Triterpene Hydrocarbon Production Engineered Into a Metabolically Versatile Host—Rhodobacter capsulatus Nymul E. Khan, 1 S. Eric Nybo, 2 Joe Chappell, 2 Wayne R. Curtis 1 1 Department of Chemical Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802; telephone: 814-863-4805; fax: 814-865-7846; e-mail: wrc2@psu.edu 2 Department of Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky 40536 ABSTRACT: Triterpene hydrocarbon biosynthesis of the ancient algae Botryococcus braunii was installed into Rhodobacter capsulatus to explore the production of C 30 hydrocarbon in a host capable of diverse growth habits—utilizing carbohydrate, sunlight or hydrogen (with CO 2 fixation) as alternative energy feedstocks. Engineering an enhanced MEP pathway was also used to augment triterpene accumulation. Despite dramatically different sources of carbon and reducing power, nearly the same level of botryococcene or squalene (5 mg oil/g-dry-weight [gDW]) was achieved in small-scale aerobic heterotrophic, anaerobic photo- heterotrophic, and aerobic chemoautotrophic growth conditions. A glucose fed-batch bioreactor reached 40 mg botryococcene/L (12mg/gDW), while autotrophic bioreactor performance with CO 2 ,H 2 , and O 2 reached 110 mg/L (16.7 mg/gDW) during batch and 60 mg/L (23 mg/gDW) during continuous operation at a dilution rate corresponding to about 10% of m max . Batch and continuous autotrophic specific productivity was found to reach 0.5 and 0.32 mg triterpene/g DW/h, comparable to prior reports for terpene production driven by heterotrophic growth conditions. This demonstrates the feasibility of alternative feedstocks and trophic modes to provide comparable routes to biochemicals that do not rely on sugar. Biotechnol. Bioeng. 2015;112: 1523–1532. ß 2015 Wiley Periodicals, Inc. KEYWORDS: CO 2 fixation; squalene; botryococcene; MEP pathway; productivity; continuous bioreactor Introduction Feedstock flexibility is being pursued to improve the economic feasibility and sustainability of the production of biofuels and chemicals. Corn-ethanol and the ongoing transitioning to cellulosic-ethanol or biobutanol are examples of allowing plants to capture photonic energy in chemical bonds, and subsequently releasing that energy to selectively produce a more convenient liquid transportation fuel. Similarly, the production of biofuels by algae reflects an even more direct use of the sun that relies on the storage of triacylglycerol lipids by these photosynthetic organisms. These oils can then be converted to biodiesel with minimum processing requirements. The use of these plant-derived energy sources, is inherently competitive with their alternative use as food and adds to the interaction of energy and food production costs. A more desirable alternative would be a host organism capable of chemical production from a variety of energy and carbon sources. To demonstrate this, we have targeted the production of two C 30 hydrocarbons, a biofuel (botryococcene), and a pharmaceutical constituent (squalene)—botryococcene and squalene, in Rhodo- bacter capsulatus—a host capable of diverse metabolism and robust growth. Nature has devised extensive options to tap into the energy of the sun, with equally diverse carbon balances. This presents alternative approaches for biofuels production, where Figure 1 illustrates the metabolic scenarios addressed in the presented work. Heterotrophic growth is the familiar consumption of carbohydrates, where aerobic growth takes advantage of metabolic efficiencies to produce CO 2 and water. When oxygen is constrained, anaerobic heterotrophic metabolism can result in the production of alcohols and organic acids and dihydrogen gas. Photoheterotrophic metabolism utilizes a carbon source such as malic or succinic acid from the TCA cycle and achieves tremendous carbon-use efficiency by generating most of its energy by ATP production from photophosphorylation. The final R. capsulatus trophism illustrated in Figure 1 is aerobic chemo- lithoautotrophic metabolism, where the photonic energy generates hydrogen (via electrolytic splitting of water), and this energy can then be used to fix carbon dioxide. In nature the CO 2 and H 2 can be derived from anaerobic metabolism. Current address of S. Eric Nybo is Department of Pharmaceutical Sciences, College of Pharmacy, Ferris State University, Big Rapids, MI, USA. Nymul Khan and S. Eric Nybo contributed equally to this work. Correspondence to: W. R. Curtis Contract grant sponsor: U.S. Department of Energy Contract grant number: ARPA-e Electrofuels, DE-AR0000092 Received 31 October 2014; Revision received 14 January 2015; Accepted 11 February 2015 Accepted manuscript online 27 February 2015; Article first published online 12 May 2015 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.25573/abstract). DOI 10.1002/bit.25573 ARTICLE ß 2015 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 112, No. 8, August, 2015 1523