Keasling Lab

Application Areas

The research in the Keasling Lab is centered on real-world applications.  We engineer microbes to turn sugar and other feedstocks into high-value chemicals that are sustainable and stimulate economic development and create jobs.  Below are some of the molecules we’ve engineered our microbes to produce.

Artemisinin

Malaria infects 300–500 million people and causes 1-2 million deaths each year, primarily children in Africa and Asia. One of the principal obstacles to addressing this global health threat is a lack of effective, affordable drugs. The chloroquine-based drugs that were used widely in the past have lost effectiveness because the Plasmodium parasite which causes malaria has become resistant to them.  The faster-acting, more effective artemisinin-based drugs — as currently produced from plant sources — are too expensive for large-scale use in the countries where they are needed most.  The Keasling lab engineered both Escherichia coli and Saccharomyces cerevisiae to produce a precursor to artemisinin, artemisinic acid, which can be readily converted into artemisinin.  Microbial production of artemisinic acid will eventually reduce the cost of artemisinin-based combination therapies significantly below their current price and stabilize the supply of artemisinin while controlling access.  Our partner in this work was Amyris Biotechnologies, a company founded to develop and optimize this technology.  Sanofi licensed the technology and scaled it.  They began shipping artemisinin combination therapies containing artemisinin produced using this microbial production process in 2014.  Approximately 51 million treatments have been shipped to Africa. Example publications include:

  • V. J. J. Martin, D. J. Pitera, S. T. Withers, J. D. Newman, and J. D. Keasling. 2003. “Engineering the mevalonate pathway in Escherichia coli for production of terpenoids.” Nat. Biotechnol. 21:796-802.
  • 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.
  • D.-K. Ro, M. Ouellet, E. M. Paradise, H. Burd, D. Eng, C. J. Paddon, J. D. Newman, and J. D. Keasling. 2008. “Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of antimalarial drug precursor, artemisinic acid.” BMC Biotechnol. 8:83 (doi:10.1186/1472-6750-8-83).
  • J. Dietrich, Y. Yoshikuni, K. Fisher, F. Woolard, D. Ockey, D. McPhee, N. Renninger, M. Chang, D. Baker, and J. D. Keasling. 2009. “A novel semi-biosynthetic route for artemisinin production using engineered substrate-promiscuous P450BM3.” ACS Chem. Biol. 4:261-267.
  • J. E. Dueber, G. C. Wu, G. R. Malmirchegini, T. S. Moon, C. J. Petzold, A. V. Ullal, K. J. Prather, and J. D. Keasling. 2009. “Synthetic protein scaffolds provide modular control over metabolic flux.” Nat. Biotechnol. 27:753-759.
  • P. J. Westfall, D. J. Pitera, J. R. Lenihan, D. Eng, F. Woolard, R. Regentin, T. Horning, Hiroko Tsuruta, D. Melis, A. Owens, S. Fickes, D. Diola, J. D. Keasling, M. D. Leavell, D. McPhee, N. S. Renninger, J. D. Newman, C. J. Paddon. 2012. “Production of Amorpha-4,11-diene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin.” Proc. Natl. Acad. Sci. USA 109:E111-E118.
  • C. J. Paddon, P. J. Westfall, D. J. Pitera, K. Benjamin, K. Fisher, D. McPhee, M. D. Leavell, A. Tai, A. Main, D. Eng, D. R. Polichuk, K. H. Teoh, D. W. Reed, T. Treynor, J. Lenihan, M. Fleck, S. Bajad, G. Dang, D. Diola, G. Dorin, K. W. Ellens, S. Fickes, J. Galazzo, S. P. Gaucher, T. Geistlinger, R. Henry, M. Hepp, T. Horning, T. Iqbal, H. Jiang, L. Kizer, B. Lieu, D. Melis, N. Moss, R. Regentin, S. Secrest, H. Tsuruta, R. Vazquez, L. F. Westblade, L. Xu, M. Yu, Y. Zhang, L. Zhao, J. Lievense, P. S. Covello, J. D. Keasling, K. K. Reiling, N. S. Renninger & J. D. Newman. 2013. “High-level semi-synthetic production of the potent antimalarial artemisinin.” Nature 496:528-532.
  • R. H. Dahl, F. Zhang, J. Alonso-Gutierrez, E. Baidoo, T. S. Batth, A. M. Redding-Johanson, C. J. Petzold, A. Mukhopadhyay, T. Soon Lee, P. D. Adams, and J. D. Keasling. 2013. “Engineering dynamic pathway regulation using stress-response promoters.” Nat. Biotechnol. 31(11):1039-1046. doi: 10.1038/nbt.2689.
  • C. J. Paddon and J. D. Keasling. 2014. “Semi-synthetic artemisinin: a model for the use of synthetic biology in pharmaceutical development.” Nat. Rev. Microbiol. 12:355-367.

