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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  • Abstract
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  • Claims
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Problems solved by technology

The genetic manipulation of rhizobia itself is complex, and it is difficult to conduct large-scale gen

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

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0029]Example 1 Carrier Pcas9-pdxH KO, Pcas9-pdxH KO-pdxSTOE And Pcas9-pdxH KO-pdx1 / 2OE Construction.

[0030](1)pdxH Construction of gene knockout vector.

[0031]pdxH The knock-out mutant strain and the wild type are used as the control strain of the present invention, so firstpdxH The gene knockout vector was constructed:

[0032]Using the primer pairs Liulx-58 and Liux-59, Liulx-60 and Liulx-61 in Table 1, respectively, to E. coliEscherichia coli The MG1655 genome is used as a template and amplified by PCR to obtainpdxH The upstream homology arm Up1 and the downstream homology arm Down of the gene were verified by electrophoresis. After the nucleic acid electrophoresis gel was recovered, 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 the Up1+Down fragment 600 bp was obtained by PCR amplification. Using the primer pairs Liulx-62 and Liulx-63, Liulx-64 and Liulx-65 in Table 1, and usin...

Example Embodiment

[0039]Example 2 Construction of engineered 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]The plasmid Pcas9- in Example 1pdxH KO, Pcas9-pdxH KO-pdxST OE, Pcas9-pdxH KO-pdx1 / 2 OE(st) was transferred into E. coli MG1655 according to the following method:

[0043](1) A single colony of freshly activated E. coli wild-type WT (MG1655) was inoculated in a 5 mL LB test tube, and cultured on a shaker at 37°C and shaking at 200 rpm for 12 h;

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

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

[0046](4) Add 30 mL of pre-chilled ddH2Resuspend the cells in O, centrifuge at 4000 rpm for 10 min at 4°C, and remov...

Example Embodiment

[0057]Example 3 Determination of growth curve of strain

[0058]The wild-type strain and the engineered strain in Example 2 were used to determine the growth curve according to the following method:

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

[0060](2) Determine the OD600 of the bacterial solution in the test tube, transfer to a 50 mL LB shake flask, three each, initial OD600=0.1, shake culture at 37°C, shake at 200 rpm;

[0061](3) Using the uninoculated LB medium as a blank control, the OD600 of the culture solution at different times was measured from 0h, and the concentration of the bacterial solution was diluted and measured. The result is as followsimage 3 Shown. Among them, the growth of LL02 is basically the same as that of LL01, and the growth of LL03 is significantly better than that of LL01, but still weaker than the wild-type strain. Therefore, the RBS sequence of related enzymes in channe...

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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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