application thereof in catalytic synthesis of D-pantolactone and belongs to the field of biocatalytic research. The genetic recombinant engineering bacterium takes escherichia coli as a host bacterium, the host bacterium contains recombinant expression plasmids containing a conjugated polyketide reductase gene and a glucose dehydrogenase gene, and the nucleotide sequence of the conjugated polyketide reductase gene is shown as SEQ ID NO.1. The engineering bacterium simultaneously expresses polyketide reductase and glucose dehydrogenase through induction; the engineering bacterium is applied to the asymmetric catalytic synthesis of D-pantolactone, the in-situ regeneration of a coenzyme NADPH is realized through a synergistic effect of the two enzymes, and the purpose of efficient production from keto-pantolactone to D-pantolactone can be achieved. A method simplifies a production process, avoids addition of extra coenzymes at the same time 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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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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