Recombinant bacterium with high biomass and/or high growth speed, and construction method and application thereof

A construction method and a technology of recombinant bacteria, applied in biochemical equipment and methods, methods based on microorganisms, microorganisms, etc., can solve the problems of lycopene and astaxanthin with complex processes, high costs, and unsuitability for large-scale production

Active Publication Date: 2015-10-28
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The process of obtaining natural lycopene and astaxanthin is comp

Method used

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  • Recombinant bacterium with high biomass and/or high growth speed, and construction method and application thereof
  • Recombinant bacterium with high biomass and/or high growth speed, and construction method and application thereof
  • Recombinant bacterium with high biomass and/or high growth speed, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1. Construction of recombinant bacteria BW25113ΔptsGΔgalR and BW25113ΔptsGΔgalRglk

[0059] The recombinant bacterium BW25113ΔptsGΔgalRglk is a recombinant bacterium obtained by transforming Escherichia coli BW25113 with A1, A2 and A3, and the recombinant bacterium BW25113ΔptsGΔgalR is a recombinant bacterium obtained by transforming Escherichia coli BW25113 with A1 and A2.

[0060] The A1 is to knock out the phosphotransferase G subunit (ptsG) gene of Escherichia coli BW25113;

[0061] The A2 is to knock out the galactose repressor (GalR) gene of Escherichia coli BW25113;

[0062] The A3 is the introduction of the glucokinase (Glk) gene into Escherichia coli BW25113.

[0063] The construction method of BW25113ΔptsGΔgalRglk includes the following 1)-3), and the construction method of BW25113ΔptsGΔgalR includes the following 1) and 2):

[0064] 1) Knockout (deletion) the phosphotransferase G subunit (ptsG) gene of the Escherichia coli BW25113, keeping other gen...

Embodiment 2

[0081] Example 2, Biomass determination of recombinant bacteria BW25113ΔptsGΔgalR and BW25113ΔptsGΔgalRglk

[0082] The growth curve of the strain was measured in a micro-bioreactor, and the biomass (biomass) of the cells was carried out in the following test medium: ZYM-5052 was used to induce transformation with autoinduction medium containing kanamycin resistance, autoinduction medium ZYM-5052 formula: 100ml A+2ml B+2ml C+200μl D+100μl E, where the solutes and concentrations of A, B, C, D and E are as follows (the following are mass percentage concentrations):

[0083] A.ZY: 1% tryptone, 0.5% yeast powder;

[0084] B.50×M: 1.25M Na 2 HPO 4 , 1.25M KH 2 PO 4 , 2.5M NH 4 Cl and 0.25M Na 2 SO 4 ;

[0085] C.50×5052: 25% glycerin, 2.5% glucose, 10% lactose;

[0086] D.1M MgSO 4 ;

[0087] E.1000×trace elements: 50mM FeCl 3 , 20mM CaCl 2 , 10mM MnCl 2 , 10mM ZnSO 4 , CoCl 2 、NiCl 2 、Na 2 Mo 4 、Na 2 SeO 3 and H 3 BO 3 2mM each.

[0088] Test medium: Take 4...

Embodiment 3

[0093] Embodiment 3, construction and performance of streptosporine recombinant bacteria

[0094] 1. Construction of streptosporine recombinant bacteria

[0095] pLY036 was introduced into Escherichia coli BW25113 and the five recipient bacteria BW25113ΔptsG, BW25113ΔgalR, BW25113ΔptsGΔgalR and BW25113ΔptsGΔgalRglk constructed in Example 1, respectively, to obtain streptorubin recombinant bacteria. The plasmid transformation method used therein is the chemical transformation method. The streptosporine recombinant strain obtained by introducing pLY036 into Escherichia coli BW25113 was named BW25113 / pLY036, and the streptosporine recombinant strain obtained by introducing pLY036 into BW25113ΔptsG constructed in Example 1 was named BW25113ΔptsG / pLY036, and The recombinant streptosporin strain obtained by introducing pLY036 into BW25113ΔgalR constructed in Example 1 was named BW25113ΔgalR / pLY036, and the recombinant strain obtained by introducing pLY036 into BW25113ΔptsGΔgalR con...

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Abstract

The invention discloses a recombinant bacterium with high biomass and/or high growth speed, and a construction method and an application thereof. The construction method of the recombinant bacterium is characterized in that the recombinant bacterium with higher biomass and/or growth speed than a receptor bacterium is obtained through A1, A2 and A3 reconstruction of the receptor bacterium or A1 and A2 reconstruction of the receptor bacterium; the A1 is characterized by knocking out phosphotransferase Gsubunit gene of the receptor bacterium; the A2 is characterized by knocking out galactose inhibitor gene of the receptor bacterium; the A3 is characterized by inducting glucose kinase gene to the receptor bacterium; and the receptor bacterium is Escherichia coli, lactobacillus, streptococcus or hemophilus. The recombinant bacterium with high biomass and/or high growth speed constructed in the invention and a neurosporene producing recombinant bacterium have high application prospects and exploitation values in the fields of biological researches and medical science.

Description

technical field [0001] The invention relates to high biomass and / or high growth rate recombinant bacteria and its construction method and application. Background technique [0002] The growth of engineered strains is the key to industrial fermentation. The slow growth rate of bacteria will prolong the production cycle, reduce efficiency and increase cost, which is not conducive to the large-scale application of bacterial strains. The final cell density achieved by bacterial cell fermentation is also crucial. Therefore be badly in need of a kind of growth rate fast, the bacterial strain that the cell density that can reach is big. [0003] Terpenoids are compounds composed of isoprene as a structural unit. The synthesis pathway of terpenoids is divided into MVA pathway and MEP pathway. The MEP pathway is divided into two stages: 1. Generation of intermediate isopentenyl pyrophosphate (Isopentenyl diphosphate IPP) and its double bond isomer dimethylpropyl pyrophosphate (Di...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/21C12R1/19C12R1/225C12R1/46C12R1/21
Inventor 陈楠刘伟丰林白雪陶勇
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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