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Method for producing 1, 3 propylene glycol safely and highly efficiently

A propylene glycol, high-efficiency technology, applied in the field of bioengineering, to achieve the effect of pro-secretion

Inactive Publication Date: 2012-09-12
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no example of the application of the Red recombination system in Klebsiella, mainly because Klebsiella has ampicillin resistance, and the selection of pKD46 and pCP20 relies on this resistance gene

Method used

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  • Method for producing 1, 3 propylene glycol safely and highly efficiently
  • Method for producing 1, 3 propylene glycol safely and highly efficiently
  • Method for producing 1, 3 propylene glycol safely and highly efficiently

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Embodiment 1, construct the Red recombinant plasmid system pKD46-Tc and pCP20-Tc applicable to Klebsiella

[0030] Through sequence search, it is found that there is an Xmn I restriction enzyme site on the Amp resistance gene of these two plasmids, and there is only one of this site in the whole plasmid, inserting other resistance genes at this position can make these two plasmids have New resistance marker, while removing the Amp resistance marker.

[0031]Using the pBR322 plasmid as a template, design primers F-tet (5'-GGAAAACGTTCCTCATGTTTGACAGCTFATCATCG-3') and R-tet (5'-GGAACATTTTCGGAGTGGTGAATCCGTTAGCG-3') for the tetracycline resistance gene, with Xmn I restriction sites on both ends , amplify the tet gene by PCR, recover the tet fragment from the gel, transform it into DH5α competent cells after being connected to the T carrier, smear LB plates (tetracycline hydrochloride 20 μg / ml), and culture overnight at 37°C for 12-16 hours; single clones are picked the next d...

Embodiment 2

[0034] Embodiment 2, knockout the capsular gene wzi of Klebsiella

[0035] 1. Two-step PCR method to amplify the fragment Δwzi::FK

[0036] Using the pKD4 plasmid as a template, primers F-FRT and R-FRT were designed to amplify the kan resistance gene fragment FK (about 1500bp) containing FRT sites at both ends. Determine that the homology arm is 250bp, design the primers Fup-wzi and Rup-wzi::FRT at both ends of the upstream 250bp sequence of the wzi fragment, and add the partial sequence of F-FRT to the 5' end of the reverse primer, and the downstream 250bp sequence of the wzi fragment A partial sequence of R-FRT was added to the 5' end of the forward primer Fdown-FRT::wzi and Rdown-wzi forward primer, and the genome of K.pneumoniae ATCC49790 was used as a template to amplify. The three fragments up-wzi, down-wzi, and FK amplified by PCR were mixed according to the substance ratio of 1:1:1 as a template, and Fup-wzi and Rdown-wzi were used as primers to amplify the fragment Δ...

Embodiment 3

[0041] Embodiment 3, the characteristic of knocking out capsular gene wzi Klebsiella

[0042] 1. Capsule staining experiment

[0043] Dip the Klebsiella strains cultured overnight and the Klebsiella bacteria with capsule knockout respectively, and spot on two glass slides, after natural drying, stain with 1% crystal violet aqueous solution for 2min, wash with 20% CuSO4, and then wash with absorbent paper Wipe off the residual liquid and observe the phenomenon under the oil immersion lens. The results of microscopic examination of the original bacterial liquid showed that the bacterial cells were stained dark purple, and the capsule around the bacterial cells was lavender. The results of microscopic examination of the capsule-knockout bacterial fluid showed that there were only dark purple bacterial cells without surrounding lavender capsules.

[0044] 2. Transformation efficiency experiment

[0045] Use 10% glycerol to prepare competent cells of original and capsulated Kleb...

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Abstract

The invention discloses a method for producing 1, 3 propylene glycol safely and high efficiently, i.e. knocking out the wzi gene, which causes the generation of capsules, from K.pneumoniae through a specifically improved Red recombination system to improve the security and product secreting performance of 1, 3-PD fermentation. Compared with the prior art, the method of the invention substantially raises the gene knockout efficiency of the K.pneumoniae. The K.pneumoniae having wzi capsule gene deletion has a high product concentration, great production intensity and high security.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, specifically: through a specific gene knockout system, the capsule production gene wzi in Klebsiella (K.pneumoniae) is knocked out, thereby removing the capsule and improving the 1,3 - The safety and product secretion performance of PD fermentation, a safe and efficient method for converting glycerol to produce 1,3-propanediol. Background technique [0002] 1,3-propanediol (1,3-porpanediol, referred to as 1,3-PD) is an important chemical raw material, which can be used as a solvent, antifreeze or protective agent, fine chemical raw material and a new type of polyester—polyester Monomer of propylene glycol phthalate (PTT) and polyurethane. PTT has been proved to be a new type of polyester material with excellent performance. The existing 1,3-PD production methods include chemical synthesis and biosynthesis. Compared with chemical synthesis, biological synthesis of 1,3-PD has milder condi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12P7/18C12R1/22
Inventor 宫衡莫隽颖周佳佳付水林
Owner EAST CHINA UNIV OF SCI & TECH
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