Method for increasing pyruvic acid accumulated in escherichia coli

A technology of Escherichia coli and pyruvate, applied in the biological field, can solve problems such as the inability to accurately discover key genes

Active Publication Date: 2018-09-14
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Based on the traditional mutagenesis method, it is difficult to quickly locate the gene locus of the strain wi

Method used

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  • Method for increasing pyruvic acid accumulated in escherichia coli
  • Method for increasing pyruvic acid accumulated in escherichia coli
  • Method for increasing pyruvic acid accumulated in escherichia coli

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1 Construction of Escherichia coli genetically engineered bacteria KLPP:

[0042] Escherichia coli engineering bacterium KLPP transformation schematic diagram of the present invention is as figure 1 shown.

[0043] 1) In the first step, the pKD46 plasmid was introduced into Escherichia coli K-12MG1655 by heat shock transformation method to obtain Escherichia coli K. The specific operation steps are as follows: absorb 1uL pKD46 plasmid and mix it with Escherichia coli K-12MG1655 competent cells, place it on ice for 20 minutes, place it in a water bath at 42°C for heat shock for 90 seconds, then place it on ice for 2 minutes, add 1 mL of LB medium, and then 30 Incubate at ℃ for 40 min, spread on Amp-resistant LB plates, and culture at 30 °C overnight, and select the grown clones as Escherichia coli K.

[0044] 2) In the second step, NCBI downloads the E. coli genome sequence, finds the ldhA gene sequence from it, and designs primers to amplify the cat-sacB sequ...

Embodiment 2

[0072] Example 2 Establishment and high-throughput screening of Escherichia coli genetically engineered KLPP mutant library

[0073] The high-throughput screening schematic diagram of the present invention is as follows figure 2 shown.

[0074] 1) A high-throughput detection method for pyruvate: pyruvate undergoes a condensation reaction with 2,4-dinitrophenylhydrazine under acidic conditions to form pyruvate dinitrophenylhydrazone. Dinitrophenylhydrazone appears red under alkaline conditions, and can sensitively reflect the content of pyruvate after colorimetry at 520 nm. Prepare 6.25 g / L pyruvate mother solution, and then dilute it into 0, 0.625, 1.250, 1.875, 2.5, 3.125, 3.75, 5 g / L solutions, take 40 μL different dilutions of pyruvate solution, 40 μL 0.033% Nitrophenylhydrazine and 40 μL of 2.81 mol / L NaOH were mixed, left to react for 10 min, and the absorbance was measured at a wavelength of 520 nm using a Molecular Device Spectra MaxM2 microplate reader. Three paral...

Embodiment 3

[0088] Example 3 Scale-up fermentation evaluation of mutant strain K30

[0089] Pick recombinant Escherichia coli K30 monoclonal into 3 ml LB test tube and culture at 37 ℃ OD 600 Grow to 3.0, transfer to a 1 L shake flask with a capacity of 200 ml LB, cultivate overnight at 37 °C, transfer to a fermenter with a 10% inoculum size, and use Shanghai Baoxing BioTech-5BG automatic 5 L fermentation in the fermenter system, filled with 2 L fermentation broth. The pH of the fermenter is controlled at 6.5, the ventilation rate is 1 v / v min, and the dissolved oxygen is controlled to not be lower than 30%. Sampling and determination of fermentation broth during fermentation OD 600 and the pyruvate content in the fermentation broth, the pyruvate output can reach 63g / L after 47 hours of fermentation (such as Figure 5 shown).

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Abstract

The invention belongs to the technical field of microorganism genetic engineering and in particular relates to an escherichia coli genetically engineered bacterium establishment method for increasingaccumulation of pyruvic acid, and production and application. The genetically engineered bacterium provided by the invention is established by using a method of the following steps: deleting encodinggenes of lactic dehydrogenase, encoding genes of pyruvic oxidase, encoding genes of phosphotransacetylase and encoding genes of acetokinase from wild derived escherichia coli, and establishing an escherichia coli genetically engineered bacterium KLPP with accumulated pyruvic acid. From KLPP, a mutation library is established through Tn5 transposons, a mutation strain with increased pyruvic acid accumulation is screened in a high flux manner, and genes for increasing pyruvic acid accumulation can be found through whole genome sequencing.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a new gene that affects pyruvate accumulation and a modified Escherichia coli ( Escherichia coli ) to improve its pyruvate accumulation method and use the method to construct pyruvate-producing bacteria. Background technique [0002] Pyruvate is a key intermediate product in the process of biological metabolism. At the same time, it provides raw materials for the glycolysis pathway and the tricarboxylic acid cycle, and provides the necessary energy for various life activities of organisms. In addition, it is also widely used in medicine, food, feed, daily chemical, pesticide, biochemical research and cell culture and other fields. As our demand for pyruvate continues to increase, how to find an environmentally friendly and sustainable method for obtaining pyruvate in large quantities is particularly important. [0003] There are three main methods for producing pyruvate:...

Claims

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Application Information

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IPC IPC(8): C12P7/40C12N1/21C12R1/19
CPCC12N9/0006C12N9/0008C12N9/1029C12N9/1217C12P7/40C12Y101/01027C12Y101/01028C12Y102/03003C12Y207/02001
Inventor 王钦宏彭彦峰史晓荣
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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