title: Optimization of amorphadiene synthesis in bacillus subtilis via transcriptional, translational, and media modulation.
abstract: Genetically engineered microbes have been intensively investigated as a means for the cost-effective production of isoprenoids. Bacillus subtilis is a promising microbial host for this purpose because of its fast growth rate and GRAS (generally regarded as safe) status. To date, development of this host has been impaired by the lack of genetic tools for modulating the expression of multiple genes. In this study, we present a novel two-promoter system which can be used to independently control the expression levels of two gene cassettes over a large dynamic range. Coupled with protein translation engineering and systematic media optimization, ~20 mg/L amorphadiene was produced in shake flask scale, a 40-fold improvement over the highest reported isoprenoid product yield in B. subtilis. As the tools and strategies developed here can be extended to the overproduction of other valuable metabolites, this proof-of-concept study lays the foundation for high level heterologous production of isoprenoids in Bacillus.
authors.affiliation: Chemical and Pharmaceutical Engineering, Singapore-MIT Alliance, 4 Engineering Drive 3, Singapore.
authors.forename: Kang
authors.initials: K
authors.lastname: Zhou
authors.forename: Ruiyang
authors.initials: R
authors.lastname: Zou
authors.forename: Congqiang
authors.initials: C
authors.lastname: Zhang
authors.forename: Gregory
authors.initials: G
authors.lastname: Stephanopoulos
authors.forename: Heng-Phon
authors.initials: HP
authors.lastname: Too
mesh_terms: D001412:Bacillus subtilis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways; D000081223:Polycyclic Sesquiterpenes; D011401:Promoter Regions, Genetic; D012717:Sesquiterpenes / Q000032:analysis / Q000378:metabolism*
chemical_list: D003470:Culture Media; D000081223:Polycyclic Sesquiterpenes; D012717:Sesquiterpenes; C515348:amorpha-4,11-diene
keywords: bacillus subtilis; deoxyxylulose phosphate pathway; isoprenoids; metabolic engineering; multiple gene expression