Biofuels

Carbon-rich fossil fuels, primarily oil, coal and natural gas, provide 85% of the energy consumed in the United States. As world demand increases, oil reserves are becoming rapidly depleted. Fossil fuel use increases CO2 emissions and raises the risk of global warming. The high-energy content of liquid hydrocarbon fuels makes them the preferred energy source for all modes of transportation. Unfortunately, existing biofuels (ethanol, butanol, and plant oil-derived biodiesel) are not compatible with our existing transportation infrastructure.  The Keasling Lab is engineering metabolic pathways in various heterologous host organisms including Escherichia coli, Saccharomyces cerevisiae, and Streptomyces venezuelae for production of advanced biofuels compatible with our existing transportation infrastructure.  These advanced biofuels have the full fuel value of petroleum-based biofuels, will be transportable using existing pipelines, and can be used in existing automobiles, trucks, trains, and airplanes.  These biofuels will be produced from biosynthetic pathways that exist in plants and a variety of microorganisms (such as engineered pathways derived from fatty acid, terpene, and polyketide biosynthesis).  Large-scale production of these fuels will reduce our dependence on petroleum and reduce the amount of carbon dioxide released into the atmosphere, while allowing us to take advantage of our current transportation infrastructure. Example publications include:

  • L. d’Espaux, A. Ghosh, W. Runguhpan, M. Wehrs, F. Xu, O. Konzock, I. Dev, M. Nhan, J. Gin, A Reider Apel, C. J. Petzold, S. Singh, B. A. Simmons, A. Mukhopadhyay, H. Garcia Martin, and J. D. Keasling. 2017. “Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks.”  Met. Eng.
  • W. Runguphan and J. D. Keasling. 2014. “Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals. Met. Eng. 21:103-113.
  • P. P. Peralta-Yahya, F. Zhang, S. B. del Cardayre, and J. D. Keasling. 2012. “Microbial engineering for the production of advanced biofuels.”  Nature 488:320-328.
  • S. Sarria, B. Wong, H. G. Martin, J. D. Keasling, and P. Peralta-Yahya. 2014. “Microbial synthesis of pinine.” ACS Synth. Biol. 3:466-475.
  • G. Bokinsky, P. Peralta-Yahya, A. George, B. M. Holmes, E. J. Steen, J. Dietrich, T. S. Lee, D. Tullman-Ercek, C. Voigt, B. A. Simmons, J. D. Keasling. 2011. “Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli.”  Proc. Natl. Acad. Sci. USA 108:19949-19954.
  • E. J. Steen, Y. Kang, G. Bokinsky, Z. Hu, A. Schirmer, A. McClure, S. B. del Cardayre, and J. D. Keasling. 2010. “Microbial production of fatty acid-derived fuels and chemicals from plant biomass.” Nature 463:559-562.
  • H. R. Beller, E.-B. Goh, and J. D. Keasling. 2010. “Genes involved in long-chain alkene biosynthesis in Micrococcus luteus.” Appl. Environ. Microbiol. 76:1212-1223.

