Bacterium for fermenting L-tryptophan from mixed saccharum and fermentation method thereof

A technology of sugar fermentation and tryptophan, applied in the field of microorganisms, can solve the problems of complicated operation and difficult realization, and achieve the effect of translation enhancement

Active Publication Date: 2014-12-03
新疆梅花氨基酸有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above method requires additional cloning of large fragments and transformation of the promoter on the basis of the production bacteria, and the operation is complicated and difficult to implement.
[0006] Wild-type E. coli can utilize pentose sugars such as L-arabinose or D-xylose as a carbon source, but the associated metabolism is tightly regulated

Method used

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  • Bacterium for fermenting L-tryptophan from mixed saccharum and fermentation method thereof
  • Bacterium for fermenting L-tryptophan from mixed saccharum and fermentation method thereof
  • Bacterium for fermenting L-tryptophan from mixed saccharum and fermentation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1c

[0057] The acquisition of embodiment 1crp* gene

[0058] Synthetic primers PW037 / PW038 (as shown in SEQ ID NO: 3, 4), using the Phusion high-fidelity polymerase kit of NEB company, using the chromosome of E.coli strain MG1655 as a template, PCR amplified to obtain a DNA fragment containing the crp gene , after purification, it was digested by BamHI / SalI enzyme digestion, connected to the pACYC184 vector that was also digested by BamHI / SalI enzyme digestion, and a new plasmid was constructed and named pW13.

[0059] Using pW13 as a template and PW071 / PW072 (as shown in SEQ ID NOs: 5 and 6) as primers, PCR amplification was performed. After the PCR product was treated with Dpn I, it was directly transformed into competent cells, and the vector pW13D containing the crp* gene fragment was obtained. The structure is shown in figure 1 .

[0060] At the same time, the cohesive ends of the pACYC184 vector digested with BamHI / SalI were completed to obtain pACYC184δTc, that is, the pA...

Embodiment 2

[0061] Growth and sugar consumption ability of embodiment 2 L-tryptophan producing bacteria in glucose and pentose mixed medium

[0062] L-tryptophan-producing bacteria E. coli strain MHZ-0800 (pMG43 / SA01) was used as a parent strain to evaluate the fermentation of a mixture of glucose and pentose to produce L-tryptophan, and its preservation number is CGMCC No.6863.

[0063] respectively by using CaCl 2 The conventional method used pW13D plasmid and vector / pACYC184δTc (pACYC184 plasmid with Tc resistance gene removed) to transform strain MHZ-0800 to obtain strains MHZ-0820 (pMG43 / SA01 / pW13D) and MHZ-0821 (pMG43 / SA01 / / pACYC184δTc) .

[0064] Take E.coli strains MHZ-0820 and MHZ-0821 from the cryopreservation tube and streak on LB plate (Tc, Cm resistance), culture at 37°C for 18-24hr; scrape off a ring of bacteria from the plate and inoculate Put 50mL of seed medium (Tc, Cm resistance) into the shake flask, 37℃, rotation speed 240rpm, culture for 5-10 hours, OD600 is control...

Embodiment 3

[0068] Example 3 Production of L-tryptophan by fermentation in a mixture of glucose and pentose sugars using L-tryptophan-producing bacteria

[0069] Take E.coli strains MHZ-0820 and MHZ-0821 from the cryopreservation tube and streak on LB plate (Tc, Cm resistance), culture at 37°C for 18-24hr; scrape off a ring of bacteria from the plate and inoculate Put 50mL of seed medium (Tc, Cm resistance) into the shake flask, 37℃, rotation speed 240rpm, culture for 5-10 hours, OD600 is controlled at 6-10; transfer 2mL of seed liquid to 20mL of fermentation medium (Tc, Cm resistance) shake flask, reciprocating shaker 37 ℃, 120rpm fermentation culture. During the cultivation process, dilute ammonia water was added to control the pH value of the fermentation broth to 6.5-7.0. Until the residual sugar was exhausted, the OD600 of the sample was measured at the end of the fermentation, and the L-tryptophan content was determined by HPLC. The results are shown in Table 1.

[0070] The compo...

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Abstract

The invention relates to the field of microorganisms, and in particular relates to a bacterium for fermenting L-tryptophan from mixed saccharum and a fermentation method thereof. The Enterobacteriaceae bacterium for producing L-tryptophan can have the capability of simultaneously utilizing hexose and pentose to produce the L-tryptophan because of containing the coding gene of a cAMP receptor protein mutant. The cAMP receptor protein mutant is obtained through mutating one or a plurality of amino acid loci of the cAMP receptor protein. The bacterium provided by the invention consumes glucose and xylose in a culture medium containing the glucose and the xylose at the same time, and the yield and conversion rate of the L-tryptophan are increased.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a bacterium and a fermentation method for producing L-tryptophan by fermenting mixed sugar. Background technique [0002] L-Tryptophan is one of the essential amino acids in 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, L-tryptophan is an important component of amino acid infusion and an important pharmaceutical intermediate. In the field of food applications, L-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, L-tryptophan becomes an important limiting amino acid in the diet. Supplementing exogenous L-tryptophan can improve the L-tryptophan in the diet of livestock, poultry and fish. Increas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P13/22C12R1/19
Inventor 毛贤军赵津津吴涛
Owner 新疆梅花氨基酸有限责任公司
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