Cheng Jintao, Chen Jiali, Chen Dingfeng, Li Baoxian, Wei Chaozhi, Liu Tao, Wang Xiao, Wen Zhengshun, Jin Yuanxiang, Sun Chenfan, Yang Guiling
Xianghu Laboratory, Hangzhou, 310027, China.
College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310032, China.
Microb Cell Fact. 2025 Jan 21;24(1):29. doi: 10.1186/s12934-025-02649-5.
Sesquiterpene ( +)-valencene is a characteristic aroma component from sweet orange fruit, which has a variety of biological activities and is widely used in industrial manufacturing of food, beverage and cosmetics industries. However, at present, the content in plant sources is low, and its yield and quality would be influenced by weather and land, which limit the supply of ( +)-valencene. The rapid development of synthetic biology has accelerated the construction of microbial cell factories and provided an effective alternative method for the production of natural products.
In this study, we first introduced the ( +)-valencene synthase into Komagataella phaffii by CRISPR/Cas9 system, and successfully constructed a ( +)-valencene producer with the initial yield of 2.1 mg/L. Subsequently, the ( +)-valencene yield was increased to 8.2 mg/L by fusing farnesyl pyrophosphate synthase with ( +)-valencene synthase using the selected ligation linker. High expression of key genes IDI1, tHMG1, ERG12 and ERG19 enhanced metabolic flux of MVA pathway, and the yield of ( +)-valencene was further increased by 27%. Besides, in-situ deletion of the promoter of ERG9 increased the yield of ( +)-valencene to 48.1 mg/L. Finally, we optimized the copy number of farnesyl pyrophosphate synthase and ( +)-valencene synthase fusion protein, and when the copy number reached three, the yield of ( +)-valencene achieved 173.6 mg/L in shake flask level, which was 82-fold higher than that of the starting strain CaVAL1.
The results obtained here suggest that K. phaffii has the potential to efficiently synthesize other terpenoids.
倍半萜（+）-瓦伦烯是甜橙果实中的一种特征性香气成分，具有多种生物活性，广泛应用于食品、饮料和化妆品行业的工业生产中。然而，目前其在植物来源中的含量较低，且其产量和质量会受到天气和土地的影响，这限制了（+）-瓦伦烯的供应。合成生物学的快速发展加速了微生物细胞工厂的构建，并为天然产物的生产提供了一种有效的替代方法。
在本研究中，我们首先通过CRISPR/Cas9系统将（+）-瓦伦烯合酶导入毕赤酵母中，并成功构建了一株初始产量为2.1mg/L的（+）-瓦伦烯生产菌株。随后，使用选定的连接接头将法尼基焦磷酸合酶与（+）-瓦伦烯合酶融合，使（+）-瓦伦烯产量提高到8.2mg/L。关键基因IDI1、tHMG1、ERG12和ERG19的高表达增强了MVA途径的代谢通量，（+）-瓦伦烯的产量进一步提高了27%。此外，原位缺失ERG9的启动子使（+）-瓦伦烯的产量提高到48.1mg/L。最后，我们优化了法尼基焦磷酸合酶和（+）-瓦伦烯合酶融合蛋白的拷贝数，当拷贝数达到3时，（+）-瓦伦烯在摇瓶水平的产量达到173.6mg/L，比出发菌株CaVAL1高82倍。
本研究结果表明，毕赤酵母具有高效合成其他萜类化合物的潜力。
