Examination of Triacylglycerol Biosynthetic Pathways via De Novo Transcriptomic and Proteomic Analyses in an Unsequenced Microalga Michael T. Guarnieri1*, Ambarish Nag2, Sharon L. Smolinski3, Al Darzins1¤, Michael Seibert4, Philip T. Pienkos1 1 National Bioenergy Center, National Renewable Energy Laboratory, Golden, Colorado, United States of America, 2 Computational Science Center, National Renewable Energy Laboratory, Golden, Colorado, United States of America, 3 Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado, United States of America, 4 Energy Sciences Directorate, National Renewable Energy Laboratory, Golden, Colorado, United States of America Abstract Biofuels derived from algal lipids represent an opportunity to dramatically impact the global energy demand for transportation fuels. Systems biology analyses of oleaginous algae could greatly accelerate the commercialization of algal- derived biofuels by elucidating the key components involved in lipid productivity and leading to the initiation of hypothesis-driven strain-improvement strategies. However, higher-level systems biology analyses, such as transcriptomics and proteomics, are highly dependent upon available genomic sequence data, and the lack of these data has hindered the pursuit of such analyses for many oleaginous microalgae. In order to examine the triacylglycerol biosynthetic pathway in the unsequenced oleaginous microalga, Chlorella vulgaris, we have established a strategy with which to bypass the necessity for genomic sequence information by using the transcriptome as a guide. Our results indicate an upregulation of both fatty acid and triacylglycerol biosynthetic machinery under oil-accumulating conditions, and demonstrate the utility of a de novo assembled transcriptome as a search model for proteomic analysis of an unsequenced microalga. Citation: Guarnieri MT, Nag A, Smolinski SL, Darzins A, Seibert M, et al. (2011) Examination of Triacylglycerol Biosynthetic Pathways via De Novo Transcriptomic and Proteomic Analyses in an Unsequenced Microalga. PLoS ONE 6(10): e25851. doi:10.1371/journal.pone.0025851 Editor: Debashish Bhattacharya, Rutgers University, United States of America Received July 1, 2011; Accepted September 12, 2011; Published October 17, 2011 Copyright: 2011 Guarnieri et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Air Force Office of Scientific Research funding, grant WFC31000, for A.D., M.S., P.T.P., and M.T.G., and by the Laboratory Directed Research and Development (LDRD) Program at the National Renewable Energy Laboratory (NREL), LDRD # 06510901 for P.T.P, S.S., M.T.G., and A.N.. NREL is a national laboratory of the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy operated by the Alliance for Sustainable Energy, LLC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: This work was funded in part by the Alliance for Sustainable Energy, LLC. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: michael.guarnieri@nrel.gov ¤ Current address: Biochemical Sciences and Engineering, DuPont Central Research and Development, Wilmington, Delaware, United States of America Introduction Oleaginous microalgae produce substantial amounts of neutral lipids, primarily comprised of triacylglycerides (TAGs) of favorable fatty acid chain length, making them an ideal feedstock for conversion to biodiesel or renewable diesel and jet fuel [1,2,3]. Many of these species can also grow rapidly under a large range of environmental conditions, such as varied light intensity, temper- ature, and nutrient availability [1,2]. Microalgae are also capable of growth on non-arable land using a variety of water sources, including fresh, brackish, saline, and waste water [1,4,5]. At current production levels, oleaginous microalgae also have the potential to produce 1–2 orders of magnitude more oil per acre than soybeans, the most common US oilseed crop (,50 gal acre21 y21) [6]. The Department of Energy funded an almost 20-year effort, known as the Aquatic Species Program (ASP), aimed at developing algal biofuels. The ASP was terminated, however, in 1996 because the projected cost of algal biofuels could not compete at that time with the low price of petroleum. Now, after more than a decade, microalgae have again risen to international prominence for their potential to contribute to the global liquid fuel demand. However, despite the great promise of algae-based fuels, our understanding of algal lipid metabolism, particularly the regulation of biosynthetic pathways of fatty acids and TAGs, as well as the metabolism affecting carbon partitioning, is still largely inadequate. Furthermore, the lack of available genome sequence information limits the development of basic biological under- standing required for strain improvement of unsequenced microalgae. Rapidly developing post-genomic, systems biology approaches such as transcriptomics, proteomics, and metabolomics have become essential for understanding how microorganisms respond and adapt to changes in their physical environment. The application of such high-throughput approaches could greatly accelerate the commercialization of algal-derived biofuels by providing the framework for hypothesis-based strain improvement programs, built on an improved fundamental understanding of the specific pathways and regulation of networks involved in algal oil production. However, a systems biology approach to investigating oleaginous microalgal metabolism remains relatively unexplored especially in unsequenced organisms. To date, the nuclear genomes of only about ten eukaryotic microalgae have been sequenced (http://genome.jgi-psf.org), and though a few of these strains may have some properties of interest for algal biofuels, PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e25851