Biochemistry and microbiology are foundational sciences for the work in the Keasling Lab. Our work in biochemistry and microbiology focuses on understanding unknown metabolic pathways, enzymes or microbial processes that will be central to be able to engineer new metabolic functions inside cells.
Elucidating unknown steps in biosynthetic pathways
The biosynthetic pathways for some of the most important natural products from plants are not known. If we want to produce these natural products using a microbial host, it is essential that we know the natural biosynthetic enzymes and be able to reconstitute those steps in the microbial host. When we are faced with unknown pathway enzymes, the Keasling Lab uses a variety of genomics tools and enzyme assays to discover the missing biosynthetic steps. Examples include the discovery of the cytochrome P450 responsible for oxidizing amorphadiene to artemisinic acid in Artemisia annua and terpene synthases responsible for producing a variety of terpenoids in plants and microorganisms. Ongoing work includes elucidating biosynthetic pathways for important plant terpenoids. Representative publications are:
- X. Xie, J. Kirby, and J. D. Keasling. 2012. “Functional characterization of four sesquiterpene synthases from Ricinus communis (Caster Bean).” Phytochemistry 78:20-28.
- R. P. McAndrew, P. P. Peralta-Yahya, A. DeGiovanni, J. H. Pereira, M. Z. Hadi, J. D. Keasling, and P. D. Adams. 2011. “Structure of a three-domain sesquiterpene synthase: a prospective target for advanced biofuels production.” Structure 19:1876-1884.
- J. Kirby, M. Nishimoto, J. G. Park, S. T. Withers, F. Nowroozi, D. Behrendt, E. J. Rutledge, J. L. Fortman, H. E. Johnson, J. V. Anderson, and J. D. Keasling. 2010. “Cloning of casbene and neocembrene synthases from Euphorbiaceae plants and expression in Saccharomyces cerevisiae.” Phytochem. 71:1466-1473.
- L. Prach, J. Kirby, J. D. Keasling, T. Alber. 2010. “Diterpene production in Mycobacterium tuberculosis.” FEBS J. 277:3588-3595.
- D-K. Ro, E. M. Paradise, M. Ouellet, K. J. Fisher, K. L. Newman, J. M. Ndungu, K. A. Ho, R. A. Eachus, R. S. Ham, J. Kirby, M. C. Y. Chang, S. T. Withers, Y. Shiba, R. Sarpong, and J. D. Keasling. 2006. “Production of the antimalarial drug precursor artemisinic acid in engineered yeast.” Nature 440:940-943.
Biochemistry of polyketide synthases
With the aim of producing important chemicals in microorganisms, we focus our efforts at investigating novel biochemical pathways and engineering microorganisms to host these pathways. The rich chemical and structural diversity of natural products make them attractive sources for biochemical pathways that can lead to novel chemicals. Multienzyme polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are molecular assembly lines that biosynthesize complex natural products, making them ideal for rational enzyme engineering. In the Keasling lab, part of our effort focuses on understanding the fundamental biochemical processes that govern the enzymatic catalysis of PKS and NRPS products. Using the most current advances in multi-omic, DNA recombination and biochemical technologies, we aim at understanding the basic mechanistic foundation of PKS and NRPS biocatalysis. We apply the knowledge gain from basic biochemistry to engineer PKS and NRPS to produce novel compounds that will have a significant impact on the petrochemical and pharmaceutical industries. Examples of these compounds include primary alcohols, diacids and hydroxyacids. Representative publications include:
- Curran, S.C., Hagen, A., Poust, S., Chan, L.J.G., Garabedian, B.M., de Rond, T., Baluyot, M.-J., Vu, J.T., Lau, A.K., Yuzawa, S., Petzold, C.J., Katz, L. and Keasling, J.D. 2018. Probing the flexibility of an iterative modular polyketide synthase with non-native substrates in vitro. ACS Chemical Biology. Article ASAP. DOI: 10.1021/acschembio.8b00422
- J. F. Barajas, R. M. Phelan, A. J. Schaub, J. T. Kliewer, P. J. Kelly, D. R. Jackson, R. Luo, J. D. Keasling and S.-C. Tsai. 2015. “Comprehensive structural analysis of the terminal myxalamid reductase domain for the engineered production of primary alcohols.” Chem. Biol. 22(8):1018-29.
- S. Poust, R. M. Phelan, K. Deng, L. Katz, C. J. Petzold, and J. D. Keasling. 2015. “Divergent mechanistic routes for the formation of gem-dimethyl groups in the biosynthesis of complex polyketides.” Angew. Chem. Int. 54:2370-2373.
- Hagen, S. Poust, T. de Rond, S. Yuzawa, L. Katz, P. D. Adams, C. J. Petzold, and J. D. Keasling. 2014. “In vitro analysis of carboxyacyl substrate tolerance in the loading and first extension modules of Borrelidin polyketide synthase.” Biochemistry 53:5975-5977.
- S. Yuzawa, C. H. Eng, L. Katz, and J. D. Keasling. 2014. “Enzyme analysis of the polyketide synthase leads to the discovery of a novel analog of the antibiotic a-lipomycin.” J. Antibiotics 67:199-201.
A longstanding goal in the Keasling Lab centers on the characterization and development of microbial hosts beyond those traditionally associated with synthetic biology and metabolic engineering (E. coli and S. cerevisiae). This includes developing detailed multi-omic understanding of select microbes and improved biological tools to manipulate these hosts. Recently, we have become ever more interested studying the complex metabolism of Streptomyces species and developing tools to allow reliable and rapid metabolic engineering in this bacterial genus. Given the immense scope of secondary metabolites produced by these bacteria and other favorable properties unique to Streptomyces venezuelae, we are committed to developing this bacterium for routine biotechnology efforts. Upcoming reports will detail the generation of improved biological tools (i.e., fluorescent reporters, improved promoters, Cas9 genome editing systems, etc.) that facilitate efforts aimed toward producing non-native products. Future efforts will integrate our superior understanding of S. venezuelae metabolism with improved biocatalytic systems to significantly impact the efficiency with which we can biologically produce petrochemicals and therapeutic agents. Representative publications include:
- R. M. Phelan, D. Sachs, S. J. Petkiewicz, J. F. Barajas, J. M. Blake-Hedges, M. G. Thompson, A. R. Apel, B. J. Rasor, L. Katz, and J. D. Keasling. 2016. “Development of next generation synthetic biology tools for use in Streptomyces venezuelae.” ACS Syn. Biol. 6(1):159–166. DOI: 10.1021/acssynbio.6b00202.
- W. D. Hollinshead, S. Rodriguez, H. Garcia Martin, G. Wang, E. E.K. Baidoo, K. L. Sale, J. D. Keasling, A. Mukhopadhyay, and Y. J. Tang. 2016. “Examining Escherichia coli glycolytic pathways, catabolite repression, and metabolite channeling using Δpfk mutants.” Biotechnology for Biofuels 9:212. DOI: 10.1186/s13068-016-0630-y
- C. J. Joshua, R. Dahl, P. I. Benke, and J. D. Keasling. 2011. “Absence of diauxie during simultaneous utilization of glucose and xylose by Sulfolobus acidocaldarius.” J. Bacteriol. 193:1293-1301.