High production of valencene in Saccharomyces cerevisiae through metabolic engineering
High production of valencene in Saccharomyces cerevisiae through metabolic engineering
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通过代谢工程在酿酒酵母中高产朱栾倍半萜
DOI：
10.1186/s12934-019-1246-2
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发表时间：
2019-11-07
影响因子：
6.4
通讯作者：
Li, Shuang
中科院分区：
文献类型：
--
作者：
Chen, Hefeng;Zhu, Chaoyi;Li, Shuang
背景利用代谢工程微生物工厂合成高价值化合物的研究近年来受到越来越多的关注。朱栾倍半萜是一种高价值的香精香料，但天然浓度低，提取成本高，限制了其应用。酿酒酵母是最常用的基因表达宿主之一，被公认为安全。S.结果将朱栾倍半萜合成酶导入到S. aeBJ5464.通过引入Cre/loxP，构建了一个可回收的CRISPR/Cas9系统，通过下调或敲除甲羟戊酸（MVA）途径中的角鲨烯合成和其他抑制因子（如aserg 9、rox 1），观察到朱栾倍半萜产量显著增加。为了增加前体法尼基焦磷酸（FPP）的补充，在酵母基因组中过量表达MVA途径上游的所有FPP基因。此外，比较了含有不同启动子和终止子的朱栾倍半萜表达盒，发现PHXT 7-VS-TTPI 1在朱栾倍半萜生产中具有优异的性能。最后，在3L生物反应器中进行补料分批发酵，朱栾倍半萜的产量达到539.3mg/L，比初始产量提高了160倍，是目前报道的最高朱栾倍半萜产量。可回收质粒的构建也可用于多基因编辑。
BackgroundThe biological synthesis of high value compounds in industry through metabolically engineered microorganism factories has received increasing attention in recent years. Valencene is a high value ingredient in the flavor and fragrance industry, but the low concentration in nature and high cost of extraction limits its application.Saccharomyces cerevisiae, generally recognized as safe, is one of the most commonly used gene expression hosts. Construction ofS. cerevisiaecell factory to achieve high production of valencene will be attractive.ResultsValencene was successfully biosynthesized after introducing valencene synthase intoS. cerevisiaeBJ5464. A significant increase in valencene yield was observed after down-regulation or knock-out of squalene synthesis and other inhibiting factors (such aserg9,rox1) in mevalonate (MVA) pathway using a recyclable CRISPR/Cas9 system constructed in this study through the introduction of Cre/loxP. To increase the supplement of the precursor farnesyl pyrophosphate (FPP), all the genes of FPP upstream in MVA pathway were overexpressed in yeast genome. Furthermore, valencene expression cassettes containing different promoters and terminators were compared, and PHXT7-VS-TTPI1was found to have excellent performance in valencene production. Finally, after fed-batch fermentation in 3 L bioreactor, valencene production titer reached 539.3 mg/L with about 160-fold improvement compared to the initial titer, which is the highest reported valencene yield.ConclusionsThis study achieved high production of valencene inS. cerevisiaethrough metabolic engineering and optimization of expression cassette, providing good example of microbial overproduction of valuable chemical products. The construction of recyclable plasmid was useful for multiple gene editing as well.