One-step construction of a food-grade expression system based on the URA3 gene in Kluyveromyces lactis

作者：Liang, Zhicheng; Deng, Mulan; Zhang, Zhi; Li, Meirong; Zhou, SuJin; Zhao, ZhengGang; Mu, YunPing; Wang, LiNa; Ning, Chengyun; Zhao, Allan Zijian; Li, Fanghong^*

来源：Plasmid, 2021, 116: 102577.

DOI：10.1016/j.plasmid.2021.102577

• Food-grade expression
• URA3 gene
• Kluyveromyces lactis
• Human serum albumin
• mCherry

Proteins from food-grade expression systems can be used in food products and medical applications. Herein, we describe a one-step method of constructing an expression vector in Kluyveromyces lactis by combining a URA3- deficient strain and a plasmid vector with no drug-resistant selection. Adjacent DNA elements of the vector were assembled in a targeted manner through a reaction with a special recombinase to form a plasmid vector using a one-step reaction. The unnecessary fragments containing the pUC origin and the ampicillin resistance gene were removed, and the vector was isolated and purified before transformation. A single transformation of the vector can produce a URA3-deficient strain. PCR assay, sequencing, and western blot analysis all indicated that the method of vector construction and target protein expression (mCherry and human serum albumin) were suc-cessful. This method may potentially be applied to any species containing the URA3 gene; this system has the potential to become a safe and powerful tool for promoting protein expression in food-safe species.

• 单位
  广东工业大学