Construction and Verification of a Ribosomal DNA-Mediated Integrative Expression Vector in Kluyveromyces lactis

doi:10.3969/j.issn.1673-1689.2019.10.013

Home > Archive>Volume 38, Issue 10, 2019 >87-97. DOI:10.3969/j.issn.1673-1689.2019.10.013

Construction and Verification of a Ribosomal DNA-Mediated Integrative Expression Vector in Kluyveromyces lactis
DOI:
                        10.3969/j.issn.1673-1689.2019.10.013
                    
CSTR:
                        [cstr]
                    
Author:
                        
                        
                    
Affiliation:
Clc Number:Q78
Fund Project:

Article

Figures

Metrics

Reference

Cited by

Materials

Comments

Abstract:

An integrative expression vector applied for Kluyveromyces lactis was constructed in this study. To verify its application，the adenylate deaminase gene（AMPD） derived from Streptomyces murinus was sucessfully expressed by this vector. The recombinant vector pTRGA-amdS that based on the plasmid pMD 19T-simple was established containing K. lactis-derived 18S rDNA sequence for homologous recombination，the Saccharomyces cerevisiae-derived galactosidase gene promoter（pScGAL7） for expression of heterologous genes，the acetamidase gene（amdS） as selection marker，and AMPD as reporter gene. The recombinant expression vector was linearized by Sac II，removing E.coli ori and Amp resistant gene，then electransformed into K. lactis GG799 to obtain transformants. Real-time quantitative PCR analysis indicated that the copy number of the integrated AMPD gene ranged from 1 to 3 and positively correlated with AMP deaminase activity. When the inducer galactose was used as carbon source，the AMP deaminase activity in the culture supernant of the recombinant with three AMPD gene copies reached to 590±13.33 U/mL，improved 32.6%. The integrants were mitoically stable for 58 generations under the non-selective pressure condition（98.58%）. We constructed a new vector with the stable expression ability of heterologous gene and confirmed the 18S rDNA as a suitable recombination site. These research results lay a foundation for further molecular modification of K. lactis.

Reference

Cited by

Get Citation

SUN Haiye, ZHANG Liang, LI Youran, GU Zhenghua, DING Zhongyang, SHI Guiyang. Construction and Verification of a Ribosomal DNA-Mediated Integrative Expression Vector in Kluyveromyces lactis[J]. Journal of Food Science and Biotechnology,2019,38(10):87-97.

Copy

Article Metrics

Abstract:
PDF:
HTML:
Cited by:

History

Received:
Revised:
Adopted:
Online: April 02,2020
Published:

Article QR Code

Copy Right：Editorial Board of Journal of Food Science and Biotechnology

Address：No. 1800, Lihu Avenue, Wuxi 214122, Jiangsu Province,China PostCode：214122

Phone：0510-85913526 E-mail：xbbjb@jiangnan.edu.cn

Supported by:Beijing E-Tiller Technology Development Co., Ltd.

Get Citation

Share

Article Metrics

History

Article QR Code

WeChat

Mobile website