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Recombinant saccharomyces cerevisiae and application thereof to glutamine dipeptide synthesis

A technology for recombining Saccharomyces cerevisiae and glutamate dipeptide, applied in the biological field, can solve the problems of long reaction time, low enzyme activity, low production efficiency, etc., and achieve the effects of avoiding the use of antibiotics, high molar conversion rate, and reducing production costs.

Active Publication Date: 2017-05-31
INNOBIO CORP LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this ligase is an ATP-dependent enzyme and needs to provide ATP from an external source, so that the production cost of the dipeptide is still high, and it cannot be widely used in the market [Tabata K, Hashimoto S.Fermentative production of L-alanyl-L-glutamine by a Metabolically engineered Escherichia coli strain expressing L-amino acidα-ligase.Applied and environmental microbiology,2007,73(20):6378-6385.]
[0004] CN105274174A and CN104480075A disclose two methods of using biological enzymes to catalyze the production of glucosinolate dipeptide, which respectively use catalyzed enzyme buffer solution or lyophilized powder of biological enzymes to convert the substrate into glucosamine dipeptide, but the cost of enzyme separation and purification is high, the enzyme activity is low, and it is difficult to Recycling, thus limiting the possibility of its industrial application
However, since the expressed transesterase is located in Escherichia coli cells, there is a large transmembrane resistance of the substrate and the product, resulting in a very low yield of glucodipeptide, only 0.49g / L, and a long reaction time, which is far from meeting industrialization requirements. need
In order to reduce the resistance of the substrate entering the cell and the product diffusing out of the cell, it has been reported that the recombinant E. coli was treated with chemical reagents, which reduced the transmembrane resistance of the substrate to a certain extent (Appl.Environ.Microbiol.2007:6378– 6385), or increase the cell density in the catalytic reaction system to increase the reaction rate, but the reaction time is still long and the production efficiency is low (Biosci, Biotechnol, Biochem.2013,77:618-628)
At the same time, dipeptide is mainly used in the fields of medical care and other fields. Using Escherichia coli to synthesize dipeptide may cause biological safety problems such as endotoxin pollution, and it is difficult to apply it in industrial production.

Method used

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  • Recombinant saccharomyces cerevisiae and application thereof to glutamine dipeptide synthesis
  • Recombinant saccharomyces cerevisiae and application thereof to glutamine dipeptide synthesis
  • Recombinant saccharomyces cerevisiae and application thereof to glutamine dipeptide synthesis

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preparation example Construction

[0025] The amino acid mixture mentioned in the preparation of the above seeds and induction medium contains the following components: Arginine 0.2g / L, Aspartic acid 1.0g / L, Glutamic acid 1.0g / L, Isoleucine Acid 0.3g / L, Lysine 0.3g / L, Valine 1.5g / L, Methionine 0.2g / L, Phenylalanine 0.5g / L, Serine 3.75g / L, Tyrosine 0.3g / L And adenine 0.4g / L.

[0026] On this basis, the present invention further provides a biosynthetic method of glucodipeptide, which consists in the step of using the transformation reaction of the above-mentioned recombinant Saccharomyces cerevisiae of the present invention to the substrate.

[0027] The biosynthesis of CG dipeptide is a relatively mature technology, and its substrate can be described as a substrate comprising a carboxyl component and an amine component according to the prior art. In the present invention, the carboxyl component is selected from amino acid esters and amino acid Amide, most preferably L-alanine methyl ester hydrochloride; said am...

Embodiment 1

[0052] Construction of recombinant yeast cells:

[0053] According to the known nucleotide sequence (SEQ ID NO.5), the upstream and downstream primers SEQ ID NO.3 / 4 were designed to be derived from the genus Sphingobacterium sp. The genome of the strain Sphingobacterium siyangensis (CGMCC strain number: 1.6855) was used as a template to amplify the biological enzyme gene (SEQ ID NO.1).

[0054] Among them, the PCR reaction system (50μL):

[0055]

[0056] PCR reaction conditions:

[0057]

[0058] After the PCR reaction, 2 μL of the PCR product was taken for agarose gel electrophoresis detection, and the amplified PCR product was purified using a PCR product purification kit (OMEGA, USA) and stored in a -20°C refrigerator for later use.

[0059] The PCR product and the expression vector pYD1 were digested and purified and ligated overnight, and the ligated product was transformed into E.coli DH5α competent cells, and the transformants were screened on the plate of LB-r...

Embodiment 2

[0061] Fermentation culture of recombinant yeast cells:

[0062] The recombinant yeast cells were inoculated into the seed medium, and the cells were activated at 30°C and 180rpm. Then transfer to the seed medium for expansion culture, 30°C, 180rpm shaking culture. After growing to a suitable biomass, they were inoculated into 1.0L induction medium and cultured at 20°C with 150rpm aeration. After 16 to 24 hours, put into the tank and collect the cells by centrifugation. It is the recombinant yeast cell used in the present invention.

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Abstract

The invention discloses a recombinant saccharomyces cerevisiae and application thereof to glutamine dipeptide synthesis. The recombinant saccharomyces cerevisiae contains an exogenous gene with the nucleotide sequence shown as SEQ ID NO.1. The invention further discloses a recombinant vector containing the gene, the recombinant saccharomyces cerevisiae and a method for glutamine dipeptide synthesis through biotransformation by using the recombinant saccharomyces cerevisiae. The condition that saccharomyces cells still have high catalytic activity after the multi-time circulation utilization is discovered; the obvious industrial advantages are realized. The method and the application provided by the invention have the advantages that the raw materials are cheap and can be easily obtained; the operation is simple and convenient and can be easily controlled; the synthesis path is green and efficient; the biosecurity is high; the reaction speed is high; the conversion rate is high, and the like; The foundation is laid for the industrial production of the glutamine dipeptide.

Description

technical field [0001] The invention relates to a simple and efficient microbial synthesis method of glucuronide dipeptide, in particular to a method for biotransforming and synthesizing glucoglutinate dipeptide using recombinant genetically engineered yeast, and belongs to the field of biotechnology. Background technique [0002] L-Ala-Gln has great application value in medical care and other fields, and its higher solubility and good thermal stability have gradually replaced glutamine (L-Gln) as the main parenteral nutrition drug. However, the known methods for synthesizing dipeptide mainly include chemical synthesis and chemical plus biological enzyme catalysis. In the process of producing glutamic dipeptide by chemical synthesis, it is generally necessary to introduce and remove protective groups, and there are problems such as many reaction steps, many by-products, high toxicity of reagents, and non-environmental friendliness [Chinese patent, synthetic method of dipepti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P21/02C12R1/865
CPCC07K5/06026C12N9/93C12N15/81C12N2800/102C12P21/02C12Y603/02009
Inventor 袁文杰李益民范超吴文忠
Owner INNOBIO CORP LTD
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