Escherichia coli integrated with amino acid oxidase and glucose dehydrogenase

A technology of glucose dehydrogenase and Escherichia coli, applied in the field of bioengineering, can solve the problems of increasing the complexity of antibiotics and inducers, and increasing the production cost of inducers, achieving strong environmental adaptability, reducing operation steps, and good application characteristics. Effect

Pending Publication Date: 2020-12-22
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, engineering strains constructed using L-amino acid oxidase or glucose dehydrogenase usually use plasmid expression systems, and plasmid expression systems require antibiotics to mainta

Method used

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  • Escherichia coli integrated with amino acid oxidase and glucose dehydrogenase
  • Escherichia coli integrated with amino acid oxidase and glucose dehydrogenase
  • Escherichia coli integrated with amino acid oxidase and glucose dehydrogenase

Examples

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Example Embodiment

[0032] Example 1: Screening of Chromosomal Integration Sites

[0033] 1. The chromosome of Escherichia coli BL21 (DE3) with a total sequence length of 4.6Mbp is divided into six regions, and two sites are selected in each region to integrate L-amino acid oxidase and glucose dehydrogenase, and then The strains with L-amino acid oxidase and glucose dehydrogenase integrated at each site were tested for activity, so as to determine the best integration site. The grouped regions and sites are 0~0.76Mbp(dkgB,eaeH), 0.76~1.53Mbp(lysO,bluF), 1.53~2.28Mbp(tam,glsB), 2.28~3.04Mbp(rcs,mntH), 3.04 ~3.8Mbp(nupG, pitB), 3.8~4.6Mbp(rbsA, pgi).

[0034] (1) Extraction of plasmid

[0035] Plasmid extraction was performed according to the instructions on the plasmid mini-extraction kit of Shanghai Jierui Company.

[0036] (2) Construction of pIn-LA-cat-RA plasmid

[0037] The primers used during the construction are listed in Table 1. Chloramphenicol resistance gene was amplified from pKD3...

Example Embodiment

[0075] Example 2

[0076] This example integrates different combinations of L-amino acid oxidase (pmaao, cmaao) and glucose dehydrogenase (gsgdh, hmgdh, bsgdh) at the optimal integration site nupG screened, and inoculates the integrated strains of different combinations in Cultivate overnight in the test tube, then inoculate 50 mL of fresh LB medium with 1% inoculum, put it into a vibrating shaker, and cultivate at 25°C and 200 rpm for 24 hours. After 24 hours, the cells were collected by centrifugation at 8000 rpm for 10 min at 4°C. Using 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate solution at pH 7.5 as the reaction buffer, the whole-cell catalytic reaction system is: 50g / L wet cells, 35°C, 3g / L L-phenylalanine, transformed The time is 5h. After conversion, the yield of phenylpyruvate was determined by high performance liquid chromatography (HPLC). Take 20mL, OD 600 3.5 bacteria solution, centrifuged at 8000 rpm for 10 min at 4°C, and then collected the c...

Example Embodiment

[0081] Example 3

[0082] This example integrates multiple copies of L-amino acid oxidase pmaao and glucose dehydrogenase bsgdh at the screened optimal integration sites glsB, nupG, and pitB, and tests the multi-site multi-copy integration of pmaao and bsgdh to transform the strain into L- Yield of phenylalanine to phenylpyruvate and activity of glucose dehydrogenase.

[0083] Genes with different copy numbers were sequentially integrated at the sites screened in Example 1, and multi-copy integration followed the FLP recombination method (rapid and reliable strategy for chromosomal integration of gene(s) with multiple copies. Scientific Reports 2015, 5.). Inoculate E.coli BL21 strains with different copy numbers of pmaao and bsgdh into test tubes for overnight culture, then inoculate 1% of the inoculum into 50 mL of fresh LB medium, put it into a shaking shaker, and incubate at 25°C 1. Cultivate for 24 hours under the condition of 200 rpm. After 24 hours, the cells were coll...

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Abstract

The invention discloses escherichia coli integrated with amino acid oxidase and glucose dehydrogenase, and belongs to the technical field of bioengineering. According to the invention, L-amino acid oxidase and glucose dehydrogenase genes are integrated into escherichia coli chromosomes in a multi-site multi-copy manner by adopting a synthetic biological technical means, so that escherichia coli has better growth characteristics, and the final quantity of strains cultured in a high-density manner can be increased by more than 30% compared with that of original strains. When the engineering bacterium is applied to production of tanshinol, the yield of tanshinol can reach 87.46 mM, hydroxytyrosol can be well produced, and the yield of hydroxytyrosol can reach 54.43 mM.

Description

technical field [0001] The invention relates to Escherichia coli integrated with amino acid oxidase and glucose dehydrogenase, belonging to the technical field of bioengineering. Background technique [0002] L-amino acid oxidase is widely found in bacteria, fungi, mammalian cells, snake venoms, insect toxins and algae (L-amino acid oxidase as biocatalyst: a dream too far? Appl.Microbiol.Biotechnol.2013,97:9323-41 ). Most of the L-amino acid oxidase will produce hydrogen peroxide when oxidizing amino acids, which will be toxic to itself or the cells in the environment. There is also a class of amino acid oxidases that do not produce hydrogen peroxide in nature, and these enzymes are derived from Proteus sp., Providencia sp., Morganella sp., etc. According to relevant literature, the oxidases in these bacteria dehydrogenate amino acids to generate corresponding ketoacids, then transfer electrons to cytochrome oxidase through the membrane respiratory chain, and finally combi...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/42C12P7/22C12R1/19
CPCC12N9/0022C12N9/0006C12Y104/03002C12Y101/9901C12N15/70C12P7/42C12P7/22Y02A50/30
Inventor 蔡宇杰熊天真丁彦蕊白亚军郑晓晖
Owner JIANGNAN UNIV
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