Niu Fu-Xing, He Xin, Wu Ya-Qin, Liu Jian-Zhong
Institute of Synthetic Biology, Biomedical Center, Guangdong Province Key Laboratory of Improved Variety Reproduction in Aquatic Economic Animals and South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, China.
Front Microbiol. 2018 Jul 31;9:1623. doi: 10.3389/fmicb.2018.01623. eCollection 2018.
α-Pinene is a natural and active monoterpene, which is widely used as a flavoring agent and in fragrances, pharmaceuticals, and biofuels. Although it has been successfully produced by genetically engineered microorganisms, the production level of pinene is much lower than that of hemiterpene (isoprene) and sesquiterpenes (farnesene) to date. We first improved pinene tolerance to 2.0% and pinene production by adaptive laboratory evolution after atmospheric and room temperature plasma (ARTP) mutagenesis and overexpression of the efflux pump to obtain the pinene tolerant strain YZFP, which is resistant to fosmidomycin. Through error-prone PCR and DNA shuffling, we isolated an geranyl pyrophosphate synthase variant that outperformed the wild-type enzyme. To balance the expression of multiple genes, a tunable intergenic region (TIGR) was inserted between and . In an effort to improve the production, an modular co-culture system was engineered to modularize the heterologous mevalonate (MEV) pathway and the TIGR-mediated gene cluster of and . Specifically, the MEV pathway and the TIGR-mediated gene cluster were integrated into the chromosome of the pinene tolerance strain YZFP and then evolved to a higher gene copy number by chemically induced chromosomal evolution, respectively. The best co-culture system of fermentation was found to improve pinene production by 1.9-fold compared to the mono-culture approach. The modular co-culture system of whole-cell biocatalysis further improved pinene production to 166.5 mg/L.
α-蒎烯是一种天然的活性单萜，广泛用作调味剂以及用于香料、制药和生物燃料。尽管它已通过基因工程微生物成功生产，但迄今为止，蒎烯的生产水平远低于半萜（异戊二烯）和倍半萜（法尼烯）。我们首先通过常压室温等离子体（ARTP）诱变和外排泵的过表达，经适应性实验室进化将蒎烯耐受性提高到2.0%并提高蒎烯产量，从而获得对磷霉素耐药的蒎烯耐受菌株YZFP。通过易错PCR和DNA改组，我们分离出一种优于野生型酶的香叶基焦磷酸合酶变体。为了平衡多个基因的表达，在[具体基因1]和[具体基因2]之间插入了一个可调基因间隔区（TIGR）。为了提高产量，构建了一个模块化共培养系统，将异源甲羟戊酸（MEV）途径以及TIGR介导的[具体基因1]和[具体基因2]基因簇模块化。具体而言，将MEV途径和TIGR介导的基因簇整合到蒎烯耐受菌株YZFP的染色体中，然后分别通过化学诱导染色体进化使其进化到更高的基因拷贝数。发现最佳的共培养发酵系统与单培养方法相比，蒎烯产量提高了1.9倍。全细胞生物催化的模块化共培养系统进一步将蒎烯产量提高到166.5 mg/L。
