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Construction method of glucose metabolic pathway in shewanella strain

A construction method, glucose technology, applied in the field of strain construction of Shewanella bacteria that uses glucose as the only carbon source for growth and metabolism, can solve the problems of narrow substrate spectrum, low electron transfer efficiency, and unutilization, etc., to improve cell Efficiency of external electron transfer, effect on broadening available substrate spectrum

Pending Publication Date: 2022-05-27
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Wild-type Shewanella generally uses lactic acid as a substrate for growth and metabolism, and cannot utilize sugars such as glucose and xylose, which makes the available substrate spectrum of the strain too narrow, and the extracellular electron transfer efficiency is low

Method used

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  • Construction method of glucose metabolic pathway in shewanella strain
  • Construction method of glucose metabolic pathway in shewanella strain
  • Construction method of glucose metabolic pathway in shewanella strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Example 1: Construction of recombinant plasmids

[0112] (1) Acquisition and sequence optimization of the target gene:

[0113] First, query the required target gene sequence information on KEGG (http: / / www.kegg.jp / kegg / genes.html), the target gene includes the promotion of glucose uptake into cells (glucose transmembrane transport module: ompC, galp, glf, glk) to promote intracellular metabolism (optimized ED pathway: zwf, pgl, edd, eda, pfkA, pykA) key genes of two modules; use Jcat (http: / / www.jcat.de / ) to code the coding sequence Sub-optimization and site-directed mutagenesis of the bio-brick restriction sites in the sequence were selected for optimization in Shewanella oneidensis MR-1, and the restriction sites EcoRI, XbaI, SpeI and PstI needed for future experiments were avoided.

[0114] (2) Using the Biobrick method, construct the recombinant plasmid:

[0115] To obtain the optimized sequence, add an XbaI restriction site at the 5' end of each target gene frag...

Embodiment 2

[0124] Example 2: Construction of recombinant Shewanella strains

[0125] ①Transformation: The recombinant plasmid obtained above was introduced into E.coliWM3064 by physical transformation method for the construction of the subsequent engineering Shewanella.

[0126] Remove 50 μL of E.coliWM3064 competent cells from the -80°C refrigerator, thaw naturally in an ice box, add 33 μL of recombinant plasmid pSH, let stand on ice for 30 min, heat shock at 42°C for 90 s, and let stand on ice for 2-3 min. Add 1mL LB+DAP liquid medium (5g / L yeast extract, 10g / L tryptone, 10g / LNaCl, 0.059g / L 2,6-diaminopimelic acid) to the tube, and place at 37°C, 220rpm. Resuscitate in a shaker for 1 h. After centrifugation, it was spread on LB+DAP+kana solid plate (5g / L yeast extract, 10g / L tryptone, 10g / LNaCl, 0.059g / L DAP, 50mg / mL kanamycin (kana, 1:1000). ), 15g / L agar powder) and finally put it upside down in a constant temperature incubator at 37°C for overnight culture, pick out positive clone...

Embodiment 3

[0139] Example 3: Shewanella engineering bacteria WT, PSH1-13 were fermented and cultured in a modified M9 medium containing a concentration of 3 g / L glucose solution (including aerobic and anaerobic)

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Abstract

The invention relates to a construction method of a glucose metabolic pathway in shewanella strains. A glucose transmembrane transport module is synthesized into a key gene for biological modification and optimization (Ompc, galp, glf and glk) and combination of the glucose transport module and an ED module to enhance glucose metabolism (zwf, pgl, edd, eda, pfkA and pykA) by means of a heterologous expression technology and genetic engineering and the like, recombinant plasmids are constructed through Biobrick and are introduced into wild shewanella MR-1, a metabolic pathway of shewanella is reconstructed, and the recombinant shewanella MR-1 is obtained. Growth metabolism can be carried out by taking glucose as a unique carbon source, and the available substrate spectrum of shewanella is widened. 13 engineering strains capable of performing glucose growth metabolism can be obtained. A practical basis is provided for the design of co-utilization of the substrate of the microbial fuel cell, and the extracellular electron transfer efficiency of the strain is favorably improved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and biological metabolism, in particular to a method for constructing a strain that enables Shewanella to use glucose as the sole carbon source for growth and metabolism. Background technique [0002] Genetic engineering, also known as gene splicing technology and DNA recombination technology, is based on molecular genetics and uses modern methods of molecular biology and microbiology as means to combine genes from different sources according to pre-designed blueprints. The operation of constructing hybrid DNA molecules in vitro, and then introducing them into living cells, so that foreign genes can be replicated, transcribed, translated and expressed in recipient cells, so as to change the original genetic characteristics of organisms, obtain new varieties, and produce new products. [0003] Glucose (chemical formula C 6 H 12 O 6 ), referred to as glucose. Chemical name: 2,3,4,5,6...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/21C12R1/01
CPCC12N15/74C12Y207/01002C07K14/245C12Y504/99009C12Y101/01049C12Y301/01031C12Y207/01011C12Y402/01012C12Y401/02014C12Y207/0104C12N9/1205C12N9/90C12N9/0006C12N9/18C12N9/88C12N2800/22Y02E60/50
Inventor 宋浩杨池张君奇李锋尹静
Owner TIANJIN UNIV