Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene
Abstract
The introduction or creation of metabolic pathways in microbial hosts has allowed for the production of complex chemicals of therapeutic and industrial importance. However, these pathways rarely function optimally when first introduced into the host organism and can often deleteriously affect host growth, resulting in suboptimal yields of the desired product. Common methods used to improve production from engineered biosynthetic pathways include optimizing codon usage, enhancing production of rate-limiting enzymes, and eliminating the accumulation of toxic intermediates or byproducts to improve cell growth. We have employed these techniques to improve production of amorpha-4,11-diene (amorphadiene), a precursor to the anti-malarial compound artemisinin, by an engineered strain of Escherichia coli. First we developed a simple cloning system for expression of the amorphadiene biosynthetic pathway in E. coli, which enabled the identification of two rate-limiting enzymes (mevalonate kinase (MK) and amorphadiene synthase (ADS)). By optimizing promoter strength to balance expression of the encoding genes we alleviated two pathway bottlenecks and improved production five fold. When expression of these genes was further increased by modifying plasmid copy numbers, a seven-fold increase in amorphadiene production over that from the original strain was observed. The methods demonstrated here are applicable for identifying and eliminating rate-limiting steps in other constructed biosynthetic pathways.
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Both Saccharomyces cerevisiae and Escherichia coli have been used as hosts when attempting to establish a viable recombinant microbial pathway for artemisinin biosynthesis.
“Both Saccharomyces cerevisiae [379,380] and Escherichia coli [381,382,383] have been used as hosts when attempting to establish a viable recombinant microbial pathway for the biosynthesis of artemisinin.”
From The Biosynthesis of Artemisinin (Qinghaosu) and the Phytochemistry of Artemisia annua L. (Qinghao) (2010) · cites this paper as [381]
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- Authors
- Anthony, J. R., Anthony, L. C., Nowroozi, F., Kwon, G., Newman, J. D., & Keasling, J. D.
- Affiliations
- University of California, Berkeley (US)
- Amyris (United States) (US)
- Lawrence Berkeley National Laboratory (US)
- Corresponding author
- Keasling, Jay D.
- ORCID
- Keasling, Jay D.
- Venue
- Metabolic Engineering
- Publisher
- Elsevier BV
- Published
- 2009-01
- Volume
- 11
- Issue
- 1
- Pages
- 13-19
- ISSN
- 1096-7176 (ISSN-L)
- OpenAlex
- W1982836457
- Cited by
- 292
- References
- 21
- Citations by year
- 2026: 5 · 2025: 5 · 2024: 9 · 2023: 8 · 2022: 13 · 2021: 12 · 2020: 15 · 2019: 14 · 2018: 14 · 2017: 17 · 2016: 30 · 2015: 23 · 2014: 30 · 2013: 29 · 2012: 28
- FWCI
- 5.46
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Metabolic Engineering (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health (1f32gm077922-01)
- National Institute of General Medical Sciences (1F32GM077922-01)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant biochemistry and biosynthesis
- Health Sciences › Medicine › Pharmacology › Microbial Natural Products and Biosynthesis
- Keywords
- mevalonate pathway, isoprenoid biosynthesis, Escherichia coli, rate-limiting enzyme, mevalonate kinase, plasmid copy number, codon optimization
- MeSH terms
- Polycyclic Sesquiterpenes, Antimalarials, Escherichia coli, Ligases, Mevalonic Acid, Prodrugs, Sesquiterpenes, Terpenes, Phosphotransferases (Alcohol Group Acceptor)
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