Commodity and specialty chemicals

Many of the commodity chemicals that are used in many of the products we encounter on a daily basis are derived from petroleum. As such, the prices of these chemicals fluctuate with the price of petroleum and will become more expensive and scarce as petroleum supplies dwindle. The Keasling Lab is modifying microbial metabolism to produce the very same chemicals that would otherwise be derived from petroleum. In the common biotechnological host E. coli, we have produced the commodity chemical adipic acid, a precursor to some forms of nylon and responsible for 10% of the man-made emissions of the greenhouse gas nitrous oxide.  In addition, the Keasling Lab is modifying metabolism to produce chemicals that could not economically be produced from petroleum. When these chemicals are introduced into polymers, they will make much more durable plastics and fibers than have been possible using the simple chemicals derived from petroleum using conventional catalysts. Example publications include:

  • J. Zhang, J. F. Barajas, M. Burdu, G. Wang, E. E. K. Baidoo, and J. D. Keasling. 2017. “Application of an acyl-CoA ligase from Streptomyces aizunensis for lactam biosynthesis.” ACS Synth. Biol. 6(5):884–890. DOI: 10.1021/acssynbio.6b00372.
  • J. Zhang, E. Kao, G. Wang, E. E. K. Baidoo, M. Chen, and J. D. Keasling. 2016. “Metabolic engineering of Escherichia coli for the biosynthesis of 2-pyrrolidone.” Metab. Eng. Commun. 3:1-7.
  • R. W. Haushalter, R. M. Phelan, K. M. Hoh, C. Su, G. Wang, E. E. Baidoo, and J. D. Keasling. 2017. “Production of odd-carbon dicarboxylic acids in Escherichia coli using an engineered biotin-fatty acid biosynthetic pathway.” J. Am. Chem. Soc. 139(10):4615-4618. DOI: 10.1021/jacs.6b11895.
  • Hagen A, Poust S, Rond Td, Fortman JL, Katz L, Petzold CJ, Keasling JD. 2016. “Engineering a polyketide synthase for in vitro production of adipic acid.” ACS. Synth. Biol. 15:21-27
  • J. Alonso-Gutierrez, R. Chan, T. S Batth, P. D Adams, J. D. Keasling, C. J Petzold, and T. S. Lee. 2013. “Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production. Met. Eng. 19:33-41.
  • S. Yuzawa, N. Chiba, L. Katz, and J. D. Keasling. 2012. “Construction of a part of a 3-hydroxypropionate cycle for heterologous polyketide biosynthesis in Escherichia coli.” Biochemistry 51:9779-9781.
  • Y. Satoh, K. Tajima, M. Munekata, J. D. Keasling, and T. S. Lee. 2012. “Engineering of a tyrosol-producing pathway, utilizing simple sugar and the central metabolite tyrosine, in Escherichia coli.” J. Agric. Food Chem. 60:979-984.
  • D. Juminaga, E. E. Baidoo, A. M. Redding-Johanson, T. S. Batth, H. Burd, A. Mukhopadhyay, C. J. Petzold, and J. D. Keasling. 2012. “Modular engineering of L-tyrosine production in Escherichia coli.” Appl. Environ. Microbiol. 78:89-98.
  • E. J. Steen, Y. Kang, G. Bokinsky, Z. Hu, A. Schirmer, A. McClure, S. B. del Cardayre, and J. D. Keasling. 2010. “Microbial production of fatty acid-derived fuels and chemicals from plant biomass.” Nature 463:559-562.

Natural product pharmaceuticals

Approximately one-half of the drugs in use today are natural products or derivatives of natural products. Generally, the therapeutic molecule is produced in low quantities in the natural producer. Production of the natural product (or a derivative) in a natural host can be an effective production method but necessitates reconstituting the biosynthetic pathway in the microbial host. To enable production of isoprenoid-based therapeutics, members of the Keasling Lab are elucidating the metabolic pathways responsible for synthesis of prostratin, a 20-carbon phorbol ester from the mamala tree and Taxol, a diterpenoid used as an anti-cancer drug.  While many natural products have become important pharmaceuticals, most natural products are not ideal for treating human disease.  To broaden the product range of metabolic pathways and develop alternatives to natural products that might be more suitable for treating human disease, we are examining and changing the substrate range of cytochrome P450 hydoxylases to oxygenate a broader range of terpenes than they would normally oxidize and to do so in different positions on the molecule than they would naturally oxidize.  The resulting oxidized terpenes might be precursors for production of pharmaceuticals or other products. Example publications include:

