Recombinant yeast and preparation method thereof

A yeast and recombinant enzyme technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial-based methods, etc., can solve problems such as easy loss, low expression level, and inability to strictly control the efficiency of exogenous promoters. Achieve the effect of low cost and simple operation

Inactive Publication Date: 2010-08-25
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Both have their own characteristics: episomal vectors can produce higher copy numbers in the host, but are unstable and easy to lose; integrative vectors can produce stable transformants because they can be in...

Method used

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  • Recombinant yeast and preparation method thereof
  • Recombinant yeast and preparation method thereof
  • Recombinant yeast and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1, the construction of recombinant yeast

[0044] 1. Construction of expression vector pKGAPGA-amy-18S

[0045] (1) Construction of the vector pGAPGA: construction methods such as literature (Houhui Song, Yong Li, Weihuan Fang, Yunfeng Geng, Xu Wang, Min Wang, Bingsheng Qiu. Development of a set of expression vectors in Hansenula polymorpha. Biotechnology Letters, 2003, 25 (23): 1999-2006.). (Institute of Microbiology, Chinese Academy of Sciences)

[0046] (2) Construction of vector pKGAPGA

[0047] In order to be able to use the loxP site and the Cre recombinase system to knock out the resistance screening marker gene sequence on the vector pGAPGA, a pair of primers loxP-F and loxP-R were designed. The nucleotide sequences of the primers are as follows:

[0048] loxP-F:

[0049] 5'-GataacttcgtataatgtatgctatacgaagttatAGATCTAGGAAAAGGTGACAACTCGCAAAGT-3' (sequence 5 in the sequence listing)

[0050] loxP-R:

[0051] 5'-TataactcgtataatgtatgctatacgaagttatGGA...

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Abstract

The invention discloses recombinant yeast and a preparation method thereof. The method comprises the following steps of: (1) transferring an expression vector I into host yeast to obtain recombinant yeast transferred into the expression vector I, and recording the recombinant yeast as yeast A; and (2) transferring an expression vector II into the yeast A to obtain recombinant yeast transferred into the expression vector I and the expression vector II, and recording the recombinant yeast of the expression vector I and the expression vector II as yeast B. Experiments prove that the recombinant yeast prepared by the preparation method does not carry any resistance genes; the transferred expression vectors have multiple copy numbers between 5 and 50, and the copies are all integrated into a genome of the host yeast; and exogenous gene products expressed by the recombinant yeast are safe food-grade products. The preparation method has the advantages of simple operation, time saving, labor saving and low cost, and thus the recombinant yeast and the preparation method thereof are suitable for popularization and application.

Description

technical field [0001] The invention relates to a recombinant yeast and a preparation method thereof. Background technique [0002] Kluyveromyces lactis, early named Saccharomyceslactis and K.lactis van der Walt, was classified as Kluyveromycesmarxianus var.lactis in 1984, belonging to the genus Kluyveromyces, referred to as Kluyveromyces lactis (K. lactis). The yeast expression system, together with the expression systems of Pichia pastoris (P. pastoris), Hansenula polymorpha and S. cerevisiae, is called "four major expression systems of yeast". Cultivation, low cost, fast reproduction, convenient for large-scale cultivation and high-density fermentation and other characteristics. [0003] As a widely used exogenous gene expression system, K.lactis has the following advantages in addition to the common characteristics of the above-mentioned yeast expression system: 1), K.lactis expression system is safe and reliable, such as lactase prepared by K.lactis and chymosin have...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/09C12N15/66C12R1/645
Inventor 邱并生牛振东宋浩雷
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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