Unlock instant, AI-driven research and patent intelligence for your innovation.

Mutant of AroG and application of mutant in amino acid producing genetically engineered bacteria

A genetically engineered bacteria, genetic engineering technology, applied in bacteria, microorganism-based methods, enzymes, etc., can solve problems such as difficulty in confirming impact, expensive raw materials, and complex processes

Active Publication Date: 2021-01-26
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
View PDF7 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical synthesis method has been gradually eliminated due to the complex process and complex product components.
Although the enzymatic conversion method and the microbial conversion method have been industrialized, there are still problems such as expensive raw materials and low conversion rates.
The microbial fermentation method of producing L-tryptophan from cheap raw materials such as glucose is the earliest developed method of L-tryptophan production, but this method cannot be industrialized for a long period of time
The reason is mainly that in the early studies, researchers only relied on traditional chemical or physical mutagenesis to breed L-tryptophan producing strains, and after multiple rounds of chemical or physical mutagenesis, it was difficult to confirm the mutagenicity The impact of secondary mutations on the strain during the mutation process, the background is relatively unclear, which hinders the further improvement of the strain

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Mutant of AroG and application of mutant in amino acid producing genetically engineered bacteria
  • Mutant of AroG and application of mutant in amino acid producing genetically engineered bacteria
  • Mutant of AroG and application of mutant in amino acid producing genetically engineered bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1, construction bacterial strain BDZ1

[0027] The wild-type genes involved in this example and their accession numbers in GenBank are: ID of aroG: 945605, ID of rpoS: 947210, ID of ppsA: 946209, ID of trpR: 948917, ID of tnaA: 948221.

[0028] Construction of PH5a-aroG-trpEDCBA plasmid: Using wild-type Escherichia coli MG1655 as a template, the aroG fragment with linker was amplified with primers aroG-F and aroG-R. Using the PH5a plasmid as a template, PH5a-M-F and PH5a-M-R amplify the PH5a-M fragment with a linker. Using the aroG fragment and the PH5a-M fragment as templates, the aroG-F and PH5a-M-R primers were amplified to obtain the aroG-M-Gibson fragment for Gibson assembly. Using MG1655 as a template, primers trpEDCBA-F and trpEDCBA-R were used to amplify the trpEDCBA-Gibson fragment with adapter. Use PH5a-ver-F and PH5a-ver-R to amplify the plasmid backbone with adapters, and use the above aroG-M-Gibson fragment and trpEDCBA-Gibson fragment to obtai...

Embodiment 2

[0039] Embodiment 2, construction bacterial strain BDZ2

[0040] DAHP synthase AroG catalyzes the formation of DAHP from two important precursors, phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), thereby entering the shikimate pathway. AroG occupies a key site in the tryptophan synthesis pathway, but AroG is subject to feedback inhibition by L-phenylalanine, so releasing the inhibitory effect of phenylalanine on AroG is critical to expanding the metabolic flux of the shikimate pathway. Aiming at the inhibitory effect of L-phenylalanine on AroG, after docking of L-phenylalanine and AroG protein (PDB: 5CKS), it was found that the 180th amino acid (S) and the 6th amino acid in the AroG protein (Q) directly formed a hydrogen bond interaction with L-phenylalanine. In order to relieve the inhibitory effect of phenylalanine, the S211 residue of AroG was replaced by F, and the benzene ring in the mutated F directly occupied part of the binding position of the inhibitor L-ph...

Embodiment 3

[0049] Example 3, construction of L-tryptophan production strain BDZ3

[0050] RpoS code σ s , is a subunit of RNA polymerase that acts as a master regulator of the adaptation of many stationary-phase genes to nutrient deprivation and other stresses in Escherichia coli. Genome-wide analysis of RpoS-dependent gene expression revealed that up to 10% of genes in E. coli are directly or indirectly regulated by RpoS. Although the RpoS regulator is a huge conserved system that is essential for adaptation to various stressful stresses, its regulatory mechanism in specific metabolic pathways, such as the L-tryptophan synthesis pathway, is still not fully understood. Therefore, the present invention introduces the mutation site Q33* to inactivate RpoS, thereby determining the influence of RpoS on the L-tryptophan synthesis pathway.

[0051] Cas9-rpoS plasmid construction: use MG1655 as template, use primers rpoS-up-F, rpoS-up-R to amplify the rpoS-up fragment with linker, and use pri...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a mutant of AroG and application of the mutant in amino acid producing genetically engineered bacteria, and discloses application of inactivation of the mutant of 3-deoxy-alpha-arabinoheptanoic acid-7-phosphate synthase AroG and a regulatory factor RpoS in construction of tryptophan producing strains. When the genetically engineered bacterium obtained by the invention is used for fermentation, effective accumulation of L-tryptophan can be obtained, the yield of the L-tryptophan can be obviously improved, and the genetically engineered bacterium has an industrial application value.

Description

technical field [0001] The invention belongs to the field of genetic engineering and microorganisms, and specifically relates to the application of AroG mutants and the inactivation of regulatory factor RpoS in the construction of tryptophan-producing genetically engineered bacteria, and the use of the same in the fermentative production of tryptophan. Background technique [0002] As an essential amino acid in the human body, L-tryptophan can synthesize important biologically active substances such as pigments, 5-hydroxytryptamine, indole, etc., and is widely used in medicine, food, feed and other industries. At present, the annual demand for tryptophan in the world market is more than 10,000 tons, and it is increasing at a rate of 10% per year. In the medical field, L-tryptophan is widely used in amino acid injections, essential amino acid drugs and additives for hydrolyzed proteins. The conversion product of L-tryptophan, 5-hydroxytryptamine, has anti-depression, improve...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N1/21C12P13/22C12R1/19
CPCC12N9/1022C12P13/227
Inventor 张大伟丁冬芹柏丹阳朱亚如
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI