A method for producing 1,3-propanediol by fermentation of glycerol by microorganisms

A technology of microorganisms and glycerin, applied in the field of bioengineering, can solve the problems of low conversion rate and high production cost, and achieve the effect of increasing conversion rate and reducing production cost

Active Publication Date: 2022-04-08
镇江百泰生物科技有限公司
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Problems solved by technology

[0006] Aiming at the problems of low conversion rate and high production cost of producing 1,3-propanediol by fermentation of glycerol by a single strain, the present invention provides a method for microbial production of 1,3-propanediol by fermentation of glycerol, that is, the establishment of biosynthesis through co-cultivation of microorganisms The upstream and downstream of the pathway, respectively realize the utilization of glycerol and the production of 1,3-propanediol, reduce the biosynthetic pathway and metabolic pressure, and improve production efficiency

Method used

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  • A method for producing 1,3-propanediol by fermentation of glycerol by microorganisms
  • A method for producing 1,3-propanediol by fermentation of glycerol by microorganisms
  • A method for producing 1,3-propanediol by fermentation of glycerol by microorganisms

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Embodiment 1

[0033] 1. Construction and expression of recombinant Escherichia coli Rosseta-dhaB1dhaB2

[0034] 1) According to the gene sequence of the related glycerol dehydratase (GDHt) and glycerol dehydratase activator (GDHt active factor) of Clostridium butyricum VPI 1718 (GenBank number: AY112989) and the characteristics of the multiple cloning site of the expression vector pANY1, using biological information Synthetic primers designed by the scientific software NTI are: upstream primer F: 5'- GGGGGATCCACTAGTAGGCCT ATGATAAGTAAAGGATTTAG-3' and downstream primer R:5'- CAGGAGCTCCCATGGAGGCCT TTAATGATGATGATGATGGTGCTCAGCTCCAATTGTGCAAG-3', the underlined part is the StuI restriction site, and the wavy part is the upstream and downstream homology arms homologous to the pANY1 plasmid;

[0035] 2) Using Clostridium butyricum YJH-09 genomic DNA as template, PCR amplifies the target gene, PCR amplification reaction parameters: pre-denaturation: 95°C for 5min, denaturation: 94°C for 2min, anne...

Embodiment 2

[0052] Determination of glycerol dehydratase activity:

[0053] 1) Take an appropriate amount of the Rosseta-dhaB1dhaB2 bacterium liquid that has been successfully expressed in Example 1, centrifuge at 4°C for 10 min, collect the bacteria sludge, wash twice with 0.1mol / L potassium phosphate buffer solution of pH 7.4, and wash with an appropriate amount of potassium phosphate after centrifugation The bacteria were resuspended in the buffer, ultrasonically disrupted in an ice bath, centrifuged at 10,000 rpm, 4°C for 10 min, and the supernatant was the crude enzyme solution.

[0054] 2) Determination method of glycerol dehydratase activity: under optimum temperature and optimum pH conditions, according to the above reaction, the amount of enzyme needed to catalyze the generation of 1 μmoL propionaldehyde per minute is defined as one enzyme activity unit. Measured by MBTH (3-methyl-2-benzothiazolinone hydrazine) method, GDHt can catalyze the substrate glycerin (or 1,2-propanediol)...

Embodiment 3

[0057] Determination of 1,3-propanediol dehydrogenase activity:

[0058] 1) Take an appropriate amount of the Rosseta-dhaT bacterial liquid successfully expressed in Example 1, centrifuge at 4°C for 10 min, collect the bacterial sludge, wash twice with 0.1mol / L potassium phosphate buffer solution of pH 7.4, and wash with an appropriate amount of potassium phosphate after centrifugation The bacteria were resuspended in the buffer, ultrasonically disrupted in an ice bath, centrifuged at 10,000 rpm, 4°C for 10 min, and the supernatant was the crude enzyme solution.

[0059] 2) Determination of 1,3-propanediol dehydrogenase reductive activity: under the optimal temperature and optimal pH conditions, according to the above reaction, the amount of enzyme required to generate 1 μmoL 1,3-PD per minute is defined as an enzyme activity unit . In a total volume of 1.0mL, mix the following components according to the final concentration: 27mmol / L propionaldehyde, 0.37mmol / L NADH, 35mmol / ...

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Abstract

The invention provides a method for producing 1,3-propanediol by fermentation of glycerol in a microbial co-cultivation system, by successfully constructing recombinant Escherichia coli Rosseta-dhaB1dhaB2 and Rosseta-dhaT, co-cultivating the above-mentioned recombinant Escherichia coli for seed culture and fermentation culture , wherein the recombinant Escherichia coli Rosseta-dhaB1dhaB2 realizes the utilization of glycerol to produce the intermediate metabolite 3-hydroxypropionaldehyde (3-HPA), and the recombinant Escherichia coli Rosseta-dhaT catalyzes 3-HPA to produce 1,3-propanediol. During the fermentation process, press Inoculate in the fermentation medium at a ratio of 1:1.5, add 60g / L substrate glycerol and 10g / L cosubstrate glucose. The method of the present invention improves the conversion rate of glycerol and the output of 1,3-propanediol, and the present invention adopts Clostridium butyricum containing independent coenzyme B 12 Glycerol dehydratase, which reduces the production cost.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for microorganisms to ferment and produce 1,3-propanediol using glycerol. Background technique [0002] Crude glycerin is one of the main by-products in the production of biodiesel. Every 10kg of biodiesel produced will produce 1kg of crude glycerin. According to reports, it is expected to reach more than 5.87 billion pounds in 2020. If crude glycerin cannot be used and processed in a timely and effective manner, It may become a new source of pollution. Therefore, using glycerol to produce high value-added products and improving the conversion rate of glycerol is a hot spot in current research. [0003] 1,3-Propanediol (1,3-PD), as a good solvent, antifreeze and protective agent, is widely used in the fields of food, medicine, cosmetics and chemical industry. In the field of food, 1,3-propanediol is easy to dissolve food additives that are difficult ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/18C12N1/21C12R1/19
CPCC12P7/18C12N9/88C12N9/0006C12Y402/0103C12Y101/01202
Inventor 齐向辉员君华张国艳袁娇张宇飞王洋
Owner 镇江百泰生物科技有限公司
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