title: Development of an efficient genetic manipulation strategy for sequential gene disruption and expression of different heterologous GFP genes in Candida tropicalis.
abstract: The diploid yeast Candida tropicalis, which can utilize n-alkane as a carbon and energy source, is an attractive strain for both physiological studies and practical applications. However, it presents some characteristics, such as rare codon usage, difficulty in sequential gene disruption, and inefficiency in foreign gene expression, that hamper strain improvement through genetic engineering. In this work, we present a simple and effective method for sequential gene disruption in C. tropicalis based on the use of an auxotrophic mutant host defective in orotidine monophosphate decarboxylase (URA3). The disruption cassette, which consists of a functional yeast URA3 gene flanked by a 0.3 kb gene disruption auxiliary sequence (gda) direct repeat derived from downstream or upstream of the URA3 gene and of homologous arms of the target gene, was constructed and introduced into the yeast genome by integrative transformation. Stable integrants were isolated by selection for Ura+and identified by PCR and sequencing. The important feature of this construct, which makes it very attractive, is that recombination between the flanking direct gda repeats occurs at a high frequency (10-8) during mitosis. After excision of the URA3 marker, only one copy of the gda sequence remains at the recombinant locus. Thus, the resulting ura3 strain can be used again to disrupt a second allelic gene in a similar manner. In addition to this effective sequential gene disruption method, a codon-optimized green fluorescent protein-encoding gene (GFP) was functionally expressed in C. tropicalis. Thus, we propose a simple and reliable method to improve C. tropicalis by genetic manipulation.
authors: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
authors: Lihua
authors: L
authors: Zhang
authors: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China. xzchen@jiangnan.edu.cn.
authors: Xianzhong
authors: X
authors: Chen
authors: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
authors: Zhen
authors: Z
authors: Chen
authors: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
authors: Zezheng
authors: Z
authors: Wang
authors: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
authors: Shan
authors: S
authors: Jiang
authors: Evonik Degussa (China) Co. Ltd., Shanghai Branch, Shanghai, 201108, China.
authors: Li
authors: L
authors: Li
authors: Evonik Degussa (China) Co. Ltd., Shanghai Branch, Shanghai, 201108, China.
authors: Markus
authors: M
authors: Pötter
authors: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
authors: Wei
authors: W
authors: Shen
authors: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
authors: You
authors: Y
authors: Fan
mesh_terms: D041022:Candida tropicalis / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D005827:Genetics, Microbial / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000172:deficiency
chemical_list: D049452:Green Fluorescent Proteins; D009964:Orotidine-5'-Phosphate Decarboxylase
keywords: Candida tropicalis; Carnitine acetyltransferase; Dodecanedioic acid; GFP; Gene disruption; Gene disruption auxiliary sequence; URA3 gene