Candida tropicalis, a diploid asporogenic yeast, is frequently utilized in industrial applications and research studies. However, the low efficiency of genetic transformation limits the strain improvement by metabolic engineering. A reliable transformation and efficient deletion of target gene are prerequisite for molecular improvement of C. tropicalis. In this study, an efficient approach for genetic transformation of C. tropicalis was developed based on the URA3 gene as a reusable selection marker and both of PDC allele genes encoding pyruvate decarboxylase were successfully deleted by this approach. Firstly, an auxotrophic mutant strain of C. tropicalis XZX which is defective in orotidine-5′-phosphate decarboxylase (URA3) was isolated by chemical mutagenesis combined with nystatin enrichment selection and 5-fluoro-orotic acid (5-FOA) resistance selection using C. tropicalis ATCC 20336 as the parent strain. Then, the first PDC deletion cassette PDC1-hisG-URA3-hisG- PDC1 (PHUHP) which contains a 1.6 kb URA3 marker gene, two copies of 1.1 kb Salmonella hisG fragments and homologous arms of target gene was constructed and transformed into C. tropicalis XZX cells. Transformants with a single copy of PDC deleted were isolated and identified by PCR and DNA sequencing, which was designated as C.tropicalis XZX02. The C.tropicalis XZX02 cells were spread on the minimal medium containing 5-FOA to generate mutant C. tropicalis XZX03 in which URA3 marker gene was excised from PHUHP fragment integrated into the PDC gene site. The second PDC gene deletion cassette PDCm-URA3-PDCm (MUM) was constructed and transformed into C. tropicalis XZX03 to generate C.tropicalis XZX04 in which both of PDC allele genes were deleted. All strains were confirmed by PCR and DNA sequencing. This efficient genetic transformation approach laid a foundation for further metabolic engineering of C. tropicalis.
Zheng Xiang, Xianzhong Chen, Lihua Zhang, Wei Shen, You Fan, Maolin Lu
. Development of a genetic transformation system for Candida tropicalis based on a reusable selection marker of URA3 gene[J]. Hereditas(Beijing), 2014
, 36(10)
: 1053
-1061
.
DOI: 10.3724/SP.J.1005.2014.1053
[1] 十二碳二元酸工业生产试验研究. 微生物学通报, 1998, 25(4): 244-244.
[2] 吴绵斌, 赵炯烽, 林建平, 杨立荣. 基因工程技术改造木糖醇生产菌株的研究进展. 中国生物工程杂志, 2012, 32(11): 124-131.
[3] S, Bravo V, García JF, Cruz N, Cuevas M. Fer-mentation of D-glucose and D-xylose mixtures by Can d-ida tropicalis NBRC 0618 for xylitol production. World J Microbiol Biotechnol , 2008, 24(5): 709-716.
[4] MR, Ajiboye EA, Akintunde JK, Odaibo A. Single cell proteins: as nutritional enhancer. Adv Appl Sci Res , 2011, 2(5): 396-409.
[5] YR, Li DP, Liu Y. Production of single cell protein from soy molasses using Candida tropicalis . Ann Microbiol , 2012, 62(3): 1165-1172.
[6] 王友玲. 热带假丝酵母菌处理马铃薯淀粉废水的研究. 安徽农业科学, 2013, 41(20): 8698-8699, 8702.
[7] 何连芳, 周景辉. 热带假丝酵母处理红麻亚铵法制浆废液的研究. 造纸科学与技术, 2007, 26(2): 26-29, 33.
[8] 付灿, 王同阳. 热带假丝酵母在木薯淀粉酒精废液治理中的应用研究. 中国酿造, 2009(7): 121-123.
[9] 郝新艳, 殷朝敏, 曾庆福. 不同氮源对热带假丝酵母处理苎麻生物脱胶废水的影响. 工业水处理, 2013, 33(3): 29-32.
[11] P, Cabral J M S. Phytosterols: applications and recovery methods. Bioresour Technol , 2007, 98(12): 2335-2350.
[12] MA, Haas LO, Cregg JM. Isolation of Candida tropicalis auxotrophic mutants. Appl Environ Microb , 1990, 56(8): 2562-2564.
[13] S, Krügener S, Hirth T, Rupp S, Zibek S. Biote-chnological synthesis of long-chain dicarboxylic acids as building blocks for polymers. Eur J Lipid Sci Tech , 2011, 113(5): 548-561.
[14] A, Arie M, Kanai T, Matsui T, Matsuda H, Furuhashi K, Ueda M, Tanaka A. Novel and convenient methods for Candida tropicalis gene disruption using a mutated hygro-mycin B resistance gene. Arch Microbiol , 2001, 176(5): 364-369.
[15] 生物催化生产十三碳二元酸中β-氧化途径的代谢调控[学位论文]. 北京: 清华大学, 2005.
[16] 蒋华, 杨胜利. 新的热带假丝酵母载体-宿主系统的建立. 生物工程学报, 1997, 13(3): 309-312.
[17] T, Suzuki T, Tokogawa T, Nishikawa K, Watanabe K. Unique structure of new serine tRNAs responsible for decoding leucine codon CUG in various Candida species and their putative ancestral tRNA genes. Biochimie , 1994, 76(12): 1217-1222.
[18] LO, Cregg JM, Gleeson MA. Development of an integrative DNA transformation system for the yeast Candida tropicalis . J Bacteriol , 1990, 172(8): 4571-4577.
[19] S, Deanda K, Mielenz J. Determination of Candida tropicalis acyl coenzyme A oxidase isozyme function by sequential gene disruption. Mol Cell Biol , 1991, 11(9): 4333-4339.
[20] E, Cao L, Kleckner N. A method for gene disruption that allows repeated use of URA3 selection in the con-struction of multiply disrupted yeast strains. Genetics , 1987, 116(4): 541-545.
[21] BS, Kim J, Kim JH. Production of xylitol from D-xylose by a xylitol dehydrogenase gene-disrupted mut-ant of Candida tropicalis . Appl Environ Microb , 2006, 72(6): 4207-4213.
[22] 林海, 王正祥, 诸葛健. 双重筛选产甘油假丝酵母营养缺陷型菌株及其发酵性能. 无锡轻工大学学报, 2000, 19(2): 95-99.
[23] 邓旭衡, 王德培, 李磊. 酿酒酵母丙酮酸脱羧酶活性测定的研究. 中国酿造, 2011(3): 128-130.
[24] C, Fleck CB, Brock M. Role of the glyoxylate cycle in pathogenesis of the yeast Candida albicans and the filamentous fungus Aspergillus fumigatus : Pyruvate decar-boxylase activity provides the key for metabolite flow. Mycoses , 2012, 55(4): 17-18.
[25] 吕 红)
