Optimization of amorphadiene synthesis in bacillus subtilis via transcriptional, translational, and media modulation.

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.

[1]  G. Stephanopoulos,et al.  Identifying gene targets for the metabolic engineering of lycopene biosynthesis in Escherichia coli. , 2005, Metabolic engineering.

[2]  Susan C. Roberts,et al.  Pharmaceutically active natural product synthesis and supply via plant cell culture technology. , 2008, Molecular pharmaceutics.

[3]  G. Stephanopoulos,et al.  Engineering Yeast Transcription Machinery for Improved Ethanol Tolerance and Production , 2006, Science.

[4]  T. Panavas,et al.  SUMO fusion technology for enhanced protein production in prokaryotic and eukaryotic expression systems. , 2009, Methods in molecular biology.

[5]  Jay D. Keasling,et al.  High-Level Production of Amorpha-4,11-Diene, a Precursor of the Antimalarial Agent Artemisinin, in Escherichia coli , 2009, PloS one.

[6]  I. Maeda Genetic modification in Bacillus subtilis for production of C30 carotenoids. , 2012, Methods in molecular biology.

[7]  J. Keasling,et al.  Engineering a mevalonate pathway in Escherichia coli for production of terpenoids , 2003, Nature Biotechnology.

[8]  Kazuyuki Yoshida,et al.  Carotenoid production in Bacillus subtilis achieved by metabolic engineering , 2009, Biotechnology Letters.

[9]  W. Schumann,et al.  Development of a strong intracellular expression system for Bacillus subtilis by optimizing promoter elements. , 2012, Journal of biotechnology.

[10]  Keith E. J. Tyo,et al.  Isoprenoid Pathway Optimization for Taxol Precursor Overproduction in Escherichia coli , 2010, Science.

[11]  G. Stephanopoulos,et al.  Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR , 2011, BMC Molecular Biology.

[12]  R. Larossa,et al.  Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli. , 2006, Metabolic engineering.

[13]  Mark A Eiteman,et al.  Simultaneous utilization of glucose, xylose and arabinose in the presence of acetate by a consortium of Escherichia coli strains , 2012, Microbial Cell Factories.

[14]  B. Seong,et al.  N‐terminal domains of native multidomain proteins have the potential to assist de novo folding of their downstream domains in vivo by acting as solubility enhancers , 2007, Protein science : a publication of the Protein Society.

[15]  G. Stephanopoulos,et al.  Metabolite Profiling Identified Methylerythritol Cyclodiphosphate Efflux as a Limiting Step in Microbial Isoprenoid Production , 2012, PLoS ONE.

[16]  Junfeng Xue,et al.  Enhancing Isoprene Production by Genetic Modification of the 1-Deoxy-d-Xylulose-5-Phosphate Pathway in Bacillus subtilis , 2011, Applied and Environmental Microbiology.

[17]  John R. Haliburton,et al.  Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases. , 2011, Metabolic engineering.

[18]  J. McCafferty,et al.  Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression , 2004, BMC biotechnology.

[19]  M. Ashiuchi,et al.  Bacillus subtilis pgsE (Formerly ywtC) stimulates poly-γ-glutamate production in the presence of zinc. , 2011, Biotechnology and bioengineering.

[20]  R. Fall,et al.  Evidence of Isoprenoid Precursor Toxicity in Bacillus subtilis , 2011, Bioscience, biotechnology, and biochemistry.