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Method for producing L-tyrosine recombinant engineering bacteria and application of recombinant engineering bacteria

A technology of recombining engineering bacteria and tyrosine, which is applied in the biological field to achieve the effects of reducing production costs, accelerating industrial production, and improving economic benefits

Inactive Publication Date: 2018-04-17
THE FRONTIER TECH RES INST OF TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to provide the recombinant engineered bacteria producing L-tyrosine and Danshensu aiming at the current production status of L-tyrosine and Danshensu

Method used

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  • Method for producing L-tyrosine recombinant engineering bacteria and application of recombinant engineering bacteria
  • Method for producing L-tyrosine recombinant engineering bacteria and application of recombinant engineering bacteria
  • Method for producing L-tyrosine recombinant engineering bacteria and application of recombinant engineering bacteria

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Embodiment 1

[0017] Embodiment 1: Construction of L-tyrosine recombinant engineering strain

[0018] Using SyBE-002447 as the starting strain, the gene fragment P was synthesized at the position of the paa gene cluster on the genome by λred homologous recombination lac -aroG-tyrA-aroE, integrate P at the position of LacI gene trc -pps-tkt-glk fragment, resulting in the final chassis strain SyBE-22002. The specific process is as follows:

[0019] (1) use P lac -aroG-tyrA-aroE replaces the paa gene cluster on the chromosome of SyBE-002447. Using the Escherichia coli SyBE-002447 genome as a template, design upstream homology arm primers F1-F and F1-R, use the sequence shown in SEQ ID No.1 as the upstream primer, and use the sequence shown in SEQ ID No.2 as the downstream primer; Design downstream homology arm primers F4-F and F4-R, with the sequence shown in SEQ ID No.9 as the upstream primer, and with the sequence shown in SEQ ID No.10 as the downstream primer;

[0020] 5'-GAAGTCGCTGCAC...

Embodiment 2

[0067] The construction of embodiment 2 Danshensu synthetic bacterial strains

[0068] A tandem tac promoter was designed (P tacs ) drives co-expression of hpaBC and ldh genes, and puts P tacs -hpaBC-ldh was integrated into the chromosome of the chassis strain SyBE-22002 obtained in Example 1, and the integration site was selected downstream of nupG on the genome. Specific steps are as follows:

[0069] (1) Construction of recombinant fragment F9-F10-F11-F12

[0070] The construction steps of the recombinant fragment F9-F10-F11-F12 were similar to those of the integrated fragment F1-F2-F3-F4 in Example 1. Using the integrated strain SyBE-22002 genome as a template, design upstream homology arm primers F9-F and F9-R, with the sequence shown in SEQ ID No.20 as the upstream primer, and with the sequence shown in SEQ ID No.21 as the downstream primer; design The downstream homology arm primers F12-F and F12-R, the sequence shown in SEQ ID No.27 is used as the upstream primer, ...

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Abstract

The invention provides a method for producing L-tyrosine recombinant engineering bacteria and application of the recombinant engineering bacteria. The method comprises the following steps: taking escherichia coli SyBE-002447 as a starting strain and integrating total chemical synthesis modules Plac-aroG-tyrA-aroE and Ptrc-pps-tkt-glk respectively, so as to construct a chassis strain SyBE-22002 ofL-tyrosine; then integrally inserting a total chemical synthesis module Ptacs-hpaBC-ldh into a nupG downstream position on a chromosome, so as to obtain a strain SyBE-22011. By adopting the method provided by the invention, the problem that antibiotics and an induction agent isopropyl-beta-D-thiopyrangalactoside need to be added in a fermentation process is solved, and the L-tyrosine and tanshinolcan be efficiently produced; a downstream separation and purification process is simplified and the industrial production cost is reduced; pollution, caused by the antibiotics and the induction agent, to the environment is avoided and the economic benefits are improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the construction of an L-tyrosine recombinant engineering bacterium and a method for producing L-tyrosine and danshensu. Background technique [0002] L-tyrosine is an important nutritional essential amino acid, which plays an important role in the metabolism, growth and development of humans and animals. It is often used as a nutritional supplement for patients with phenylketonuria, as well as raw materials for the preparation of polypeptide hormones, antibiotics, L-dopa, melanin, p-hydroxycinnamic acid, p-hydroxystyrene, etc., and is widely used in food, feed, and medicine. and chemical industries. Based on this, the synthesis of L-tyrosine and its derivatives has attracted the attention of more and more researchers in recent years. Because the ability of natural microorganisms to synthesize and accumulate L-tyrosine is very low, even if the recombinant engineered bac...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12R1/19
CPCC12P7/42C12P13/225
Inventor 赵广荣周亮丁琦王海燕
Owner THE FRONTIER TECH RES INST OF TIANJIN UNIV
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