Application of recombinant klebsiella pneumoniae in producing 1,3-propylene glycol
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A Klebsiella, linking product technology, applied in the field of genetic engineering, can solve the problems of inability to improve substrate tolerance and decrease in biomass
Active Publication Date: 2019-02-15
JIANGNAN UNIV
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However, the existing Klebsiella transformation strategies often result in a decrea
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Embodiment 1
[0029] The preparation of embodiment 1 recombinant Klebsiella
[0030] The mexF gene sequence was obtained according to the genome sequence of Pseudomonas aeruginosa, and the fragment was amplified. The primers used are: mexF-F (sequence information is shown in SEQ ID No. 4) and mexF-R (sequence information is shown in SEQ ID No. 5).
[0031] The reaction system is as follows (50 μL): 25 μL Prime STAR Max Premix (2×), 15 pmol each of upstream and downstream 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) , the amplification reaction conditions are as follows: 98°C, 10s; 55°C, 15s, 72°C, 1kb / min, 30 cycles.
[0032] The obtained PCR amplification products were verified by electrophoresis, and after the verification was successful, UNIQ-10 Column DNA Gel Recovery Kit (Shanghai Sangon Bioengineering Co., Ltd.) was used for rubber tapping recovery....
Embodiment 2
[0038] Example 2 Fermentation of recombinant Klebsiella expressing the mexF gene derived from Pseudomonas aeruginosa
[0039] The bacterial solution was inoculated into a 5L fermenter with 4% inoculum amount, anaerobic, fed-batch fermentation, and glycerol was fed 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. like figure 2 As shown, the gene recombinant bacteria overexpressing Pseudomonas aeruginosa mexF were fed-batch fermented in a 5L fermenter, and the production of 1,3-propanediol was as high as 74g / L. Compared to wild type (see figure 1 ), the production of 1,3-propanediol increased by about 37%, and the highest biomass increased by 22.7%.
[0040] It is confirmed by Example 2 that overexpressing the Pseudomonas aeruginosa gene mexF in the recombinant Klebsiella can enhance the ability of Klebsiella to produce 1,3-propanediol.
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Abstract
The invention discloses application of recombinant klebsiella pneumoniae in producing 1,3-propylene glycol and particularly discloses a method for improving the yield of the 1,3-propylene glycol by using mexF gene in heterologously-expressed pseudomonase aeruginosa of the klebsiella pneumoniae and application thereof, and belongs to the technical field of genetic engineering. According to the method disclosed by the invention, by applying a genetic engineering method, plasmid with the mexF gene in the pseudomonas aeruginosa is transferred to the klebsiella pneumoniae for enhancing the transportation efficiency of the klebsiella pneumoniae to short chain alcohol, so that the yield of the 1,3-propylene glycol is improved. The yield of the 1,3-propylene glycol reaches as high as 74g/L by carrying out fed-batch fermentation on arecombinant strain in a 5L fermentation tank. The method has the advantages that the transportation capacity of the klebsiella pneumoniae to the 1,3-propylene glycol is successively improved, provides a novel method for improving the yield of the 1,3-propylene glycol and provides a new idea for breeding high-yielding strains of the 1,3 propylene glycol.
Description
technical field [0001] The present invention relates to the application of a recombinant Klebsiella in the production of 1,3-propanediol, in particular to the heterologous expression of the mexF gene in Pseudomonas aeruginosa 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 is one of the six new petrochemical products recognized internationally. It has broad application prospects in textiles, plastics and food packaging. Its main function is as a synthetic polyester, polyether and polyurethane. Monomer, polytrimethylene terephthalate (PTT), which is polymerized with terephthalic acid, is the precursor material 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...
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