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Recombinant saccharomyces cerevisiae utilizing starch and secreting antibacterial peptide

A technology of Saccharomyces cerevisiae and antimicrobial peptides, which is applied in the fields of genetic engineering, fermentation engineering and evolutionary engineering, and can solve problems such as drug resistance of bacterial pathogens

Inactive Publication Date: 2017-04-26
SHANDONG YISHENG LIVESTOCK & POULTRY BREEDING CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the indiscriminate use of antibiotics has been shown to lead to widespread resistance in bacterial pathogens

Method used

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  • Recombinant saccharomyces cerevisiae utilizing starch and secreting antibacterial peptide
  • Recombinant saccharomyces cerevisiae utilizing starch and secreting antibacterial peptide
  • Recombinant saccharomyces cerevisiae utilizing starch and secreting antibacterial peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Construction of Saccharomyces cerevisiae polygene co-expression vector

[0072] 1. Construction of integrated expression vector pTEGC-BsmBI

[0073] 1) Acquisition of G418 resistance gene

[0074] The target gene was amplified by PCR, and the G418 resistance gene was amplified using the G418F-MscI and G418R-EcoRV primers (Table 1) using the vector pPIC9k as a template. PCR reaction conditions: 98°C for 10s, 55°C for 15s, 72°C for 50s, 30 cycles, 72°C for 10min. It was verified by 2% agarose gel electrophoresis.

[0075] The target gene is recovered, purified, transformed into E. coli, verified, and sent for sequencing. The target fragment was recovered and purified, and stored at -20°C for future use. Connect the obtained G418 resistance gene to the T vector, transform Escherichia coli DH5α strain, culture at 37°C, extract its plasmid DNA, use G418F-MscI and G418R-EcoRV primers to screen positive strains by colony PCR, and send the positive clones to Yingw...

Embodiment 2

[0127] Example 2 A recombinant Saccharomyces cerevisiae that can efficiently utilize starch and secrete antimicrobial peptides

[0128] 1. Screening and verification of recombinant yeast transformants

[0129]Before the electrotransformation of Saccharomyces cerevisiae, the sensitivity test of the resistance screening marker G418 was carried out on Saccharomyces cerevisiae. It was found that the yeast was inhibited and could not grow on the YPD plate with a G418 concentration of 200 μg / ml, and the resistance concentration above 200 μg / ml was selected. Screening is performed, eg, 300 μg / ml.

[0130] The Saccharomyces cerevisiae polygene co-expression vector pTEGC-amy-ga-cath-T constructed in Example 1 was linearized with the restriction endonuclease HpaI, and transferred into Saccharomyces cerevisiae by electroporation transformation mediated by lithium acetate. The concentration was 300 μg / ml on the YPD plate and cultured for more than 48 hours, and the single colony that gre...

Embodiment 3

[0136] Example 3 Construction of Saccharomyces cerevisiae polygene co-expression vector

[0137] In this embodiment, the method for constructing a Saccharomyces cerevisiae multi-gene co-expression vector is the same as in Example 1, except that the antimicrobial peptide gene inserted into the carrier is an optimized gene of Plectasin (base sequence as shown in SEQ ID NO: 3), Others are the same as in Example 1, and the Saccharomyces cerevisiae polygene co-expression vector constructed in this example is named pTEGC-amy-ga-plec.

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Abstract

The invention discloses a recombinant saccharomyces cerevisiae utilizing starch and secreting antibacterial peptide. The recombinant saccharomyces cerevisiae contains a multigene coexpression vector capable of utilizing starch and secreting antibacterial peptide. The vector contains an alpha-amylase gene, a glucoamylase gene and an antibacterial peptide gene. During expression of the antibacterial peptide gene, the recombinant saccharomyces cerevisiae plays a synergistic role of starch degradation through coexpression of amylase and glucoamylase in starch degradation enzyme, thus realizing degrading utilization of starch matrix by recombinant bacteria to reach thallus growth, ethanol production or antibacterial peptide secretion, etc. Therefore, the recombinant yeast constructed by the invention can be utilized to realize starch degradation utilization, yeast thallus growth, alcohol conversion, sundry bacteria growth inhibition and other purposes synchronously in starch matrix based feed adding, industrial alcohol production and kitchen waste degradation.

Description

technical field [0001] The invention relates to the fields of genetic engineering, fermentation engineering and evolutionary engineering, and more specifically relates to a genetically recombined Saccharomyces cerevisiae capable of utilizing starch base materials and secreting antimicrobial peptides. Background technique [0002] Most of the feed materials are starch-containing base materials, such as cereals (corn, rice, wheat), roots and tubers (such as cassava, sweet potato, etc.), which contain a large amount of starch. Under the action of amylase, sucrase, etc. in the animal body, starch is finally decomposed into glucose, which is used by animals as energy. Although animals can secrete starch-degrading enzymes, it is generally necessary to add enzymes such as amylase and glucoamylase to help animals digest and utilize starch in animal breeding. For example, early weaned piglets often need to add enzymes such as amylase and protease to make up for the lack of digestive...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12N15/66C12N15/56C12N15/12C12R1/865
CPCC07K14/461C07K14/463C07K14/79C12N9/2414C12N9/2428C12N15/66C12N15/81C12N2800/102C12Y302/01001C12Y302/01003
Inventor 祝永华巩新民原新廷殷铭斌唐友康小龙张宏刚
Owner SHANDONG YISHENG LIVESTOCK & POULTRY BREEDING CO LTD
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