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A method for increasing the expression level of 5-aminolevulinic acid

A technology of aminolevulinic acid and expression amount, applied in the field of genetic engineering, can solve the problems of host cell metabolic imbalance, reducing the synthesis level of target products, etc.

Active Publication Date: 2022-05-31
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The abundance of key enzymes and the amount of cofactors in the metabolic process need to be properly balanced, and the synthesis of cofactors also needs to be increased when the enzyme protein is overexpressed; however, when the concentration of cofactors is too high, it will cause metabolic imbalance in the host cell, which will reduce the production of the target product. Synthesis level (Lv Xueqin, Acta Bioengineering, 2021, 37(5): 1619-1636)

Method used

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  • A method for increasing the expression level of 5-aminolevulinic acid
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  • A method for increasing the expression level of 5-aminolevulinic acid

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Experimental program
Comparison scheme
Effect test

Embodiment 1

1. the plasmid construction of pykA overexpression

1.1pykA codon optimization

[0024] The pykA sequence was optimized, and 2 new sequences of pykA were screened, named respectively pykA1 and pykA2, and synthesized.

The construction of 1.2 pykA overexpression elements

Take C. glutamicum ATCC 13032 chromosome as template, 2 pairs of primers (P

tuf

‑F: 5‑GTTCCTGGGCTTT

GAGCATGGCCGTTACCCTGCGA‑3 and P

tuf

[0031] The plasmid pYJ1 was digested with endonucleases ApaI and AarI, and large fragments of DNA were recovered. Applied DNA Seamless Recombination Reagent

CCTGGACTTC‑3, T

sod

‑F: 5‑ GTTTAAACATCTCAGCATTAATTAAGCATTTTTAGTACGTGCAATAACC‑3 and

T

sod

‑R: 5‑ GCCCATCCGTAAATTCTCGCTTGCCACAGCTGGCCT‑3 respectively amplifies the strong promoter P

tuf

, terminator

Tsod

, and the PCR products were named as P

tuf

, T

sod

. with P

tuf

‑F and T

sod

‑R is a primer, and the PCR product is separated by Overlap‑PCR

P

tuf

, T

sod

Fusion is perf...

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Abstract

The invention relates to the field of genetic engineering, in particular to a method for improving the expression level of 5-aminolevulinic acid. The method comprises the steps of: overexpressing ALA synthase gene in Corynebacterium glutamicum hemA and the gene encoding pyridoxal kinase pykA . The present invention optimizes hemA sequence, which improves the level of ALA synthesis; at the same time optimizes key genes in the synthesis pathway of cofactors required by ALA synthase pykA and overexpressed the sequence of the pykA sequence.

Description

A kind of method for improving 5-aminolevulinic acid expression amount technical field The present invention relates to the field of genetic engineering, be specifically related to a kind of method that improves 5-aminolevulinic acid expression amount. Background technique 5-aminolevulinic acid (5-Aminolevulinic acid, 5-ALA) is a kind of widely existing in animals, plants and micro-organisms. Non-protein amino acids in biological cells are the precursors of various compounds (chlorophyll, heme, porphyrin and vitamin B12, etc.). body. 5‑ALA has important applications in many fields and has broad application and market development prospects. In the feed industry, it can be used as As an animal feed additive to promote animal health and growth; in agriculture, as a biological fertilizer additive to promote crop growth Disease resistance and production increase; in the pharmaceutical industry, it is used in tumor localization and photodynamic diagnosis and treatment of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/53C12N15/54C12R1/15
CPCC12N9/0016C12N9/1029C12N9/1205C12Y104/01002C12Y203/01037C12Y207/01035C12N2800/22C12N2800/101
Inventor 黄火清刘波张红莲徐欣欣王苑杨浩萌涂涛柏映国罗会颖姚斌
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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