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High secretion heat-resisting yeast genetically engineered bacterial strain and applications thereof

A high-secretion, Pichia pastoris technology, applied in the biological field, can solve problems such as high energy consumption and lower temperature, and achieve the effect of high trehalose content

Active Publication Date: 2016-10-26
无锡优普克生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the lower induction temperature leads to the consumption of a large amount of energy to reduce the temperature in industrial production. Therefore, it is urgent to develop Pichia expression strains that can efficiently induce the expression of foreign proteins under high temperature conditions.

Method used

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  • High secretion heat-resisting yeast genetically engineered bacterial strain and applications thereof
  • High secretion heat-resisting yeast genetically engineered bacterial strain and applications thereof
  • High secretion heat-resisting yeast genetically engineered bacterial strain and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: bacterial strain mutagenesis method

[0050] Mutation methods such as ultraviolet mutagenesis, chemical reagent mutagenesis, and normal pressure room temperature plasma mutagenesis are used.

[0051] References of ultraviolet radiation, diethyl sulfate, and nitrosoguanidine mutagenesis methods [The mutagenic effect of different mutagenesis methods on hyaluronic acid-producing strain Sz560, Ding Yong, Shi Xiaoyong, Zhang Xinming, Food Research and Development, 2012,12:50 -53]. Atmospheric pressure and room temperature plasma mutagenesis method reference [ARTP mutagenesis breeding of low-purine Saccharomyces cerevisiae, Kang Fushuai, Yan Bing, Lu Nanquan, Zhou Shishui, Modern Food Science and Technology, 2014, 2: 188-191]. During the research process, the strains were mutagenized by the above methods and different combinations of the above methods.

Embodiment 2

[0052] Embodiment 2: the domestication of bacterial strain

[0053] The strains obtained by different mutagenesis methods were mixed (mixed strains) for high-temperature acclimatization. The mixed bacteria were inoculated into fresh YPD medium for temperature gradient acclimatization culture at 32°C, 34°C, 36°C, 38°C, 40°C. The mixed bacteria were inoculated into 100 mL of fresh YPD medium for cultivation at 32°C, and the original starter bacteria were simultaneously inoculated into 100 mL of fresh YPD medium for cultivation at 30°C, with an initial OD of 0.1. Cultivate for 72 hours to acclimate to one generation. Measure the OD after the end of the first generation. If the ratio of the OD value under high temperature conditions to the OD value of the original starting bacteria at 30°C is greater than 1, then enter the next temperature gradient acclimatization, otherwise, continue to transfer to fresh medium for this process. The second acclimatization of temperature until t...

Embodiment 3

[0054] Example 3: Monoclonal isolation of temperature-resistant and high-secretion domesticated strains

[0055] 1) Evenly spread the mutagenized and acclimated strains on the screening medium, and culture them in a 40°C incubator for 3 days.

[0056] 2) Pick 28 single clones with the largest colony and the most obvious red transparent circle and culture them overnight in 5 mL of fresh YPD medium. The larger the colony, the better the activity at high temperature, and the more obvious the red transparent circle, the higher the activity of the lipase secreted and expressed.

[0057] 3) Inoculate 28 single clones in 100mL fresh YPD medium, culture at 40°C and 200rpm for 3 days at a constant temperature, and screen out 5 strains with the best growth conditions for follow-up experiments. The 5 strains are respectively named 6, D, N, J, H.

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Abstract

The invention discloses a high secretion heat-resisting yeast genetically engineered bacterial strain and applications of the high secretion heat-resisting yeast genetically engineered bacterial strain, belonging to the field of biotechnology. According to the high secretion heat-resisting yeast genetically engineered bacterial strain and the applications, the yeast genetically engineered bacterial strain capable of expressing lipase genes with high secretion amount at high temperature is obtained, the yeast genetically engineered bacterial strain is preserved in the China Center for Type Culture Collection (CCTCC), and is assigned with the accession number of CCTC NO: M 2016278. Through the sequencing analysis, the lipase genes and promoter sequences of the lipase genes have no mutation, which illustrates that the lipase gene expression at high secretion amount of the mutant strain is caused by the mutation of other gene sequences in a genome. The lipase genes are knocked out through a homologous double-crossover manner, and the constructed knock-out bacterial strain can be taken as an expression host of other foreign protein, can be applied for improving the expression level of other foreign genes, and can be fermented under the high-temperature fermentation condition.

Description

technical field [0001] The invention relates to a high-secretion heat-resistant yeast genetically engineered bacterium and its application, belonging to the field of biotechnology. Background technique [0002] Yeast is an important production strain in the alcohol and food industries. With the continuous strengthening of human energy crisis and environmental protection awareness, it has gradually become a development trend to replace gasoline with fuel alcohol. High-temperature-resistant yeast can reduce the trouble and cost caused by cooling in alcohol production, and ensure that industrial fermentation can be carried out normally at high temperature. Therefore, the screening of high-temperature-resistant yeast has become a research hotspot. In recent years, many strains of artificially bred high-temperature-resistant yeast have been put into production, and good economic benefits have been achieved. [0003] As early as the middle of the 20th century, Chinese microbiolog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/16C12N9/20C12N9/18C12N9/50C12R1/84
CPCC12N9/18C12N9/20C12N9/50C12Y301/01003C12Y301/01004C12R2001/84C12N1/165C12N9/58C12N1/16
Inventor 喻晓蔚徐岩
Owner 无锡优普克生物科技有限公司
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