The genes encoding the mevalonate-based farnesyl pyrophosphate (FPP) biosynthetic pathway were encoded in two operons and expressed in Escherichia coli to increase the production of sesquiterpenes. Inefficient translation of several pathway genes created bottlenecks and led to the accumulation of several pathway intermediates, namely, mevalonate and FPP, and suboptimal production of the sesquiterpene product, amorphadiene. Because of the difficulty in choosing ribosome binding sites (RBSs) to optimize translation efficiency, a combinatorial approach was used to choose the most appropriate RBSs for the genes of the lower half of the mevalonate pathway (mevalonate to amorphadiene). RBSs of various strengths, selected based on their theoretical strengths, were cloned 5′ of the genes encoding mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase, and amorphadiene synthase. Operons containing one copy of each gene and all combinations of RBSs were constructed and tested for their impact on growth, amorphadiene production, enzyme level, and accumulation of select pathway intermediates. Pathways with one or more inefficiently translated enzymes led to the accumulation of pathway intermediates, slow growth, and low product titers. Choosing the most appropriate RBS combination and carbon source, we were able to reduce the accumulation of toxic metabolic intermediates, improve growth, and improve the production of amorphadiene approximately fivefold. This work demonstrates that balancing flux through a heterologous pathway and maintaining steady growth are key determinants in optimizing isoprenoid production in microbial hosts.
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This work was supported by the Joint BioEnergy Institute (http://www.jbei.org) through a contract between Lawrence Berkeley National Laboratory and the US Department of Energy, Office of Science, Office of Biological and Environmental Research (DE-AC02-05CH11231) and the Synthetic Biology Engineering Research Center (http://www.synberc.org) through a grant from the National Science Foundation (BES-0439124). We thank Chris Anderson (Department of Bioengineering, University of California, Berkeley, CA, USA) for the gift of pBca9145.
Anthony JR, Anthony LC, Nowroozi F, Kwon G, Newman JD, Keasling JD (2009) Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metab Eng 11:13–19. doi:10.1016/j.ymben.2008.07.007PubMedCrossRefGoogle Scholar
Barbirato F, Grivet JP, Soucaille P, Bories A (1996) 3-Hydroxypropionaldehyde, an inhibitory metabolite of glycerol fermentation to 1,3-propanediol by enterobacterial species. Appl Environ Microbiol 62:1448–1451PubMedCentralPubMedGoogle Scholar
Carrier T, Jones KL, Keasling JD (1998) mRNA stability and plasmid copy number effects on gene expression from an inducible promoter system. Biotechnol Bioeng 59:666–672PubMedCrossRefGoogle Scholar
Harcum SW, Bentley WE (1999) Heat-shock and stringent responses have overlapping protease activity in Escherichia coli. Implications for heterologous protein yield. Appl Biochem Biotechnol 80:23–37PubMedCrossRefGoogle Scholar
Harker M, Bramley PM (1999) Expression of prokaryotic 1-deoxy-d-xylulose-5-phosphatases in Escherichia coli increases carotenoid and ubiquinone biosynthesis. FEBS Lett 448:115–119PubMedCrossRefGoogle Scholar
Hui A, Hayflick J, Dinkelspiel K, de Boer HA (1984) Mutagenesis of the three bases preceding the start codon of the beta-galactosidase mRNA and its effect on translation in Escherichia coli. EMBO J 3:623–629PubMedGoogle Scholar
Jones KL, Keasling JD (1998) Construction and characterization of F plasmid-based expression vectors. Biotechnol Bioeng 59:659–665PubMedCrossRefGoogle Scholar
Ma SM, Garcia DE, Redding-Johanson AM, Friedland GD, Chan R, Batth TS, Haliburton JR, Chivian D, Keasling JD, Petzold CJ, Lee TS, Chhabra SR (2011) Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases. Metab Eng 13:588–597. doi:10.1016/j.ymben.2011.07.001PubMedCrossRefGoogle Scholar
Martin VJJ, Pitera DJ, Withers ST, Newman JD, Keasling JD (2003) Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol 21:796–802. doi:10.1038/nbt833PubMedCrossRefGoogle Scholar
Matthews PD, Wurtzel ET (2000) Metabolic engineering of carotenoid accumulation in Escherichia coli by modulation of the isoprenoid precursor pool with expression of deoxyxylulose phosphate synthase. Appl Microbiol Biotechnol 53:396–400PubMedCrossRefGoogle Scholar
Nakamura CE, Whited GM (2003) Metabolic engineering for the microbial production of 1,3-propanediol. Curr Opin Biotechnol 14:454–459PubMedCrossRefGoogle Scholar
Newman JD, Marshall J, Chang M, Nowroozi F, Paradise E, Pitera D, Newman KL, Keasling JD (2006) High-level production of amorpha-4,11-diene in a two-phase partitioning bioreactor of metabolically engineered Escherichia coli. Biotechnol Bioeng 95:684–691. doi:10.1002/bit.21017PubMedCrossRefGoogle Scholar
Steen EJ, Kang Y, Bokinsky G, Hu Z, Schrimer A, McClure A, Del Cardayre SB, Keasling JD (2010) Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 463:559–562. doi:10.1038/nature08721PubMedCrossRefGoogle Scholar
Zhu MM, Lawman PD, Cameron DC (2002) Improving 1,3-propanediol production from glycerol in a metabolically engineered Escherichia coli by reducing accumulation of sn-glycerol-3-phosphate. Biotechnol Prog 18:694–699. doi:10.1021/bp020281PubMedCrossRefGoogle Scholar