title: Combinatorial engineering of hybrid mevalonate pathways in Escherichia coli for protoilludene production.
abstract: BACKGROUND
  Protoilludene is a valuable sesquiterpene and serves as a precursor for several medicinal compounds and antimicrobial chemicals. It can be synthesized by heterologous expression of protoilludene synthase in Escherichia coli with overexpression of mevalonate (MVA) or methylerythritol-phosphate (MEP) pathway, and farnesyl diphosphate (FPP) synthase. Here, we present E. coli as a cell factory for protoilludene production.
  
  
  RESULTS
  Protoilludene was successfully produced in E. coli by overexpression of a hybrid exogenous MVA pathway, endogenous FPP synthase (IspA), and protoilludene synthase (OMP7) of Omphalotus olearius. For improving protoilludene production, the MVA pathway was engineered to increase synthesis of building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) by sequential order permutation of the lower MVA portion (MvL), the alteration of promoters and copy numbers for the upper MVA portion (MvU), and the coordination of both portions, resulting in an efficient entire MVA pathway. To reduce the accumulation of mevalonate observed in the culture broth due to lower efficiency of the MvL than the MvU, the MvL was further engineered by homolog substitution with the corresponding genes from Staphylococcus aureus. Finally, the highest protoilludene production of 1199 mg/L was obtained from recombinant E. coli harboring the optimized hybrid MVA pathway in a test tube culture.
  
  
  CONCLUSIONS
  This is the first report of microbial synthesis of protoilludene by using an engineered E. coli strain. The protoilludene production was increased by approx. Thousandfold from an initial titer of 1.14 mg/L. The strategies of both the sequential order permutation and homolog substitution could provide a new perspective of engineering MVA pathway, and be applied to optimization of other metabolic pathways.
authors.affiliation: Division of Applied Life Science (BK21 Plus Program), PMBBRC, Gyeongsang National University, Jinju, 660-701, Korea. yangyang8028121@hotmail.com.
authors.forename: Liyang
authors.initials: L
authors.lastname: Yang
authors.affiliation: Division of Applied Life Science (BK21 Plus Program), PMBBRC, Gyeongsang National University, Jinju, 660-701, Korea. wangchonglong@outlook.com.
authors.forename: Chonglong
authors.initials: C
authors.lastname: Wang
authors.affiliation: Division of Applied Life Science (BK21 Plus Program), PMBBRC, Gyeongsang National University, Jinju, 660-701, Korea. jiazhou@hyit.edu.cn.|Faculty of Life Science and Food Engineering, Huaiyin Institute of Technology, Huai'an, 223003, The People's Republic of China. jiazhou@hyit.edu.cn.
authors.forename: Jia
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Division of Applied Life Science (BK21 Plus Program), PMBBRC, Gyeongsang National University, Jinju, 660-701, Korea. swkim@gnu.ac.kr.
authors.forename: Seon-Won
authors.initials: SW
authors.lastname: Kim
mesh_terms: D004926:Escherichia coli / Q000378:metabolism*; D051228:Geranyltranstransferase / Q000378:metabolism; D045782:Hemiterpenes / Q000378:metabolism; D008798:Mevalonic Acid / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism
chemical_list: D045782:Hemiterpenes; D009943:Organophosphorus Compounds; C043060:3,3-dimethylallyl pyrophosphate; D051228:Geranyltranstransferase; D008798:Mevalonic Acid