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Pichia pastoris engineering bacteria for producing inositol and fermentation method

A technology of Pichia pastoris and engineering bacteria, which is applied in the field of bioengineering to achieve the effect of enhancing inositol synthesis capacity and improving supply

Active Publication Date: 2021-06-15
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problems existing in the production of inositol by the existing microbial fermentation method, the object of the present invention is to provide a Pichia engineering bacterium producing inositol

Method used

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  • Pichia pastoris engineering bacteria for producing inositol and fermentation method
  • Pichia pastoris engineering bacteria for producing inositol and fermentation method
  • Pichia pastoris engineering bacteria for producing inositol and fermentation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Construction of Pichia pastoris engineering strains overexpressing endogenous and exogenous inositol synthesis key enzyme genes and knocking out inositol transporter genes

[0038] Such as figure 1 As shown, glucose is transported into cells to generate glucose-6-phosphate, and then glucose-6-phosphate is catalyzed by inositol-1-phosphate synthase and inositol monophosphatase to generate inositol, and finally excess inositol will be secreted outside the cell. Extracellular myo-inositol can be absorbed into the cell through two inositol transporters (PpITR1 and PpITR2), while excessive intracellular myo-inositol will affect the expression of myo-inositol-1-phosphate synthase and myo-inositol monophosphatase and activity. Therefore, the ability of Pichia pastoris to accumulate inositol can be effectively improved by overexpressing endogenous and exogenous inositol synthesis key enzyme genes and knocking out inositol transporter genes.

[0039] use mazF - ...

Embodiment 2

[0055] Example 2 Increased inositol production by reducing glycolytic flux

[0056] The precursor of inositol synthesis is glucose-6-phosphate, and glucose-6-phosphate can also enter the glycolysis and pentose phosphate pathway for metabolism, so reducing the glycolytic metabolic flow helps to increase the glucose-6-phosphate supply. fructose-6-phosphate kinase gene pfk1 and pfk2 It is a key gene in glycolysis. Knocking out one of the genes can reduce the carbon metabolism flow of glycolysis while ensuring the normal growth of bacteria.

[0057] 1. Construction pfk2 gene editing plasmid

[0058] Amplification of fructose-6-phosphate kinase 2 gene by PCR pfk2 For the homologous arm fragments on both sides, the target gene after gel electrophoresis was gel recovered, and the obtained fragment was ligated with the enzyme-cut JQ vector, transformed into Escherichia coli Trans10 by heat shock, recovered at 37°C for 30 min, and coated Cloth LB plates (containing ampicillin...

Embodiment 3

[0063] Example 3 Further improving inositol production by dynamically regulating glycolysis and pentose phosphate pathways

[0064] It is proved by Example 2 that reducing the glycolytic metabolic flux helps to increase the supply of glucose-6-phosphate, thereby increasing the production of inositol. Therefore, in order to further increase the supply of glucose-6-phosphate, glycerol-inducible promoters are used to regulate the expression of the gene encoding glucose-6-phosphate isomerase in the glycolytic pathway. pgi , the gene encoding fructose-6-phosphate kinase 1 pfk1 and the gene encoding glucose-6-phosphate dehydrogenase in the pentose phosphate pathway zwf , the resulting strains were unaffected when grown in glycerol as a carbon source medium, but were affected by carbon metabolism inhibition when grown in a medium containing glucose pgi , pfk1 and zwf The expression of is blocked, thereby reducing the metabolic flux of the glycolysis and pentose phosphate pa...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to pichia pastoris engineering bacteria for producing inositol and a fermentation method. Endogenous and exogenous inositol synthesis key enzyme genes are over-expressed in pichia pastoris, and an inositol transporter protein gene is knocked out, so that the inositol accumulation capability of the pichia pastoris is improved; a fructose-6-phosphate kinase 2 (Pfk2) gene is knocked out, and a glycerol-induced promoter is utilized to regulate and express a glucose-6-phosphate isomerase (Pgi) gene, a fructose-6-phosphate kinase 1 (Pfk1) gene and a glucose-6-phosphate dehydrogenase (Zwf) gene, so that the purpose of weakening glycolysis and phosphopentose pathways when glucose is used as a carbon source is achieved; therefore, the supply of inositol synthesis precursor glucose-6-phosphoric acid is improved. In high-density fermentation, the growth of bacterial cell and the production of inositol are controlled by using the adding time of glycerol and the glucose in a culture medium, so that the production capacity of the inositol of a pichia pastoris engineering strain is greatly improved.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a Pichia pastoris engineering bacterium producing inositol and a fermentation method. Background technique [0002] Myo-inositol, also known as cyclohexyl alcohol, is an isomer of glucose. As a water-soluble vitamin, inositol belongs to the B family. Inositol theoretically has nine isomers, of which inositol is the most common one. Inositol is not only widely distributed in animals and plants, but also can be synthesized in various organs of animals such as heart and liver. Due to its biological activity, inositol has been widely used in medical, food, feed industry and other fields. In the medical field, inositol is mostly used as an auxiliary drug, not only to maintain normal metabolic activities in the human body, but also to treat neurological diseases, diabetes, vitamin deficiency, liver cirrhosis and other metabolic or neurological diseases. Studies have shown tha...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/53C12N15/54C12N15/61C12N15/31C12N15/113C12P7/18C12R1/84
CPCC07K14/39C12N1/16C12N9/0006C12N9/1205C12N9/16C12N9/90C12N9/92C12P7/18C12Y101/01049C12Y207/01011C12Y207/01105C12Y503/01009C12Y505/01
Inventor 张杰张齐全王晓璐姚斌罗会颖黄火清苏小运柏映国涂涛王苑王亚茹秦星
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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