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Improved biosynthesis method for 1, 3-propylene glycol

A technology of biosynthesis and propylene glycol, which is applied in the field of bioengineering, can solve the problems of no reports, etc., and achieve the effect of fast bacterial growth and high product concentration

Active Publication Date: 2016-12-07
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the specific role of the above genes in Klebsiella has not been studied in detail, and there is no report on the reduction of 2,3-butanediol and the increase of 1,3-PD production by knocking out the pck gene

Method used

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  • Improved biosynthesis method for 1, 3-propylene glycol
  • Improved biosynthesis method for 1, 3-propylene glycol
  • Improved biosynthesis method for 1, 3-propylene glycol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1. Knocking out pck reduces the formation of the by-product 2,3-BD and promotes the growth of bacteria and the synthesis of 1,3-PD

[0022] M2014574 strain was cultured overnight at 37° C. in LB medium (0.5% yeast extract, 1% tryptone, 1% NaCl, pH 7.0), and the genome was extracted. Using the extracted M2014574 genome as a template, primers were designed based on the pck gene (locus_tag: KPN_03773) on the genome sequence of Klebsiella pneumoniae MGH 78578 registered in NCBI (LOCUS: NC_009648), and the target item was recovered after the PCR reaction. The band was sequenced, and the gene analysis was performed on the target band after sequencing, and the similarity with the pck gene on MGH 78578 was 100%. The pck gene (1623bp) of the M2014574 genome was knocked out by means of homologous recombination, and the obtained recombinant strain was M2014574△pck.

[0023] M2014574 and M2014574△pck were inoculated in 250ml Erlenmeyer flasks at 37°C for anaerobic and micr...

Embodiment 2

[0027] Example 2. Knocking out the ppc gene has almost no effect on the growth of the bacteria, and the synthesis of 2,3-butanediol and 1,3-PD

[0028]M2014574 strain was cultured overnight at 37° C. in LB medium (0.5% yeast extract, 1% tryptone, 1% NaCl, pH 7.0), and the genome was extracted. Using the extracted M2014574 genome as a template, primers were designed based on the ppc gene (locus_tag: KPN_04245) on the genome sequence of Klebsiella pneumoniae MGH 78578 registered in NCBI (LOCUS: NC_009648), and the target item was recovered after the PCR reaction. Bands were sequenced, and the target bands were analyzed for genes after sequencing. The similarity with the ppc gene on MGH 78578 was 100%. The pck gene (2676bp) of the M2014574 genome was knocked out by means of homologous recombination, and the obtained recombinant strain was M2014574△ppc.

[0029] M2014574 and M2014574△ppc were respectively inoculated in 250ml Erlenmeyer flasks for anaerobic and microaerobic cultur...

Embodiment 3

[0033] Example 3: After knocking out the pck gene, the generation of the by-product 2,3-butanediol was significantly reduced and the synthesis of the product 1,3-PD was promoted on the 5L reactor

[0034] The 5L reactor fermentation experiment was as follows: the strains (M2014574△pckc, M2014574) were inserted into a 250ml shake flask (50ml liquid volume) for seed cultivation for 20 hours, and then inserted into a 5L fermenter (2L fermentation liquid volume), The fermentation process was controlled according to the process conditions shown below.

[0035] The initial glycerol concentration is 60g / L, the fermentation temperature is 35°C; the ventilation rate is 1.0vvm; the stirring speed is 20rpm; the pH value is controlled by adding NaOH solution to 5.5-7.5 during the fermentation process. In each period of fermentation, the glycerin concentration is controlled at 10-60 g / L by supplementing glycerin solutions with different concentrations, and the fermentation ends in 30 hours...

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Abstract

The invention discloses an improved biosynthesis method for 1, 3-propylene glycol. The improved biosynthesis method includes knocking out genes pck in Klebsiella pneumoniae to efficiently convert glycerin so as to produce the 1, 3-propylene glycol. The improved biosynthesis method has the advantages that synthesis of 2, 3-propylene glycol which is a byproduct can be reduced to a great extent after the genes pck are knocked out, and accordingly the level of production for converting the glycerin by the aid of the Klebsiella pneumonia to generate the 1, 3-propylene glycol can be upgraded.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, specifically, knocking out the gene encoding phosphoenolpyruvate carboxykinase synthesis in Klebsiella pneumoniae, thereby improving the production level of 1,3-propanediol fermentation. Background technique [0002] 1,3-propanediol (1,3-porpanediol, referred to as 1,3-PD) is an important chemical raw material, and its most important use is as a monomer to synthesize a new type of polyester—polytrimethylene terephthalate ( PTT). Studies have shown that PTT is a polyester material with particularly excellent properties, so the industrial value of 1,3-PD has attracted increasing attention from various countries. The current research shows that the biosynthesis method in the production method of 1,3-PD has more advantages in industrial application than the chemical synthesis method because of its mild conditions, simple operation, less by-products, and environmental protection. Especially w...

Claims

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Application Information

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IPC IPC(8): C12P7/18C12N15/74C12R1/22
Inventor 宫衡张永强傅水林
Owner EAST CHINA UNIV OF SCI & TECH
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