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Rapid optimization method based on different microbial cell factory heterologous expression enzymes

A microbial cell and heterologous expression technology, which is applied in the field of rapid optimization of heterologous expression enzymes based on different microbial cell factories, can solve problems such as the stability of strains to be verified, and achieve the effect of rapid jump

Pending Publication Date: 2021-06-18
JILIN COFCO BIOCHEM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Later, the team formed a direct synthesis process of D-psicose through whole pathway metabolic engineering, but the stability of the strain needs to be verified

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Bacillus subtilis WB800 was used as the host strain, and DPEase was derived from Clostridium boschii (Genebank No. A8RG82), which was constitutively expressed without induction, and Bacillus subtilis WB800-Bs03 was constructed. Optimizing medium composition by response surface method, determining temperature and pH conditions by orthogonal experiment, and combining fermentation cycle and other factors, determined the induction culture process for high expression of DPEase in Bacillus subtilis WB800: 20g / L glucose as carbon source, 20g / L yeast powder is the nitrogen source, and 1g / L sodium sulfate is added at the same time, the temperature is 37°C, pH 7.2, and 600g / L glucose is added to the process flow.

[0042]Under this condition, the enzyme expression level reached 96% of the enzyme reference level, and the host cell growth level reached 83% of the cell reference level. After 36 hours of fermentation and cultivation, the biomass of Bacillus subtilis reached above 10...

Embodiment 2

[0045] Escherichia coli BW25113 was used as the host strain, DPEase was derived from Clostridium cellulolyticum (Genebank No. B8I944.1), and Escherichia coli BW25113-Ec01 was constructed by using the Lac promoter. The effects of factors such as induction temperature, pH and the amount of inducer IPTG on host cell growth and enzyme expression were investigated through single factor experiments. The scope of the test comprehensively considered the environmental parameters of bacterial growth and protein folding, and determined the DPEase in Escherichia coli BW25113 High-efficiency expression induction culture process: 10g / L glucose as carbon source, 5g / L yeast powder as nitrogen source, 0.6g / L magnesium sulfate added, initial culture temperature 37°C, induction temperature 25°C, constant pH 7.0, IPTG final The concentration is 0.5mM, and 600g / L glucose and 1g / L magnesium sulfate are added during the process.

[0046] Under this condition, the enzyme expression level reaches 99% ...

Embodiment 3

[0049] Pichia pastoris GS115 was used as the host strain, and DPEase was derived from Agrobacterium tumefaciens (Genebank No. ANH56792.1), and Pichia pastoris GS115-Pp01 was constructed. The expression was induced by methanol, and the environmental parameters of the growth period and product synthesis period were optimized through single factor experiments, and factors such as medium composition, temperature, pH and starvation time were determined, and the induction culture process for the efficient secretion and expression of DPEase in Pichia pastoris GS115 was determined: With 60g / L glycerol as the carbon source, 10g / L amino-free yeast (YNB) as the nitrogen source, temperature 30°C, pH 7.2, starvation culture for about 3 hours, constant flow of 100% methanol until the end of fermentation.

[0050] Under this condition, the enzyme expression level reaches 93% of the enzyme reference level, and the host cell growth level reaches 70% of the cell reference level. The conversion ...

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Abstract

The invention relates to the field of microbial engineering, in particular to a method for optimizing heterologous expression enzyme in a microbial cell factory. The method comprises the following steps that enzyme expression levels and microbial cell factory growth levels are correspondingly determined under different external environmental factors by designing a programmed experiment method, the highest enzyme expression level is used as the enzyme reference level, and the highest microbial cell factory growth level is used as the cell reference level; and the conditions when the enzyme expression level reaches at least 90% of the enzyme reference level and the microbial cell factory growth level reaches at least 70% of the cell reference level are selected as optimization conditions for heterologous expression of the enzyme in the microbial cell factory. The method for optimizing the heterologous expression enzyme in the microbial cell factory has the advantages that enzyme expression characteristics and host cell growth characteristics are comprehensively considered, a qualitative and quantitative production expression method is determined, and rapid jump from strain construction to a biological catalysis process can be realized.

Description

technical field [0001] The present invention relates to the field of microbial engineering, in particular to a method and application for optimizing the heterologous expression of enzymes in microbial cell factories, especially the induced expression and culture process development of conventional model bacteria or model fungi, comprehensively considering the enzyme expression characteristics and host cell growth characteristics , to determine the qualitative and quantitative production and expression methods, and realize the rapid jump from strain construction to biocatalytic process. Background technique [0002] D-psicose is the epimer of D-fructose at the C-3 position. It is a rare monosaccharide with very little content in nature. It has an extremely low calorie value and is similar to sucrose in sweetness but absorbs The rate is only 0.3% of sucrose, so it can be used as a low-calorie sweetener to replace sucrose. Studies have shown that D-psicose has a variety of nut...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12R1/125C12R1/19C12R1/84C12R1/685
CPCC12N9/90C12Y501/03
Inventor 佟毅周卫强何太波唐堂沈雪梅彭超王小艳陈博李义
Owner JILIN COFCO BIOCHEM
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