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Composite promoter and application thereof in increasing yield of 3-hydroxypropionic acid of Klebsiella pneumoniae

A technology of Klebsiella and compound initiation, which is applied to bacteria, using vectors to introduce foreign genetic material, enzymes, etc., can solve the problems of low conversion rate in the production process, lack of high-efficiency expression of aldehyde dehydrogenase, etc., and achieve the goal of increasing production Effect

Active Publication Date: 2018-12-07
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

K.pneumoniae is a non-model organism, and lacks a suitable strong promoter for high-efficiency expression of aldehyde dehydrogenase, resulting in low conversion rate of 3-HP production process

Method used

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  • Composite promoter and application thereof in increasing yield of 3-hydroxypropionic acid of Klebsiella pneumoniae
  • Composite promoter and application thereof in increasing yield of 3-hydroxypropionic acid of Klebsiella pneumoniae
  • Composite promoter and application thereof in increasing yield of 3-hydroxypropionic acid of Klebsiella pneumoniae

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] The preparation of embodiment 1 recombinant vector pET-Pntac

[0060] According to the reported tac promoter core sequence (29bp), a composite promoter was designed and chemically synthesized to obtain a gene fragment Pntac (n=2-5) containing 2-5 tac promoter core sequences. Since the composite promoter contains many repetitive sequences and has a small molecular weight, it is difficult to control the conditions when using PCR for gene amplification. Ligated with the vector pET-28a to obtain the recombinant vector pET-Pntac (n=2-5), wherein the underlined part is the restriction site, and the black bold font is marked as the core sequence of the tac promoter. Between the two core sequences is a spacer sequence (TGTGGAATTGTG), which gives enough space for RNA polymerase to bind. The sequence refers to the partial sequence after the promoter of the vector pET28a; the last tac promoter core sequence is followed by a spacer sequence (TGTGGAATTGTG), Then there is the lac op...

Embodiment 2

[0094] The preparation of embodiment 2 recombinant Klebsiella pneumoniae

[0095] Primers were designed according to the puuC gene sequence published by GenBank (as shown in Seq ID No.6), and Sac I and EcoR I restriction sites were introduced into both ends of the gene, and PCR amplification was performed using the K.pneumoniae DSM 2026 genome as a template. The puuC gene was connected to the vector pET-Pntac to obtain a recombinant plasmid (pET-Pntac-puuC), which was transformed into Escherichia coli BMTop10 for sequencing verification. Extract and sequence the correct plasmid and transform it into wild-type Klebsiella pneumoniae K.pneumoniae DSM 2026 by electroporation, culture and extract genomic DNA, perform PCR, and then electrophoresis to verify whether the puuC gene has been successfully transformed. The primers for PCR are general-purpose pET28a vectors primers, results such as Figure 2A ~ Figure 2E shown. Recombinant bacteria K.pneumoniae (Pntac-puuC) (n=1-5) with...

Embodiment 3

[0109] The activity measurement of embodiment 3 aldehyde dehydrogenase

[0110] Recombinant bacteria are carried out shake flask fermentation respectively, and culture medium adopts the fermentation medium (basic) of Klebsiella pneumoniae: 250mL Erlenmeyer flask fermentation medium 100mL, cotton plug sealing, inoculum size is 1% (volume fraction), 37 Cultivate recombinant bacteria with shaking at 150r / min. Take 10 mL of the fermentation broth at 24 hours, collect the cells by centrifugation at 12000 r / min, resuspend the cells with 5 mL of PBS buffer (pH7.0), and break the cells by ultrasonic. That is the crude enzyme solution. Enzyme activity reaction system is 2mL, including 150mM n-propanal 200μL, 20mM NAD + 200μL, 200μL of crude enzyme solution, 1.4mL of PBS buffer (pH7.0). In a water bath at 37°C for 5 minutes, the amount of NADH generated in the reaction system was determined by detecting the increase in absorbance at 340 nm. Definition of enzyme activity unit: at 37°...

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Abstract

The invention provides a composite promoter. The composite promoter is formed by arranging 2 to 5 tac promoter core sequences. The invention also provides a carrier with the composite promoter, engineering recombinant bacteria and a preparation method of the engineering recombinant bacteria. The engineering recombinant bacteria with the complex promoter can express aldehyde dehydrogenase efficiently, thereby rapidly catalyzing 3-HPA to produce 3-HP, preventing the accumulation of 3-HPA from weakening the growth and metabolism of the cells on the one hand, and improving the yield of 3-HP and onthe other hand.

Description

technical field [0001] The invention belongs to the field of biochemical production, and relates to a composite promoter and its application in improving the 3-hydroxypropionic acid production of Klebsiella pneumoniae. Background technique [0002] 3-Hydroxypropionic acid (3-HP) is an important platform compound with broad market prospects. The chemical synthesis of 3-HP is difficult, the separation and purification are difficult, and it pollutes the environment, which restricts its large-scale application. Biological production of 3-HP has the advantages of low cost, easy operation, mild conditions, and environmental friendliness. Klebsiella pneumoniae has high glycerol tolerance, strong glycerol metabolism ability, and can synthesize coenzyme B by itself 12 and other characteristics, so it becomes the dominant host bacteria for the production of 3-HP by glycerol biological method. K.pneumoniae utilizes glycerol dehydratase (DhaB) to catalyze glycerol to 3-hydroxypropana...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/74C12N15/66C12N1/21C12P7/42
CPCC12N1/20C12N9/0008C12N15/66C12N15/74C12P7/42C12Y102/01004
Inventor 田平芳赵鹏马春路
Owner BEIJING UNIV OF CHEM TECH
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