Metabolic engineering strain for producing vitamin B6 as well as construction method and application of metabolic engineering strain

A construction method and metabolic engineering technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve problems such as limiting vitamin B6 production research, difficult large-scale genetic research, and complex genetic manipulation of rhizobia

Active Publication Date: 2021-02-19
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The genetic manipulation of rhizobia itself is complex, and it is difficult to conduct large-scale genetic research, which greatly limits the research on rhizobia as chassis cells for vitamin B6 production

Method used

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  • Metabolic engineering strain for producing vitamin B6 as well as construction method and application of metabolic engineering strain
  • Metabolic engineering strain for producing vitamin B6 as well as construction method and application of metabolic engineering strain
  • Metabolic engineering strain for producing vitamin B6 as well as construction method and application of metabolic engineering strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Vector Pcas9- wxya KO, Pcas9- wxya KO- wxya and Pcas9- wxya KO- pdx1 / 2OE build.

[0030] (1) wxya Construction of gene knockout vectors.

[0031] wxya The gene knockout mutant strain is used as the control bacterial strain of the present invention together with the wild type, so at first to wxya Gene knockout vectors were constructed:

[0032] Using the primers in Table 1 to Liulx-58 and Liulx-59, Liulx-60 and Liulx-61, respectively, to Escherichia coli Escherichia coli The MG1655 genome was used as a template and amplified by PCR to obtain wxyaThe upstream homology arm Up1 and the downstream homology arm Down of the gene were verified by electrophoresis, and after recovery from the nucleic acid electrophoresis gel, purified Up1 and Down fragments of 300 bp each were obtained. The Up1 and Down fragments were fused with the primer pair Liulx-58 and Liulx-61, and a 600 bp Up1+Down fragment was obtained by PCR amplification. Using the primer ...

Embodiment 2

[0039] Example 2 Construction of engineering strains containing plasmid vectors.

[0040] Medium formula:

[0041] LB medium: sodium chloride 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, solid medium plus agar powder 2%.

[0042] With the plasmid Pcas9- in embodiment 1 wxya KO, Pcas9- wxya KO- wxya OE, Pcas9- wxya KO- pdx1 / 2 OE (st) was transferred into Escherichia coli MG1655 according to the following method:

[0043] (1) Inoculate a single colony of freshly activated Escherichia coli wild-type WT (MG1655) into a 5 mL LB test tube, and incubate in a shaker at 37°C at 200 rpm for 12 h;

[0044] (2) Transfer 1% of the inoculum to a 50 mL LB shaker flask, culture in a 37°C incubator, shake at 200 rpm until the OD600 is around 0.4~0.6;

[0045] (3) Transfer the bacterial solution to a 50 mL centrifuge tube, and after 30 min in ice bath, centrifuge at 4000rp for 10 min at 4°C, and remove the supernatant;

[0046] (4) Add 30 mL pre-cooled ddH 2 O Resuspend the cel...

Embodiment 3

[0057] The growth curve determination of embodiment 3 bacterial strains

[0058] Wild-type bacterial strain and engineering bacterial strain in embodiment 2 measure growth curve according to the following method:

[0059] (1) Take freshly activated WT, LL01, LL02, and LL03 in 5 mL LB test tubes, shake at 37°C, and culture at 200 rpm for 15 h;

[0060] (2) Measure the OD600 of the bacterial liquid in the test tube, transfer to 50 mL LB shake flasks, three for each, initial OD600=0.1, shake at 37°C, shake at 200 rpm;

[0061](3) Use the uninoculated LB medium as a blank control, measure the OD600 of the culture solution at different times from 0h, and measure the bacterial solution with a high concentration after dilution. The results are as follows: image 3 shown. Among them, the growth of LL02 is basically the same as that of LL01, and the growth of LL03 is obviously better than LL01, but still weaker than that of the wild-type strain. Therefore, the RBS sequence of the rel...

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Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a metabolic engineering strain for producing vitamin B6 as well as a construction method and application ofthe metabolic engineering strain. Two ways for synthesizing vitamin B6 from the beginning, which do not exist in the same organism in the nature, are organically integrated into escherichia coli, besides, enzyme PdxH playing a bridge role is knocked out, two parallel biosynthesis channels are formed, and the channel I is an endogenous way and is used for producing a product form of the vitamin B6;and the channel II is an exogenous way for producing the active form of the vitamin B6 for the vital movement of cells. In the constructed engineering strain, the expression of an exogenous pathway is moderately improved through a strong promoter and / or an optimized RBS sequence, so that the engineering strain has the same growth rate as an original strain. The capacity of the engineering strainfor producing the vitamin B6 through fermentation is greatly improved, and the engineering strain has high application and popularization value.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a metabolic engineering bacterial strain for producing vitamin B6, its construction method and application. Background technique [0002] Vitamin B6 is widely used in medicine, food and feed industry. It is also called pyridoxine, which is an indispensable vitamin for humans or other animals, including three natural forms: pyridoxine, pyridoxal and pyridoxamine. Exist in the body as phosphate derivatives. Vitamin B6 participates in nearly a hundred enzyme reactions in its active form - pyridoxal phosphate, most of which are related to amino acid metabolism, such as transamination, decarboxylation, dehydration and transsulfurization reactions. The product form of vitamin B6 is pyridoxine hydrochloride. At present, the oxazole chemical synthesis method is mainly used in the market for artificial total synthesis. The intermediate oxazole synthesis process uses hi...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P17/12C12R1/19
CPCC12N9/0022C12N9/78C12N15/70C12P17/12C12Y104/03005
Inventor 张大伟刘林霞王岩岩
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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