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Escherichia coli engineering bacteria capable of realizing high yield of L-tryptophan

A technology of Escherichia coli and engineering bacteria, applied in the field of microbial breeding and microbial fermentation, can solve the problems of large energy consumption, unfavorable industrialization, waste of intracellular resources, consumption of intracellular resources, etc., and achieves easy industrialization control, simple breeding scheme, and fermentation. short time effect

Active Publication Date: 2012-05-16
SHANDONG LUKANG SHELILE PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some may express metabolic burden due to excessive expression of enzymes, some may have too high a plasmid copy number, consuming too much intracellular resources; The conversion is not high; some may have high yields, but the fermentation time is too long and the energy consumption is large, which is not conducive to industrialization

Method used

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  • Escherichia coli engineering bacteria capable of realizing high yield of L-tryptophan
  • Escherichia coli engineering bacteria capable of realizing high yield of L-tryptophan
  • Escherichia coli engineering bacteria capable of realizing high yield of L-tryptophan

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction of Escherichia coli Engineering Bacteria with High Production of L-Tryptophan

[0029] 1. tnaA, trpR, tyrR gene knockout

[0030] Using the Red-mediated recombination engineering system, Escherichia coli W3110 was used as the starting bacterium to introduce the heat-sensitive plasmid pKD46, and the kan-sacB reverse selection system was used to knock out the tnaA gene. The method is as follows: figure 1 As shown, the specific method is:

[0031] 1) tnaA gene knockout

[0032] Primers and gene fragments used:

[0033] ptnapuc-1 5'-ATTGAGCCAGTAAAACGTACCACTCGCGCTTCTGACACATGCAGCTCCCGGAGACG-3'

[0034] ptnapuc-2 5'-CGCCAGCATATCGGCATATTTGTAGGTTTCGTGAGCGGATAACAATTTCACACAGGAAAC-3'

[0035] ptnaA 5'-ATTGAGCCAGTAAAACGTACCACTCGCGCTGAAACCTACAAAATATGCCGATATGCTGGCG-3'

[0036] Using ptnapuc-1 and ptnapuc-2 as primers, and using the plasmid pUC18-KS as the template, the PCR product was electrotransformed into Escherichia coli W3110 (pKD46), cultured on a 50m...

Embodiment 2

[0118] Example 2 Fermentation of Escherichia coli Engineering Bacteria with High Production of L-Tryptophan

[0119] Take the four L-tryptophan-producing strains obtained in Example 1 as an example.

[0120] (1) Medium:

[0121] Shake flask seed medium (1L): Yeast powder 5g, peptone 10g, NaCl 10g, add water to prepare;

[0122] Primary seed medium (1L): glucose 10g, ammonium sulfate 5g, MgSO 4 1g, trisodium citrate 1g, potassium dihydrogen phosphate 5g, yeast extract 5g, CaCl 2 0.02g, FeSO4 7H 2 O 0.05g, trace elements 1ml, pH 6.8;

[0123] Among them, trace element solution (1L): Na 2 MoO 4 2H 2 O 0.15g, H 3 PO 3 1g, CoCl 0.5g, CuSO 4 0.5g, MnCl 0.3g, ZnSO 4 1.2g.

[0124] Fermentation medium (1L): glucose 20g, ammonium sulfate 10g, MgSO 4 2g, trisodium citrate 1g, potassium dihydrogen phosphate 10g, yeast extract 8g, CaCl 2 0.2g, FeSO 4 ·7H 2 O 0.1g, trace elements 2ml, antifoaming agent 5 drops, pH 6.8.

[0125] (2) Determination of tryptophan conten...

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Abstract

The invention provides escherichia coli engineering bacteria capable of realizing high yield of L-tryptophan, which are characterized by: using escherichia coli engineering bacteria W3110 as starting bacteria; connecting expression trpEfbrDCBA genes on a gene group in series by using tac promoter mutants; meanwhile, inactivating or knocking out tnaA, trpR and tyrR genes in the escherichia coli engineering bacteria W3110, mutating the tyrA and pheA genes so as to weaken enzyme activities expressed by the tyrA and pheA genes; and, by serial technologies of transferring AroFfbr or AorGfbr, tktA and ppsA genes and medium and low copy plasmids into modified bacterial strains, and the like, industrial escherichia coli engineering bacteria, which are capable of realizing high yield of the L-tryptophan, high in glucose conversion rate and short in production and fermentation time, are combined and screened, wherein the AroFfbr or AorGfbr, tktA and pps genes have serial expressions and are from escherichia coli engineering bacteria K12, and the medium and low copy plasmids are from SerA genes of bacillus subtilis.

Description

technical field [0001] The invention belongs to the field of microbial breeding and microbial fermentation, and in particular relates to the selection and breeding of Escherichia coli strains for fermenting and high-yielding L-tryptophan. Background technique [0002] L-Tryptophan is the only aromatic amino acid with an indole branch, and its chemical name is 2-amino-3-indole propionic acid. L-Tryptophan is one of the essential and limiting amino acids in human and animal bodies. It is synthesized very little in the body and needs to be taken from the outside. It plays an important role in the growth, development and metabolism of humans and animals. At present, it is widely used in the fields of medicine, food and feed. [0003] Traditional tryptophan production methods mainly include proteolytic extraction, chemical synthesis, enzymatic conversion and microbial fermentation. The first three methods have shortcomings such as limited material sources, multi-step synthesis...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12R1/19
Inventor 于传军左良成徐宝兴徐洪利赵体金李水仙王东杰戴晓燕丁贞科
Owner SHANDONG LUKANG SHELILE PHARMA
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