产瓦伦西亚烯酿酒酵母的表达载体适配及发酵碳氮源优化

doi:10.13560/j.cnki.biotech.bull.1985.2019-0564

摘要/Abstract

摘要： 瓦伦西亚烯是一种倍半萜类化合物,广泛应用于香水、香皂、食品和饮料等工业制造上。但由于其自然含量极低,且目前获取瓦伦西亚烯的方法较为麻烦且花费高,因而构建细胞工厂进行瓦伦西亚烯的生物合成是更为高效和环保的方法。选取酿酒酵母（Saccharomyces cerevisiae）作为宿主构建细胞工厂,先在酿酒酵母基因组上引入黄扁柏的瓦伦西亚烯合成酶（Valencene synthase from Callitropsis nootkatensis,CnVS）,实现瓦伦西亚烯的初步合成,初始产量为4.16 mg/L。随后利用CRISPR/Cas9系统对酿酒酵母中Mevalonate（MVA）途径的erg9和rox1基因进行敲除,提高通往瓦伦西亚烯合成的碳流量。不同碳氮源浓度发酵的结果表明,细胞生长积累过高可能不利于瓦伦西亚烯的积累。最后探究了不同CnVS表达载体对瓦伦西亚烯产量的影响,并获得17.54 mg/L的最高产量,是出发菌株的4.2倍。

关键词: 酿酒酵母, 瓦伦西亚烯, 基因敲除, 表达载体

Abstract: Valencene,a terpenoid existing in various citrus fruits,was widely used in perfume,soap,food,and beverage industry. However,the concentration of valencene in nature is extremely low,and current strategy to obtain valencene is complicated and expensive,thus construction of cell factory for valencene biosynthesis will be a more efficient and environmentally friendly. Saccharomyces cerevisiae was chosen as host to construct cell factory in this study. Firstly,the valencene synthase from Callitropsis nootkatensis（CnVS）was introduced into S. cerevisiae strain,resulting in the preliminary synthesis of valencene,and the initial yield of this strain was 4.16 mg/L. Then the gene erg9 and rox1 in Mevalonate（MVA）pathway of S. cerevisiae were knocked out using CRISPR/Cas9 system,improving carbon flux towards valencene synthesis. The results of different carbon/nitrogen source concentrations in fermentation indicated that the high accumulation of cell growth may not be conducive to valencene accumulation. Finally,the influences of different CnVS expression vectors on valencene yield were examined and the highest yield of 17.54 mg/L was obtained,which was 4.2 times higher than the original strain.

Key words: Saccharomyces cerevisiae, valencene, gene knockout, expression vector

陈和锋, 朱晁谊, 李爽. 产瓦伦西亚烯酿酒酵母的表达载体适配及发酵碳氮源优化[J]. 生物技术通报, 2020, 36(1): 209-219.

CHEN He-feng, ZHU Chao-yi, LI Shuang. Expression Vector Adaptation of Valencene-producing Saccharomyces cerevisiae and Optimization of Fermentation Carbon and Nitrogen Sources[J]. Biotechnology Bulletin, 2020, 36(1): 209-219.

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