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L-tryptophan fermentation strain and method for fermentation production of L-tryptophan by using L-tryptophan fermentation strain

The technology of a fermentation strain and a production method, applied in the field of microorganisms, can solve problems such as affecting the progress of fermentation, and achieve the effects of improving yield and production efficiency, improving anthranilic acid tolerance, and satisfying large-scale industrial production.

Active Publication Date: 2015-03-04
MEIHUA BIOTECH LANGFANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the feeding amount of anthranilic acid must be basically balanced or slightly surplus with the conversion rate of tryptophan. A slight inappropriate feeding will affect the fermentation.

Method used

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  • L-tryptophan fermentation strain and method for fermentation production of L-tryptophan by using L-tryptophan fermentation strain
  • L-tryptophan fermentation strain and method for fermentation production of L-tryptophan by using L-tryptophan fermentation strain
  • L-tryptophan fermentation strain and method for fermentation production of L-tryptophan by using L-tryptophan fermentation strain

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1: NTG mutagenesis to obtain anthranilic acid tolerant strains

[0045] The starting strain is according to the method described in patent WO 87 / 01130 and Mascarenhas D et al. The constructed tryptophan-producing strain MHZ-0800 (CGMCC NO.6863), whose host strain SA01 is E. coli K-12 CICC 10303 tnaAserA derived from E. coli K-12 (CICC 10303), contains plasmid pMG43, which is pBR322 Derived from a plasmid containing serA, and feedback suppression removed aroG and trpEDCBA operons.

[0046] Streak the starting strain SA01 on the LB plate, culture at 37°C for 16-24hr, pick a single colony from the plate, inoculate it into a Erlenmeyer flask containing 5mL of LB medium, and cultivate it at 37°C with shaking at 150-240rpm for 2- 6hr, until the OD600 value is between 0.1-0.8, centrifuge at 6000rpm for 5-10min, discard the supernatant, resuspend and wash twice in 0.9% normal saline, centrifuge at 6000rpm for 5-10min, discard the supernatant, the final concentration is...

Embodiment 2

[0048] Example 2: Anthranilic acid tolerance screening of NTG mutagenized strains

[0049] The obtained mutagenized strains were cultured in M9 liquid medium containing 5g / L anthranilic acid to further verify their tolerance. Activate it on an LB plate, culture it at 37°C for 16-24hr, pick a single colony and inoculate it into a Erlenmeyer flask containing 5mL of LB medium, cultivate overnight at 37°C with shaking at 150-240rpm, transfer to In the M9 medium containing 5g / L anthranilic acid, culture at 37°C with shaking at 150-240rpm, measure the OD every 4hr 600 value, according to OD 600 The growth curve of each strain was drawn. Dozens of mutagenic strains with significant growth advantages were screened and obtained, among which strain SA16 with better tolerance had a growth curve as follows: figure 2 shown.

[0050] Depend on figure 2 It can be seen that compared with the starting strain SA01, the strain SA16 obtained by mutagenesis has obvious growth advantages in ...

Embodiment 3

[0051] Example 3: Production of L-tryptophan by shake flask fermentation of anthranilic acid tolerant strains

[0052] Take the anthranilic acid-tolerant strain SA16 from the cryopreservation tube and activate it on the LB plate to make competent cells, transform the plasmid pMG43 into the anthranilic acid-tolerant strain SA16, and obtain dozens of strains producing tryptophan bacteria. For the preparation and transformation methods of competent cells, refer to page 96, Chapter 1 of "Molecular Cloning Experiment Guide III".

[0053] Among them, the engineering strain MHZ-0830 (pMG43 / SA16) is preserved in China Microorganism Culture Collection Center, and the preservation number is CGMCC No.7941.

[0054] Its tryptophan production ability was verified by shake flask fermentation method. Take the E.coli strain MHZ-0800 and the anthranilic acid tolerance engineering strain MHZ-0830 from the cryopreservation tube to activate on the LB plate (10μg / mL tetracycline), culture at 37°...

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Abstract

The present invention relates to the field of microbiology, and discloses an L-tryptophan fermentation strain and a method for fermentation production of L-tryptophan by using the L-tryptophan fermentation strain. According to the present invention, the L-tryptophan fermentation strain with the preservation number of CGMCC No.7941 has anthranilic acid tolerance; and compared with the original strain, the strain of the present invention has the better anthranilic acid tolerance, and can make the more anthranilic acid be converted into the tryptophan, such that the high concentration tryptophan can be accumulated so as to increase the yield and the production efficiency of the L-tryptophan and meet the large-scale industrial production of the L-tryptophan.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to an L-tryptophan fermentation strain and a method for producing L-tryptophan by fermentation. Background technique [0002] L-Tryptophan is one of the essential amino acids for human and animal life activities. It plays an important role in the growth, development and metabolism of humans and animals. It is widely used in medicine, food and feed, etc. In the field of medicine, tryptophan is an important component of amino acid infusion and an important pharmaceutical intermediate. In the field of food applications, tryptophan can be used to strengthen food, improve flavor, and can also be used in bread to promote fermentation. In the field of feed addition, when lysine and methionine are satisfied, tryptophan becomes an important limiting amino acid in diets. Supplementing exogenous tryptophan can increase the content of tryptophan in livestock, poultry and fish diets, and improve ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P13/22C12R1/19
CPCC12P13/227C12N1/205C12R2001/19
Inventor 毛贤军赵津津吴涛
Owner MEIHUA BIOTECH LANGFANG CO LTD
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