title: Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production.
abstract: Microbial production can be advantageous over the extraction of phytoterpenoids from natural plant sources, but it remains challenging to rationally and rapidly access efficient pathway variants. Previous engineering attempts mainly focused on the mevalonic acid (MVA) or methyl-d-erythritol phosphate (MEP) pathways responsible for the generation of precursors for terpenoids biosynthesis, and potential interactions between diterpenoids synthases were unexplored. Miltiradiene, the product of the stepwise conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP) catalyzed by diterpene synthases SmCPS and SmKSL, has recently been identified as the precursor to tanshionones, a group of abietane-type norditerpenoids rich in the Chinese medicinal herb  Salvia miltiorrhiza . Here, we present the modular pathway engineering (MOPE) strategy and its application for rapid assembling synthetic miltiradiene pathways in the yeast  Saccharomyces cerevisiae . We predicted and analyzed the molecular interactions between SmCPS and SmKSL, and engineered their active sites into close proximity for enhanced metabolic flux channeling to miltiradiene biosynthesis by constructing protein fusions. We show that the fusion of SmCPS and SmKSL, as well as the fusion of BTS1 (GGPP synthase) and ERG20 (farnesyl diphosphate synthase), led to significantly improved miltiradiene production and reduced byproduct accumulation. The MOPE strategy facilitated a comprehensive evaluation of pathway variants involving multiple genes, and, as a result, our best pathway with the diploid strain YJ2X reached miltiradiene titer of 365 mg/L in a 15-L bioreactor culture. These results suggest that terpenoids synthases and the precursor supplying enzymes should be engineered systematically to enable an efficient microbial production of phytoterpenoids.
authors.affiliation: Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian 116023, People's Republic of China.
authors.forename: Yongjin J
authors.initials: YJ
authors.lastname: Zhou
authors.forename: Wei
authors.initials: W
authors.lastname: Gao
authors.forename: Qixian
authors.initials: Q
authors.lastname: Rong
authors.forename: Guojie
authors.initials: G
authors.lastname: Jin
authors.forename: Huiying
authors.initials: H
authors.lastname: Chu
authors.forename: Wujun
authors.initials: W
authors.lastname: Liu
authors.forename: Wei
authors.initials: W
authors.lastname: Yang
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Zhu
authors.forename: Guohui
authors.initials: G
authors.lastname: Li
authors.forename: Guofeng
authors.initials: G
authors.lastname: Zhu
authors.forename: Luqi
authors.initials: L
authors.lastname: Huang
authors.forename: Zongbao K
authors.initials: ZK
authors.lastname: Zhao
mesh_terms: D000595:Amino Acid Sequence; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004224:Diterpenes / Q000737:chemistry* / Q000494:pharmacology*; D015195:Drug Design; D006801:Humans; D008798:Mevalonic Acid / Q000737:chemistry*; D008956:Models, Chemical; D008517:Phytotherapy / Q000379:methods; D010936:Plant Extracts / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D027544:Salvia / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D004224:Diterpenes; D010936:Plant Extracts; C573600:miltiradiene; D004247:DNA; D008798:Mevalonic Acid