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.
A longstanding goal in the Keasling Lab centers on the characterization and development of microbial hosts to better enable biosynthesis of target compounds. Our aim is to move beyond hosts that are traditionally associated with synthetic biology and metabolic engineering (i.e., E. coli and S. cerevisiae) to develop both a detailed, multi-omic understanding of select microbes and improved biological tools to manipulate these hosts. While E. coli and S. cerevisiae have many favorable properties, it would be beneficial to enable engineering in any given host, thus allowing one to work in a bacterial chassis that could, for instance, catabolize cellulose, fix carbon dioxide or produce specific compound classes at multi-gram-per-liter titers.
While previous efforts in our lab and associated groups the Joint BioEnergy Insitiute have focused on underutilized bacteria such as Sulfolobus and Ralstonia, we have become ever more interested studying the complex metabolism of Streptomyces 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, Cas-9 genome editing systems, etc.) that facilitate efforts aimed toward producing non-native products. To highlight the tools we have developed, recent publications detail the production and rational improvement of a heterologous biosynthetic pathway to produce the D2 diesel alternative bisabolene in S. venezuelae from varying carbohydrate feedstocks. 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.
Department of Energy, National Science Foundation
- Barajas, J. F., 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 and Biochemical Analysis of the Terminal Myxalamid Reductase Domain for the Engineered Production of Primary Alcohols’, Chem Biol, 22: 1018-29.
- Eng, C. H., S. Yuzawa, G. Wang, E. E. Baidoo, L. Katz, and J. D. Keasling. 2016. ‘Alteration of Polyketide Stereochemistry from anti to syn by a Ketoreductase Domain Exchange in a Type I Modular Polyketide Synthase Subunit’, Biochemistry, 55: 1677-80.
- Hagen, A., S. Poust, Td Rond, J. L. Fortman, L. Katz, C. J. Petzold, and J. D. Keasling. 2016. ‘Engineering a Polyketide Synthase for In Vitro Production of Adipic Acid’, ACS Synth Biol, 5: 21-7.
- Phelan, R. M., O. N. Sekurova, J. D. Keasling, and S. B. Zotchev. 2015. ‘Engineering terpene biosynthesis in Streptomyces for production of the advanced biofuel precursor bisabolene’, ACS Synth Biol, 4: 393-9.
- Poust, S., 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 Ed Engl, 54: 2370-3.
- Poust, S., I. Yoon, P. D. Adams, L. Katz, C. J. Petzold, and J. D. Keasling. 2014. ‘Understanding the role of histidine in the GHSxG acyltransferase active site motif: evidence for histidine stabilization of the malonyl-enzyme intermediate’, PLoS One, 9: e109421.
- Poust, S., Hagen, A., Katz, L., Keasling J.D., 2014 ‘Narrowing the gap between the promise and reality of polyketide synthases as a synthetic biology platform’, Curr. Opin. Biotechnol. 1153-57