BACKGROUND The 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 of S. cerevisiae cell factory to achieve high production of valencene will be attractive. RESULTS Valencene was successfully biosynthesized after introducing valencene synthase into S. cerevisiae BJ5464. A significant increase in valencene yield was observed after down-regulation or knock-out of squalene synthesis and other inhibiting factors (such as erg9, 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-TTPI1 was 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. CONCLUSIONS This study achieved high production of valencene in S. cerevisiae through 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.

中文翻译：

---

通过代谢工程在酿酒酵母中高产朱栾倍半萜。

背景技术通过代谢工程微生物工厂在工业中生物合成高价值化合物近年来受到越来越多的关注。朱栾倍半萜是香精香料行业中的高价值成分，但天然浓度低和提取成本高限制了其应用。酿酒酵母被普遍认为是安全的，是最常用的基因表达宿主之一。建设酿酒酵母细胞工厂以实现朱栾倍半萜的高产量将是有吸引力的。结果将朱栾倍半萜合酶引入酿酒酵母BJ5464后，成功生物合成朱栾倍半萜。使用本研究构建的可回收CRISPR/Cas9系统，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erg9、rox1）后，朱栾倍半萜产量显着增加。 Cre/loxP。为了增加前体法呢基焦磷酸（FPP）的补充，MVA途径上游FPP的所有基因在酵母基因组中过表达。此外，对含有不同启动子和终止子的朱栾倍半萜表达盒进行了比较，发现PHXT7-VS-TTPI1在朱栾倍半萜生产中具有优异的性能。最后，在3 L生物反应器中补料分批发酵后，朱栾倍半萜生产滴度达到539.3 mg/L，与初始滴度相比提高了约160倍，这是报道的最高朱栾倍半萜产率。结论本研究通过代谢工程和表达盒优化，在酿酒酵母中实现了朱栾倍半萜的高产量，为微生物过量生产有价值的化学产品提供了很好的例子。可回收质粒的构建对于多基因编辑也很有用。