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A genetically engineered strain using sucrose to produce succinic acid and its fermentation method for producing succinic acid

A technology of genetically engineering bacteria and producing succinic acid, applied in the field of bioengineering, can solve problems such as inability to utilize sucrose, lack of sucrose utilization genes, etc.

Active Publication Date: 2017-05-10
态创生物科技(广州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Escherichia coli is widely used in industry, but currently most Escherichia coli cannot utilize sucrose due to the lack of sucrose utilization genes

Method used

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  • A genetically engineered strain using sucrose to produce succinic acid and its fermentation method for producing succinic acid
  • A genetically engineered strain using sucrose to produce succinic acid and its fermentation method for producing succinic acid
  • A genetically engineered strain using sucrose to produce succinic acid and its fermentation method for producing succinic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] This example illustrates the method for constructing E. coli BA500. Expression plasmid expressing exogenous sucrose permease, sucrose hydrolase and fructokinase genes.

[0042] (1) Using LB medium, cultivate Escherichia coli AFP111 to OD at 37°C under aerobic conditions 600 =0.5~0.6, using CaCl 2 Preparation of Escherichia coli AFP111 competent strain lacking lactate dehydrogenase gene (ldhA), pyruvate formate lyase gene (pflB) activity and ptsG gene chromosome spontaneous mutation of phosphotransferase system;

[0043] The formula of LB medium is: peptone 10g / L, yeast powder 5g / L, NaCl 5g / L.

[0044] (2) Synthesize primers without restriction sites,

[0045] Upstream primer: 5'-CCGGTTGAGGGATATAGAGCTATCGAC-3';

[0046] Downstream primer: 5'-CTGTTGATCCGTTGTTCCACCTGAT-3'.

[0047] Extract the E.coli W genome, and use the E.coli W genome as a template to amplify the target gene fragment by PCR. The specific reaction conditions are: 94°C, 10min; (94°C for 45s, 60°C for...

Embodiment 2

[0052] This example illustrates a method for continuously cultivating and acclimating Escherichia coli (Escherichia coli) BA500.

[0053] The Escherichia coli BA500 constructed in Example 1 was used as the starting strain. Transfer 1% (v / v) inoculum from the cryopreservation tube into the test tube, incubate overnight at 37°C, 200rpm, then transfer 1% (v / v) inoculum into the Erlenmeyer flask, incubate at 37°C, 200rpm for 6-8h Obtain the bacterium liquid of logarithmic growth phase; The bacterium liquid of logarithmic growth phase is inoculated in the 500mL continuous culture device that 300mL fermentation medium is housed with the inoculum size of 10% (v / v), 37 ℃ of water bath heating, pass into Filter-sterilized CO 2 Maintain the anaerobic environment, and feed fresh fermentation medium into the culture device at a rate of 1.5 mL / h. Regularly take samples to detect the density of bacteria in the culture device, when the density of bacteria in the reactor reaches OD 600 =2~...

Embodiment 3

[0057] This example illustrates the method for screening and obtaining excellent Escherichia coli (Escherichia coli) BA501. Screening steps:

[0058] 1. Solid plate primary screening

[0059] Under aseptic conditions, take out 2-4mL bacterial liquid from the continuous culture device and dilute it with sterile water 1×10 4 Take 100 μL and spread it on a solid plate containing 2% sucrose, incubate at 37°C for 12 hours, and select a single colony of the mutant strain with a fast growth rate and relatively plump.

[0060] 2. Solid flat plate re-screening

[0061] The screened mutant strains were repeatedly transferred and cultured on the plate, and finally the strains BA501, BA516 were obtained, and BA528 showed strong growth rate and growth stability.

[0062] 3. Shake flask fermentation screening

[0063] The mutant strains BA500, BA501, BA516 and BA528 were cultured in the seed medium, 37°C, 200r / min, cultured for 12h, and then inserted into the Erlenmeyer flask containing...

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Abstract

Belonging to the technical field of bioengineering, the invention relates to a genetic engineering strain producing succinic acid and a method for production of succinic acid by fermenting the strain, particularly a recombinant strain efficiently utilizing cane sugar and molasses to grow and produce succinic acid. The genetic engineering strain producing succinic acid is classified and named as Escherichia coli BA501, and has a preservation registration number of CCTCC NO:M2014014. The construction process of the strain mainly includes: taking Escherichia coli AFP111 that lacks lactate dehydrogenase gene and pyruvate formate lyase activity and has the chromosome ptsG gene undergoing spontaneous mutation as the starting strain, expressing exogenous sucrose permease, sucrose hydrolase and fructokinase genes, and then carrying out continuous domestication cultivation to obtain the strain efficiently utilizing cane sugar and molasses to grow and produce succinic acid. Thus, the synthesis efficiency of succinic acid is greatly improved. The fermentation method adopts a two-stage fermentation way, in the aerobic stage the biomass is improved oxygen, and in the anaerobic stage, fermentation and acid production are achieved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a succinic acid-producing genetically engineered bacterium and a method for producing succinic acid by fermentation, in particular to a recombinant bacterial strain that efficiently utilizes sucrose and molasses to grow and produce succinic acid, and uses the bacterial strain to ferment Process for producing succinic acid. Background technique [0002] Succinic acid, also known as succinic acid, is widely used in industries such as medicine, pesticides, dyes, spices, paints, food and plastics. As a C4 platform compound, it can be used to synthesize 1,4-butanediol, tetrahydrofuran, Organic chemicals such as γ-butyrolactone and biodegradable materials such as polybutylene succinate (PBS) are considered by the U.S. Department of Energy to be among the 12 most valuable biorefinery products in the future. [0003] The production method of succinic acid mainly includes chemical s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/63C12P7/46C12R1/19
Inventor 姜岷李凤刘嵘明梁丽亚马江锋陈可泉韦萍欧阳平凯
Owner 态创生物科技(广州)有限公司
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