Abstract
Heterologous biosynthesis of natural products is meant to enable access to the vast array of valuable properties associated with these compounds. Often motivated by limitations inherent in native production hosts, the heterologous biosynthetic process begins with a candidate host regarded as technically advanced relative to original producing organisms. Given this requirement, E. coli has been a top choice for heterologous biosynthesis attempts as associated recombinant tools emerged and continue to develop. However, success requires overcoming challenges associated with natural product formation, including complex biosynthetic pathways and the need for metabolic support. These two challenges have been heavily featured in cellular engineering efforts completed to position E. coli as a viable surrogate host. This chapter outlines steps taken to engineer E. coli with an emphasis on genetic manipulations designed to support the heterologous production of polyketide, nonribosomal peptide, and similarly complex natural products.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Zhang H, Boghigian BA, Armando J et al (2011) Methods and options for the heterologous production of complex natural products. Nat Prod Rep 28:125–151
Ongley SE, Bian X, Neilan BA et al (2013) Recent advances in the heterologous expression of microbial natural product biosynthetic pathways. Nat Prod Rep 30:1121–1138
Pfeifer BA, Admiraal SJ, Gramajo H et al (2001) Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli. Science 291:1790–1792
Golomb M, Chamberlin M (1974) Characterization of T7-specific ribonucleic acid polymerase. IV. Resolution of the major in vitro transcripts by gel electrophoresis. J Biol Chem 249:2858–2863
McAllister WT, Morris C, Rosenberg AH et al (1981) Utilization of bacteriophage T7 late promoters in recombinant plasmids during infection. J Mol Biol 153:527–544
Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130
Lambalot RH, Gehring AM, Flugel RS et al (1996) A new enzyme superfamily—the phosphopantetheinyl transferases. Chem Biol 3:923–936
Quadri LE, Weinreb PH, Lei M et al (1998) Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases. Biochemistry 37:1585–1595
Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645
Hradecna Z, Wild J, Szybalski W (1998) Conditionally amplifiable inserts in pBAC vectors. Microb Comp Genomics 3:58
Wild J, Hradecna Z, Szybalski W (2002) Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones. Genome Res 12:1434–1444
Wild J, Szybalski W (2004) Copy-control pBAC/oriV vectors for genomic cloning. Methods Mol Biol 267:145–154
Wild J, Hradecna Z, Szybalski W (2001) Single-copy/high-copy (SC/HC) pBAC/oriV novel vectors for genomics and gene expression. Plasmid 45:142
Le Borgne S, Palmeros B, Valle F et al (1998) pBRINT-Ts: a plasmid family with a temperature-sensitive replicon, designed for chromosomal integration into the lacZ gene of Escherichia coli. Gene 223:213–219
Phue JN, Lee SJ, Trinh L et al (2008) Modified Escherichia coli B (BL21), a superior producer of plasmid DNA compared with Escherichia coli K (DH5alpha). Biotechnol Bioeng 101:831–836
Rodriguez E, Gramajo H (1999) Genetic and biochemical characterization of the alpha and beta components of a propionyl-CoA carboxylase complex of Streptomyces coelicolor A3(2). Microbiology 145:3109–3119
Chan YA, Podevels AM, Kevany BM et al (2009) Biosynthesis of polyketide synthase extender units. Nat Prod Rep 26:90–114
Zhang H, Wang Y, Wu J, Skalina K et al (2010) Complete biosynthesis of erythromycin A and designed analogs using E. coli as a heterologous host. Chem Biol 17:1232–1240
Armando JW, Boghigian BA, Pfeifer BA (2012) LC-MS/MS quantification of short-chain acyl-CoA’s in Escherichia coli demonstrates versatile propionyl-CoA synthetase substrate specificity. Lett Appl Microbiol 54:140–148
Acknowledgement
This work was supported by grants from the NIH (AI074224) and NSF (0712019 and 0924699) in collaboration with EarthGenes Pharmaceuticals. Guidance in strain design and assessment was provided by Drs. Asuncion Martinez, David Rothstein, and Lance Davidow of EarthGenes.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Zhang, H., Fang, L., Osburne, M.S., Pfeifer, B.A. (2016). The Continuing Development of E. coli as a Heterologous Host for Complex Natural Product Biosynthesis. In: Evans, B. (eds) Nonribosomal Peptide and Polyketide Biosynthesis. Methods in Molecular Biology, vol 1401. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3375-4_8
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3375-4_8
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3373-0
Online ISBN: 978-1-4939-3375-4
eBook Packages: Springer Protocols