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Genetic engineering acetic acid bacteria of overexpressing coenzyme PQQ (pyrroloquinoline quinone) synthetic proteins and application of bacteria

A genetic engineering, overexpression technology, applied in the direction of bacteria, vinegar preparation, microbial-based methods, etc., can solve undiscovered problems

Active Publication Date: 2014-04-23
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, no genetically engineered acetic acid bacterium that utilizes the alcohol dehydrogenase promoter to overexpress the coenzyme PQQ synthetic protein has been found, and the method of using the genetically engineered acetic acid bacterium to carry out acetic acid fermentation

Method used

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  • Genetic engineering acetic acid bacteria of overexpressing coenzyme PQQ (pyrroloquinoline quinone) synthetic proteins and application of bacteria
  • Genetic engineering acetic acid bacteria of overexpressing coenzyme PQQ (pyrroloquinoline quinone) synthetic proteins and application of bacteria
  • Genetic engineering acetic acid bacteria of overexpressing coenzyme PQQ (pyrroloquinoline quinone) synthetic proteins and application of bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1 prepares the genetically engineered Acetobacter pasteurianus that contains recombinant plasmid pBBR-Padh-pqq

[0062] (1) Construction of recombinant plasmid pBBR-Padh

[0063] The genome of Acetobacter pasteurianus CGMCC3089 (purchased from the General Microbiology Center of China Microbiological Culture Collection Management Committee, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postcode 100101. Authorized patent number: ZL200810053141) is Template, using primers Padh-1 and Padh-2 for PCR, amplified to obtain the Padh sequence of the alcohol dehydrogenase promoter, the PCR reaction system is as follows:

[0064]

[0065] The PCR reaction conditions were: pre-denaturation at 95°C for 5 minutes, 30 seconds at 94°C, 20 seconds at 56°C, 30 seconds at 72°C, and 10 minutes at 72°C after 26 cycles.

[0066] Alcohol dehydrogenase promoter Padh sequence and plasmid pBBR1MCS-4 (Ko...

Embodiment 2

[0078] Embodiment 2 utilizes the Acetobacter pasteurianus CGMCC3089 genetic engineering bacterium containing recombinant plasmid pBBR-Padh-pqq to produce acetic acid by fermentation

[0079] (1) Preparation of seed solution

[0080] Acetobacter pasteurianus CGMCC3089 genetically engineered bacteria and Acetobacter pasteurianus CGMCC3089 original bacteria containing the recombinant plasmid pBBR-Padh-pqq were taken from the slant and inoculated into the seed medium, and cultured on a shaker at 30°C and 160 rpm for 24 hours. According to the 10% (v / v) inoculum size, transfer to fresh seed medium for scale-up culture.

[0081] The composition of the seed medium: glucose 20g / L, yeast extract 15g / L, ethanol 3.5% (v / v), and the rest is water.

[0082] (2) Acetic fermentation

[0083] According to the inoculum amount of 10%, inoculate the seed liquid into the fermenter containing the fermentation medium, and carry out acetic acid fermentation under the condition of 30°C.

[0084] C...

Embodiment 3

[0088] Example 3 Production of Apple Cider Vinegar by Fermentation of Acetobacter pasteurianus CGMCC3089 Genetic Engineering Bacteria Containing Recombinant Plasmid pBBR-Padh-pqq

[0089] (1) Preparation of seed solution

[0090] Take the Acetobacter pasteurianus CGMCC3089 genetically engineered bacteria containing the recombinant plasmid pBBR-Padh-pqq from the slant in the seed medium, and culture it on a shaker at 30°C and 160 rpm for 25 hours. According to the 10% (v / v) inoculum size, transfer to fresh seed medium for scale-up culture.

[0091] The composition of seed medium: glucose 20g / L, yeast extract 15g / L, ethanol 3.5% (v / v), and the rest is water.

[0092] (2) Apple cider vinegar fermentation

[0093] According to the inoculum amount of 10% (v / v), it was inoculated into the fermenter containing cider, and the apple cider vinegar was fermented at 30°C. The ethanol content in cider is 8% (v / v).

[0094] The Acetobacter pasteurianus CGMCC3089 genetically engineered b...

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Abstract

The invention relates to a building method of genetic engineering acetic acid bacteria of recombinantly expressing coenzyme PQQ (pyrroloquinoline quinone) synthetic proteins. The PQQ biosynthetic proteins are PqqA, PqqB, PqqC, PqqD, PqqE, and functional equivalents thereof. An alcohol dehydrogenase promoter from acetobacter pasteurianus and a coenzyme PQQ synthetic protein gene cluster pqqABCDE sequence are orderly connected into plasmids which can be stably copied in the acetic acid bacteria, so as to recombine pBBR-padh-pqq; recombinant plasmid pBBR-padh-pqq is transferred into the acetic acid bacteria, so as to obtain the genetic engineering acetic acid bacteria of overexpressing the coenzyme PQQ synthetic proteins. Thus, the concentration of in vivo coenzyme PQQ of the acetic acid bacteria in the fermentation process of acetic acid is improved. Acetic acid fermentation is carried out by using the genetic engineering bacteria disclosed by the invention, the fermentation delay phase can be shortened, and the fermentation rate of the acetic acid is increased. Thus, the production cost is reduced.

Description

technical field [0001] The invention relates to a genetically engineered acetic acid bacterium overexpressing coenzyme PQQ to synthesize protein and a construction method thereof. Using the strain to ferment and produce acetic acid with ethanol as a substrate has high production efficiency and belongs to the field of biotechnology. Background technique [0002] Acetic acid is one of the compounds widely used in food, medicine, chemical industry, textile and other fields. Vinegar, with acetic acid as the main component, is one of the most consumed condiments in the world and one of the most important accessories in the food industry. Acetic acid used in the food industry is generally produced by fermentation. Acetic acid bacteria (Acetic acid bacteria) is a kind of bacteria that can oxidize ethanol into acetic acid and other products, and some can also oxidize glucose into gluconic acid, which is widely used in medicine, food and other fields. Acetic acid bacteria are all G...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12P7/54C12J1/04C12R1/02
Inventor 郑宇王敏申雁冰牛丹丹骆健美聂志强殷海松
Owner TIANJIN UNIV OF SCI & TECH
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