Abstract
BackgroundProtoilludene 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 Escherichiacoli 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.ResultsProtoilludene was successfully produced in E. coli by overexpression of a hybrid exogenous MVA pathway, endogenous FPP synthase (IspA), and protoilludene synthase (OMP7) of Omphalotusolearius. 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 Staphylococcusaureus. 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0409-7) contains supplementary material, which is available to authorized users.
Highlights
Protoilludene is a valuable sesquiterpene and serves as a precursor for several medicinal compounds and antimicrobial chemicals
O. olearius protoilludene synthase (OMP7) exhibits the highest catalytic efficiency which is higher than its homologs OMP6 and Stehi1|73029 by 10 and 30 times, respectively
In order to synthesize protoilludene in E. coli, a codon-optimized OMP7 gene was assembled with E. coli farnesyl diphos‐ phate (FPP) synthase gene to construct plasmid pTAO (Fig. 2a)
Summary
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 diphos‐ phate (FPP) synthase. The most brilliant potential anticancer agent illudin S, which is first isolated from Omphalotus olearius mushroom, has been studied extensively owing to its cytotoxicity to various tumor cell types [4]. These biological properties and medicinal potential have attracted considerable attention since the Protoilludene biosynthesis begins with the formation of the universal precursors, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which can be generated. O. olearius protoilludene synthase (OMP7) exhibits a superior catalytic efficiency (Kcat/Km) of (13.0 ± 2.0) × 104 M−1 s−1 among those protoilludene synthases (Additional file 1: Table S1) [8]
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