  • 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. Poust, 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(10):e109421 doi:10.1371/journal.pone.0109421.
  • 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.
  • S. Yuzawa, C. Eng, L. Katz, and J. D. Keasling. 2013. “Broad substrate specificity of the loading didomain of the lipomycin polyketide synthase.” Biochemistry 52:3791-3793.
  • L. Prach, J. Kirby, J. D. Keasling, T. Alber. 2010. “Diterpene production in Mycobacterium tuberculosis.” FEBS J. 277:3588-3595.
  • Y. J. Tang, W. Shui, S. Myers, X. Feng, C. Bertozzi, J. D. Keasling. 2009. “Central metabolism in Mycobacterium smegmatis during the transition from O(2)-rich to O (2)-poor conditions as studied by isotopomer-assisted metabolite analysis.” Biotechnol. Lett. 31:1233-1240.
  • M. C. Y. Chang, R. A. Eachus, W. Trieu, D.-K. Ro, and J. D. Keasling. 2007. “Engineering Escherichia coli for production of functionalized terpenoids using plant P450s.” Nature Chem. Biol. 3:274-277.

Bioremediation

Not only does biology excel at synthesizing molecules, it also works effectively to degrade unwanted (and sometimes wanted) molecules and accumulate toxic metals.  Our laboratory has examined microorganisms resident in soils and groundwater aquifers contaminated with man-made chemicals to understand their growth and metabolism with the idea that these organisms could be used to remediate contaminants. This work has resulted in technologies that are currently being practiced in the field for remediating groundwater contaminated with trichloroethene and tetrachloroethene. More recently, we have rationally engineered microorganisms to degrade nerve agents for application to degrading chemical warfare agent stockpiles. Furthermore, structural similarity of nerve agents to prevalently-used pesticides may allow a microbial platform for environmental decontamination of pesticides. Example publications include:

  • M. E. Brown, A. Mukhopadhyay, and J. D. Keasling. 2016. “Engineering bacteria to catabolize the carbonaceous component of sarin: teaching E. coli to eat isopentanol.” ACS Synth. Biol. 5(12):1485-1496. DOI: 10.1021/acssynbio.6b00115.
  • Zhou, E. Baidoo, Z. He, A. Mukhopadhyay, J. K. Baumohl, P. Benke, M. P Joachimiak, M. Xie, R. Song, A. P. Arkin, T. C. Hazen, J. D. Keasling, J. D. Wall, D. A. Stahl and J. Zhou. 2013. “Characterization of NaCl tolerance in Desulfovibrio vulgaris Hildenborough through experimental evolution.” ISME J. 7(9):1790-1802.
  • B. Walker, A. M. Redding-Johanson, E. Baidoo, L. Rajeev, Z. He, E. L Hendrickson, M.P. Joachimiak, S. Stolyar, A. P. Arkin, J. A. Leigh, J. Zhou, J. D. Keasling, A. Mukhopadhyay, and D. A. Stahl. 2012. “Functional response of methanogenic archaea to syntrophic growth.” ISME J. 6:2045-2055.
  • M. Redding, A. Mukhopadhyay, D. Joyner, T. C. Hazen, and J. D. Keasling. 2006. “Study of nitrate stress in Desulfovibrio vulgaris Hildenborough using iTRAQ proteomics.” Brief. Funct. Genom. Proteom. 5:133-43.
  • M. Mattozzi, S. Tehara, and J. D. Keasling. 2006. “Mineralization of paraoxon and use as a sole C and P source by a rationally designed catabolism in Pseudomonas putida.” Appl. Environ. Microbiol. 72:6699-6706.

 

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