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Application of recombinant klebsiella peneumoniae in preparation of 1,3-propanediol

A technology of Klebsiella and Lactobacillus fermentum, applied in the field of genetic engineering, can solve the problems of low conversion rate, low yield, and inability to improve substrate transport efficiency, and achieve the effect of changing and improving transport capacity

Active Publication Date: 2019-02-22
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 1,3-propanediol is synthesized in Klebsiella as a growth-coupled metabolite, and when Klebsiella uses glycerol as a carbon source to produce 1,3-propanediol, there is a lower yield and lower conversion and other disadvantages, and the accumulation of 1,3-propanediol will inhibit the transport of substrates during the fermentation production of Klebsiella
At present, the research on improving the production of 1,3-propanediol through the modification of Klebsiella bacteria mainly focuses on the two aspects of metabolic pathway and regeneration of reducing power, but the existing modification methods cannot improve the transport efficiency of the substrate.

Method used

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  • Application of recombinant klebsiella peneumoniae in preparation of 1,3-propanediol

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Experimental program
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Embodiment 1

[0028] The preparation of embodiment 1 recombinant Klebsiella

[0029] Use primers P1-XhoI (sequence information as shown in SEQ ID NO.4) and P2-SalI (sequence information as shown in SEQ ID NO.5) PCR amplification to obtain Lactobacillus fermentum glpF gene sequence fragment according to the genome sequence of Lactobacillus fermentum .

[0030] The PCR reaction system is as follows (50 μL): 25 μL Prime STAR Max Premix (2×), 15 pmol primers, 150 ng template, add double distilled water to 50 μL (primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd., and the rest were purchased from TaKaRa Company), expanded The reaction conditions are as follows: 98°C, 10s; 55°C, 15s, 72°C, 1kb / min, 30 cycles.

[0031] The obtained PCR amplification products were verified by electrophoresis. After the verification was successful, the UNIQ-10 Column DNA Gel Recovery Kit (Shanghai Sangon Bioengineering Co., Ltd.) was used for gel cutting and recovery.

[0032] The recovery proced...

Embodiment 2

[0037] Example 2 Fermentation of recombinant Klebsiella overexpressing the glpF gene derived from Lactobacillus fermentum

[0038] The bacterial solution obtained in Example 1 was inoculated into a 5L fermenter with 4% inoculum amount, anaerobic, fed-batch fermentation, feeding glycerol to keep it at 15-30g / L, 37°C, 150rpm, and fermented for 48h . Sampling from time to time to measure OD by spectrophotometer 600 And measure the fermentation product by HPLC. Such as figure 1 and figure 2 As shown, compared with the wild type, the 1,3-propanediol concentration of Klebsiella overexpressing the glpF gene of Lactobacillus fermentum was increased from 55g / L to 76g / L, and the conversion rate of glycerol was increased from 56% to 60%.

[0039] The results showed that overexpressing the glycerol channel protein gene glpF of Lactobacillus fermentum in Klebsiella can improve the ability of Klebsiella to produce 1,3-propanediol and the efficiency of glycerol transport.

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Abstract

The invention discloses application of recombinant klebsiella peneumoniae in preparation of 1,3-propanediol, particularly relates to application of recombinant klebsiella peneumoniae overexpressing lactobacillus fermenti glycerin channel protein gene glpF in production of 1,3-propanediol and belongs to the field of genetic engineering. By applying a genetic engineering method, an overexpression vector of the lactobacillus fermenti glycerin channel protein gene glpF is transferred to klebsiella peneumoniae, accordingly the glycerin transfer capability of the gene is improved, and the 1,3-propanediol yield of the klebsiella peneumoniae is improved. The recombinant strain is subjected to material supplementation and batched fermentation with a 5L fermentation tank, the yield of 1,3-propanediol is as high as 76g / L. By adopting the application, the glycerin transfer capability of the klebsiella peneumoniae is successively changed, and a new way of thinking is provided for breeding of high-yield 1,3-propanediol strains.

Description

technical field [0001] The invention relates to the application of a recombinant Klebsiella in the preparation of 1,3-propanediol, in particular to the heterologous expression of the glycerol channel protein gene glpF of Lactobacillus fermentum in Klebsiella to increase the production of 1,3-propanediol The method and application of the invention belong to the technical field of genetic engineering. Background technique [0002] 1,3-propanediol (1,3-PDO) is one of the six new petrochemical products recognized internationally, and has broad application prospects in textiles, plastics and food packaging. Polytrimethylene terephthalate (PTT), a monomer of ether and polyurethane, which is polymerized with terephthalic acid, is a precursor for the synthesis of new high-quality polyester materials. [0003] The production methods of 1,3-propanediol include chemical synthesis and biotransformation. The production of 1,3-propanediol by biotransformation has the advantages of relat...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12N15/31C12P7/18C12R1/22
CPCC07K14/335C12N15/74C12P7/18
Inventor 诸葛斌李玉石滕宇陆信曜宗红宋健
Owner JIANGNAN UNIV
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