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 successful. 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.

来自食品级表达系统的蛋白质可用于食品和医疗应用。在此，我们描述了一种通过结合URA3缺陷菌株和无耐药性选择的质粒载体在乳酸克鲁维酵母中构建表达载体的一步法。载体的相邻DNA元件通过与特殊重组酶反应以靶向方式组装，使用一步反应形成质粒载体。去除含有pUC来源和氨苄青霉素抗性基因的不需要的片段，并在转化前分离纯化载体。载体的单一转化可以产生一个URA3- 应变不足。PCR检测、测序和蛋白质印迹分析均表明载体构建和目标蛋白表达（mCherry和人血清白蛋白）的方法是成功的。这种方法可能适用于任何含有URA3基因的物种；该系统有可能成为促进食品安全物种蛋白质表达的安全而强大的工具。