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Recombinant bacterium and construction and application thereof

A technology of recombinant bacteria and recombinant plasmids, applied in the field of genetic engineering, can solve unseen problems, and achieve the effect of simplifying steps, solving feedback inhibition, and expanding the way of development

Active Publication Date: 2021-07-13
SHANTOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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  • Recombinant bacterium and construction and application thereof
  • Recombinant bacterium and construction and application thereof
  • Recombinant bacterium and construction and application thereof

Examples

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Effect test

Embodiment 1

[0047] The construction of embodiment 1 recombinant bacteria

[0048] Synthesis of gene fragments eglA (gene cluster), Aga0283 (agarase), NH2780 (new agarobiohydrolase): the codon optimization and gene synthesis of fragments eglA, Aga0283, NH2780 were all assisted by Nanjing GenScript Biotechnology Co., Ltd. , the method is as follows: design specific primers (eglA-F: 5'-GGGTTT CATATG TTTTATATATATT TG-3' (SEQ ID NO.1, the underlined part is Nde I restriction site), eglA-R:5'-ACC GAGCTC AGCTTCAGCTTTATAA-3' (SEQ ID NO.2, the underlined part is the Sac I restriction site), Aga0283-F:5'-GTA CCCGGG ATGACAAGTTGTCAAA-3' (SEQ ID NO.3, the underlined part is the Xma I restriction site), Aga0283-R:5'-GAC GTC GAC TTATTTTGCTGATCTT-3'(SEQ ID NO.4, the underlined part is the Sal I restriction site), NH2780-F:5'-CAT CCCGGG ATGTTTTCACAAAAT-3' (SEQ ID NO.5, the underlined part is the Xma I restriction site), NH2780-R:5'-GAC GTC GAC TTATTGTTTAACAAA-3' (SEQ ID NO.6, the underlined part...

Embodiment 2

[0052] Example 2 Enzyme Activity Analysis of Agarase Secreted by Recombinant Bacteria Clostridium sp.WK-AN1

[0053] The recombinant bacteria Clostridium sp.WK-AN1 seed liquid was inoculated into the basal medium containing 250mg / mL spectinomycin (ACM, it comprises: glucose, 10g / L; Yeast extract, 10g / L; NaHCO 3 , 2.52g / L; 2-(N-morpholino)ethanesulfonic acid, 2.132g / L; 100×salt solution, 10mL / L; 1000×trace element solution, 1mL / L; deoxidizing agent: disulfide Threitol, 0.077g / L; L-cysteine, 0.0242g / L; Na 2 S·9H 2 O, 0.0156g / L; and anaerobic indicator: resazurin, 0.001g / L; where 100× salt solution includes NaCl, 1.0g / L; MgCl 2 ·6H 2 O, 0.5g / L; KH 2 PO 4 , 0.2g / L; NH 4 Cl, 0.3g / L; KCl, 0.3g / L; CaCl 2 2H 2 O, 0.015g / L; 1000× trace element solution including FeCl 2 4H 2 O, 1.5g / L; CoCl 2 ·6H 2 O, 0.19g / L; MnCl 2 4H 2 O, 0.1g / L; ZnCl 2 , 0.07g / L; H 3 BO 3 , 0.006g / L; Na 2 MoO 4 2H 2 O, 0.036g / L; NiCl 2 ·6H 2 O, 0.024g / L; CuCl 2 2H 2 O, 0.002g / L), incubate at ...

Embodiment 3

[0060] Embodiment 3 takes agar polysaccharide as substrate fermentation biobutanol

[0061] In this example, by optimizing the agar fermentation medium, a mixed carbon source fermentation mode was implemented to realize the process of effectively utilizing agar polysaccharides to convert bio-butanol by engineered strains. Configuration takes 10g / L glucose and 15g / L agar polysaccharide as the fermentation medium of carbon source (being the fermentation medium containing 250mg / mL spectinomycin in embodiment 2) 50mL, subpackage in the anaerobic serum bottle, Fill the bottle with nitrogen for 10 minutes to remove the air in the bottle, close the rubber stopper and aluminum cap tightly, inject 1mL of 1M HCl solution filled with nitrogen into the medium; sterilize the prepared medium at 121°C, cool to room temperature and adjust the pH to 6.0 , for subsequent fermentation experiments.

[0062] The engineering bacterial strain Clostridium sp.WK-AN1 bacterium liquid obtained in Examp...

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Abstract

The invention belongs to the technical field of genetic engineering, and discloses a recombinant bacterium and construction and application thereof. The recombinant bacterium comprise recombinant plasmids, and the recombinant plasmids comprise the following gene segments: agarase genes, gene clusters and new agarobiose hydrolase genes. According to the recombinant bacterium, the agarase system is introduced into clostridium and secretory expression of the clostridium is realized, an extracellular expression system of agarase is established in the clostridium for the first time, and the capability of converting biobutanol by directly utilizing agaro-polysaccharide is improved; according to the recombinant bacterium, biomass enzymolysis and butanol fermentation are combined, enzymolysis and fermentation are synchronously carried out, the capacity of degrading agaro-polysaccharide which is not originally achieved by wild type solventogenic clostridium is endowed, the feedback inhibition effect in the enzymolysis process is also achieved, the steps are greatly simplified, the production cost is reduced, and the effect is obviously better than that of physical mixing treatment of in-vitro enzyme.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant bacterium and its construction and application. Background technique [0002] With the rapid development of today's world economy, human beings' demand for energy is increasing day by day. However, non-renewable fossil fuel energy sources such as oil and natural gas are not only facing the danger of depletion, but also the impact of their exploitation and utilization on environmental pollution cannot be ignored. Therefore, The development and utilization of new energy has become an urgent need to deal with energy problems. Biofuel has become the best choice to replace fossil fuels due to its recyclability, environmental protection and renewable advantages. As a member of biofuel, biobutanol can be used for its low evaporation, high calorific value and low corrosion. It is transported through pipelines and has many advantages such as no nitrog...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/56C12N1/21C12P7/16C12R1/145
CPCC12N15/74C12N9/2468C12N9/2402C12P7/16C12Y302/01081C12Y302/01158C12Y302/01159Y02E50/10
Inventor 吴奕瑞白圣凯谢薇李潮
Owner SHANTOU UNIV
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