Khalid Ammara, Takagi Hiroshi, Panthee Suresh, Muroi Makoto, Chappell Joe, Osada Hiroyuki, Takahashi Shunji
Chemical Biology Research Group, RIKEN Centre for Sustainable Resource Science , Hirosawa, 2-1, Wako, Saitama 351-0198, Japan.
Graduate School of Science and Engineering, Saitama University , Saitama 338-8570, Japan.
ACS Synth Biol. 2017 Dec 15;6(12):2339-2349. doi: 10.1021/acssynbio.7b00249. Epub 2017 Oct 11.
Terpenoids represent the largest class of natural products, some of which are resources for pharmaceuticals, fragrances, and fuels. Generally, mass production of valuable terpenoid compounds is hampered by their low production levels in organisms and difficulty of chemical synthesis. Therefore, the development of microbial biosynthetic platforms represents an alternative approach. Although microbial terpenoid-production platforms have been established in Escherichia coli and yeast, an optimal platform has not been developed for Streptomyces species, despite the large capacity to produce secondary metabolites, such as polyketide compounds. To explore this potential, we constructed a terpenoid-biosynthetic platform in Streptomyces reveromyceticus SN-593. This strain is unique in that it harbors the mevalonate gene cluster enabling the production of furaquinocin, which can be controlled by the pathway specific regulator Fur22. We simultaneously expressed the mevalonate gene cluster and subsequent terpenoid-biosynthetic genes under the control of Fur22. To achieve improved fur22 gene expression, we screened promoters from S. reveromyceticus SN-593. Our results showed that the promoter associated with rvr2030 gene enabled production of 212 ± 20 mg/L botryococcene to levels comparable to those previously reported for other microbial hosts. Given that the rvr2030 gene encodes for an enzyme involved in the primary metabolism, these results suggest that optimized expression of terpenoid-biosynthetic genes with primary and secondary metabolism might be as important for high yields of terpenoid compounds as is the absolute expression level of a target gene(s).
萜类化合物是最大的一类天然产物，其中一些是药物、香料和燃料的来源。一般来说，有价值的萜类化合物的大规模生产受到其在生物体中低产量水平以及化学合成困难的阻碍。因此，开发微生物生物合成平台是一种替代方法。尽管已经在大肠杆菌和酵母中建立了微生物萜类化合物生产平台，但对于链霉菌属物种，尚未开发出最佳平台，尽管它们具有产生聚酮化合物等次生产物的巨大能力。为了探索这种潜力，我们在逆转链霉菌SN-593中构建了一个萜类化合物生物合成平台。该菌株的独特之处在于它含有甲羟戊酸基因簇，能够产生呋喃喹诺菌素，其可以由途径特异性调节因子Fur22控制。我们在Fur22的控制下同时表达甲羟戊酸基因簇和随后的萜类化合物生物合成基因。为了实现fur22基因表达的改善，我们从逆转链霉菌SN-593中筛选了启动子。我们的结果表明，与rvr2030基因相关的启动子能够产生212±20mg/L的葡萄藻烯，其水平与先前报道的其他微生物宿主相当。鉴于rvr2030基因编码一种参与初级代谢的酶，这些结果表明，萜类化合物生物合成基因与初级和次级代谢的优化表达对于萜类化合物的高产可能与靶基因的绝对表达水平同样重要。
