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Engineering bacterium for producing caffeine by fermentation, and establishment method and application thereof

A technology of engineering bacteria and caffeine, which is applied in the field of genetic engineering and microbial fermentation, can solve the problems that Corynebacterium glutamicum caffeine has not been found, and achieve the effect of less separation and purification steps and high yield

Active Publication Date: 2016-08-17
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there has not been any report on the production of caffeine by fermentation of Corynebacterium glutamicum

Method used

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  • Engineering bacterium for producing caffeine by fermentation, and establishment method and application thereof
  • Engineering bacterium for producing caffeine by fermentation, and establishment method and application thereof
  • Engineering bacterium for producing caffeine by fermentation, and establishment method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Construction of Caffeine-producing Corynebacterium glutamicum Recombinant Engineering Bacteria

[0027] 1. Cloning of key enzyme genes in the biosynthetic pathway of tea tree caffeine

[0028] The original sequences of the four target genes in this example are from GenBank, wherein the accession number of gene TAMPD is AB480359963, the accession number of gene TIDH is AB156763654, the accession number of gene CaXMT is AB048793, and the accession number of gene TCS1 is AB031280.

[0029] First, in order to enable the normal expression of the inserted TAMPD, TIDH, CaXMT and TCS1 genes in Corynebacterium glutamicum, it is necessary to construct four expression elements containing promoter, ribosome binding site (RBS sequence), operator and terminator expression box. At the same time, according to the codon usage frequency of Corynebacterium glutamicum, the codons of the four target gene sequences were optimized, and then a section of RBS sequence was designed in...

Embodiment 2

[0035] Example 2 Production of caffeine by fermentation of recombinant Corynebacterium glutamicum

[0036] 1. Preparation of seed solution

[0037] The formula of the seed medium used was: peptone 10g / L, yeast extract 5g / L, NaCl 10g / L, pH value 7.0, high temperature sterilization at 121°C for 20min.

[0038] The preparation method of the seed solution: pick and inoculate the activated fresh recombinant engineered bacteria into the seed medium containing 25 μg / mL kanamycin, shake and culture at 30° C. and 200 rpm for 12 hours to obtain the seed solution.

[0039] 2. Fermentation and cultivation of recombinant Corynebacterium glutamicum

[0040] The fermentation medium formula used is: (NH 4 ) 2 SO 4 20g / L, K 2 HPO 4 1g / L, KH 2 PO 4 1g / L, MgSO 4 ·7H 2 O 250mg / L, MOPS 42g / L, urea 5g / L, vitamin H 0.2mg / L, thiamine 500μg / L, trace elements 0.2mg / L, glucose 25g / L, pH 7.0-7.2. Among them, the composition of trace elements is as follows: FeSO 4 ·7H 2 O 10g / L, MnSO 4 ·7...

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Abstract

The invention provides an engineering bacterium for producing caffeine by fermentation. The engineering bacterium is a recombinant corynebacterium glutamicum which is established by guiding a key enzyme gene in a caffeine biosynthetic pathway into a corynebacterium glutamicum. The key enzyme gene in the caffeine biosynthetic pathway is over-expressed, so that the corynebacterium glutamicum is capable of producing the caffeine; and moreover, a large number of caffeine as products can be secreted out of cells, separation and purification steps are few, yield of the caffeine is high, and a flask fermentation result shows that the yield of the caffeine reaches 3.75 mg / L, so that the recombinant engineering bacterium has a good industrial application prospect, and lays a theoretical foundation for producing food safety-level caffeine by fermentation by using the corynebacterium glutamicum.

Description

technical field [0001] The invention relates to the field of genetic engineering and microbial fermentation, in particular to an engineering bacterium for fermenting and producing caffeine, its construction method and application. Background technique [0002] Caffeine, namely 1,3,7-trimethylxanthine, is the main component of purine bases in the three major soft drink plants (tea tree, coffee and cocoa) that are popular in the world. Studies have found that at least 60 kinds of plants in the world contain caffeine, among which tea (Camellia sinensis) 2%-5%, coffee beans (Coffea arabica) 1%-2%, cocoa (Theabronma cacao) 0.03%, Sudan Kola nut (Cola acuminata) 1.5%, Paullinia capana (Paullinia capana) 4% or more, Yerba mate (Ilex paraguariensis) 0.7%, etc. [0003] As an important plant secondary metabolite, caffeine has physiological functions such as antibacterial and biological invasion resistance. Caffeine is an indispensable substance for the growth of caffeine-rich plants...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/77C12P17/18C12R1/15
CPCC12N9/0006C12N9/1007C12N9/78C12P17/182C12Y101/01205C12Y201/0116C12Y305/04006
Inventor 张正竹李萌萌邓威威宁井铭邓骋
Owner ANHUI AGRICULTURAL UNIVERSITY
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