Genetically engineered bacterium for co-production of 3-hydroxypropionic acid and 1,3-propylene glycol as well as construction method and application of genetically engineered bacterium

A technology of genetically engineered bacteria and hydroxypropionic acid, applied in the field of bioengineering, can solve the problems of affecting the reaction, unbalanced coenzymes, low yield, etc., and achieve the effects of efficient metabolism, increased yield, and removal of cytotoxicity

Active Publication Date: 2021-01-12
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]Currently, since the biosynthesis of 3-hydroxypropionate needs to consume the coenzyme NAD+ to produce NADH, the production of 1,3-propanediol is exactly On the contrary, converting NADH to NAD+ can only separate the production of 3-hydroxypropionic acid and 1,3-propanediol, otherwise it will cause an imbalance of coenzyme in the microorganism and affect

Method used

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  • Genetically engineered bacterium for co-production of 3-hydroxypropionic acid and 1,3-propylene glycol as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for co-production of 3-hydroxypropionic acid and 1,3-propylene glycol as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for co-production of 3-hydroxypropionic acid and 1,3-propylene glycol as well as construction method and application of genetically engineered bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Recombination E. coli Construction and expression of BL21 / pANY-GabD4

[0032] (1) According to the gene encoding succinic semialdehyde dehydrogenase of Copper-loving hookworm GabD4 Sequence and characteristics of gene elements on the vector pANY1, using Oligo7.0 software to design primers: PANY-F: 5'-atgtatatctccttcttaaagt-3', PANY-R: 5'-cctccatgggagctcctg-3', GabD4-F1: 5'-taactttaagaaggagatatacatatgtaccaagatctggcactgt- 3' and GabD4-R1: 5'-tgcaggagctcccatggaggttacgcttgggtgatgaact-3'.

[0033] (2) Use PANY-F and PANY-R primer pairs to amplify the pANY1 vector backbone (excluding 6×His tag and ccdB expression cassette), PCR reaction parameters: pre-denaturation, 98°C 1min; denaturation, 98°C 10s; annealing, 55°C for 10s; extension, 72°C for 30s; stop extension, 72°C for 5min; pANY1 vector backbone was obtained after 32 cycles.

[0034] GabD4-F1 and GabD4-R1 primer pairs were used to amplify homology-arm containing GabD4 Gene fragments, PCR reaction par...

Embodiment 2

[0039] Example 2: Construction and expression of recombinant E.coli BL21 / pANY-PduQ

[0040] (1) According to the gene pduQ sequence of Lactobacillus reuteri encoding 1,3-propanediol oxidoreductase and the characteristics of the gene elements on the vector pANY1, use Oligo7.0 software to design primers: PduQ-F1: 5'- taactttaagaaggagatatacatatggaaaaatttagtatgccaac-3' and PduQ-R1: 5'-tgcaggagctcccatggaggttaacgaattattgcttcgtaaat-3'.

[0041] (2) Use PANY-F and PANY-R primer pairs to amplify the pANY1 vector backbone (excluding 6×His tag and ccdB expression cassette), PCR reaction parameters: pre-denaturation, 98°C for 1 min; denaturation, 98°C for 10s; annealing , 55°C for 10s; extension, 72°C for 30s; stop extension, 72°C for 5min; pANY1 vector backbone was obtained after 32 cycles.

[0042] Use the PduQ-F1 and PduQ-R1 primer pairs to amplify the pduQ gene fragment containing the homology arm, PCR reaction parameters: PCR reaction parameters: pre-denaturation, 98°C 1min; denatu...

Embodiment 3

[0047] Example 3: Construction and expression of recombinant E.coli BL21 / pANY-GabD4-PduQ

[0048] (1) Use Oligo7.0 software to design primers according to the sequences of vectors pANY-gabD4 and pANY-pduQ successfully constructed in Examples 1 and 2: GabD4-F2: 5'- cctccatgggagctcctg-3', GabD4-R2: 5'- ttacgcttgggtgatgaactt-3', PduQ-F2: 5'-agttcatcacccaagcgtaatggccttttgctgg-3' and PduQ-R2: 5'-tgcaggagctcccatggaggttaacgaattattgcttc-3'.

[0049] (2) E. coli GabD4 and E. coli PduQ plasmids pANY-gabD4 and pANY-pduQ were extracted using the plasmid mini-extraction kit, respectively. Using the plasmid pANY-gabD4 as a template, use the primer pair GabD4-F2 and GabD4-R2 to linearize pANY-gabD4. PCR reaction parameters: pre-denaturation, 98°C 1min; denaturation, 98°C 10s; annealing, 55°C 10s; extension , 72°C for 30s; stop extension, 72°C for 5min; pANY-gabD4 backbone was obtained after 32 cycles.

[0050] Using the plasmid pANY-pduQ as a template, use PduQ-F2 and PduQ-R2 primers to am...

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Abstract

The invention provides a genetically engineered bacterium for co-production of 3-hydroxypropionic acid and 1,3-propylene glycol as well as a construction method and application of the genetically engineered bacterium, and belongs to the technical field of bioengineering. According to the invention, a genetic engineering means is utilized to construct the genetically engineered bacterium for combined expression of succinate semialdehyde dehydrogenase (GabD4) and 1,3-propylene glycol oxidordeuctase (PduQ) on the basis of E.coli BL21, and the expression quantity of the balanced double enzymes isimproved through an UTR engineering technology; and moreover, the constructed genetically engineered bacterium and lactobacillus reuteri are subjected to multi-bacterium mixing to convert glycerin, sothat efficient co-production of the 3-hydroxypropionic acid and the 1,3-propylene glycol is realized.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a genetically engineered bacterium co-producing 3-hydroxypropionic acid and 1,3-propanediol, its construction method and application. Background technique [0002] 3-Hydroxypropionic acid and 1,3-propanediol are two important platform compounds in industry, which are widely used as precursors of biodegradable polymers and food additives. The production of 3-hydroxypropionic acid and 1,3-propanediol has two methods: chemical synthesis and biological method. Most of the chemical methods use non-renewable resources as raw materials. The production process consumes a lot of energy, and many by-products are difficult to separate and purify. The production process produces immeasurable environmental pollution. The biosynthesis of 3-hydroxypropionic acid and / or 1,3-propanediol mostly uses glucose and glycerol as substrates, and glycerol is used as a substrate to prod...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/02C12N9/04C12N15/53C12N15/70C12P7/42C12P7/18C12R1/19
CPCC12N9/0008C12N9/0006C12N15/70C12P7/42C12P7/18C12Y102/01016C12Y101/01202
Inventor 齐向辉张宇飞员君华张国艳袁娇王洋
Owner JIANGSU UNIV
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