Remodeling of Chlamydomonas Metabolism Using Synthetic Inducers Results in Lipid Storage During Growth

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Biochemistry -- Faculty Publications Biochemistry, Department of 11-2019 Remodeling of Chlamydomonas Metabolism Using Synthetic Inducers Results in Lipid Storage during Growth Nishikant Wase Boqiang Tu Girish Kumar Rasineni Ronald Cerny Ryan Grove See next page for additional authors Follow this and additional works at: https://digitalcommons.unl.edu/biochemfacpub Part of the Biochemistry Commons, Biotechnology Commons, and the Other Biochemistry, Biophysics, and Structural Biology Commons This Article is brought to you for free and open access by the Biochemistry, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Biochemistry -- Faculty Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors Nishikant Wase, Boqiang Tu, Girish Kumar Rasineni, Ronald Cerny, Ryan Grove, Jiri Adamec, Paul N. Black, and Concetta DiRusso Remodeling of Chlamydomonas Metabolism Using Synthetic Inducers Results in Lipid Storage during Growth1[OPEN] Nishikant Wase,a Boqiang Tu,a Girish Kumar Rasineni,a Ronald Cerny,b Ryan Grove,a Jiri Adamec,a Paul N. Black,a and Concetta C. DiRussoa,2,3 aDepartment of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588 bDepartment of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588 ORCID IDs: 0000-0002-8955-3300 (N.W.); 0000-0002-3999-1986 (B.T.); 0000-0002-7821-8021 (G.K.R.); 0000-0002-3765-2470 (R.C.); 0000-0002-1309-3360 (R.G.); 0000-0003-4899-4406 (J.A.); 0000-0002-6272-6881 (P.N.B.); 0000-0001-7388-9152 (C.C.D.). Microalgae accumulate lipids during stresssuchasthatofnutrientdeprivation,concomitantwithcessationofgrowth and depletion of chloroplasts. By contrast, certain small chemical compounds selected by high-throughput screening in Chlamydomonas reinhardtii can induce lipid accumulation during growth, maintaining biomass. Comprehensive pathway analyses using proteomics, transcriptomics, and metabolomics data were acquired from Chlamydomonas cellsgrowninthe presence of one of two structurally distinct lipid activators. WD10784 stimulates both starch and lipid accumulation, whereas WD30030-treated cells accumulate only lipids. The differences in starch accumulation are largely due to differential effects of the two compounds on substrate levels that feed into starch synthesis and on genes encoding starch metabolic enzymes. The compounds had differential effects on photosynthesis, respiration, and oxidative stress pathways. Cells treated with WD10784 showed slowed growth over time and reduced abundance of photosynthetic proteins, decreased respiration, and increased oxidative stress proteins, glutathione, and reactive oxygen species specific to this compound. Both compounds maintained central carbon and nitrogen metabolism, including the tricarboxylic acid cycle, glycolysis, respiration, and the Calvin–Benson–Bassham cycle. There were few changes in proteins and transcripts related to fatty acid biosynthesis, whereas proteins and transcripts for triglyceride production were elevated, suggesting that lipid synthesis is largely driven by substrate availability. This study reports that the compound WD30030 and, to a lesser extent WD10784, increases lipid and lipid droplet synthesis and storage without restricting growth or biomass accumulation by mechanisms that are substantially different from nutrient deprivation. Green algae hold great promise as a feedstock for biomass produced in standard growth conditions has biofuel production due to their ability to synthesize and rendered them economically impractical for biofuel store triacylglycerides (TAGs) in lipid droplets (LDs; production (Wijffels and Barbosa, 2010). Therefore, Guschina and Harwood, 2006; Li-Beisson et al., 2019). much effort has gone into devising methods to in- However, the relatively low lipid accumulation per unit crease both biomass and lipid yield, and to reduce the induction of stress response pathways. The uni- cellular green alga Chlamydomonas reinhardtii has 1 This work was supported by the National Science Foundation emerged as a valuable model system to study both (EPSCoR EPS-1004094, EPS-1264409, and CBET-1402896) and the Ne- photosynthesis and metabolic processes that are im- braska Center for Energy Science Research. portant for the biological production of economically 2Author for contact: [email protected]. 3Senior author. valuable products. The annotated genome sequence The author responsible for distribution of materials integral to the in Phytozome facilitates gene-based comparative stud- findings presented in this article in accordance with the policy de- ies to understand lipid biochemistry. Although several scribed in the Instructions for Authors (www.plantphysiol.org) is: strategies have been attempted to improve lipid pro- Concetta C. DiRusso ([email protected]). duction, such as targeted strain design and improve- C.C.D. and N.W. conceived the original research plan and were ments in growth and harvesting techniques, in most responsible for execution of the work, data analysis, and interpreta- cases the green algae accumulate lipids only when tion; N.W., B.T., G.K.R., and R.G. conducted biochemical, RNA se- subjected to severe stresses such as nutrient deple- quencing, and metabolomics experiments; R.C. and J.A. provided tion, particularly nitrogen starvation (Wang et al., oversight for proteomic and metabolomics analyses, respectively; 2009; Miller et al., 2010; Schmollinger et al., 2014). P.N.B. assisted in data presentation and preparation of figures; all authors contributed to final manuscript preparation; C.C.D. agrees However, nutrient starvation arrests growth, which to serve as the author responsible for contact and ensures is disadvantageous for large-scale production. There- communication. fore, to improve lipid yield for biofuel production, it is [OPEN]Articles can be viewed without a subscription. important to design methods to decouple lipid accu- www.plantphysiol.org/cgi/doi/10.1104/pp.19.00758 mulation from growth arrest. Plant PhysiologyÒ, November 2019, Vol. 181, pp. 1029–1049, www.plantphysiol.org Ó 2019 American Society of Plant Biologists. All Rights Reserved. 1029 Wase et al. Numerous previous studies have examined lipid deprivation was conducted by this laboratory using accumulation in green algae for cells grown under a Chlamydomonas (Wase et al., 2017). More than 43,000 variety of stressors such as nitrogen, sulfur, phospho- compounds were screened, and 243 active compounds rus, or iron deprivation (Wang et al., 2009; Miller et al., were identified and verified using dose-response 2010; Nguyen et al., 2011; Lee et al., 2012; Blaby et al., curves to estimate lipid accumulation levels during 2013; Schmollinger et al., 2014; Wase et al., 2014; Ajjawi growth. Among the primary hits were two compounds et al., 2017). A subset of these studies employed data- called “WD10784” and “WD30030,” which represented rich omics technologies to identify key alterations in two different chemical structural classes that act to expression of metabolic networks or regulatory com- increase lipid storage in algae. Secondary screens con- ponents that might contribute to lipid production firmed both compounds increased lipid storage 4- to (Boyle et al., 2012; Blaby et al., 2013; Goodenough et al., 5-fold over 72 h of compound treatment in a dose- 2014; Schmollinger et al., 2014; Wase et al., 2014; Ajjawi dependent manner. Importantly, lipid induction also et al., 2017; Gargouri et al., 2017). Classical hallmarks of occurred during growth in three additional freshwa- nitrogen starvation include downregulation of protein ter algae including Chlorella vulgaris UTEX395, Chlorella synthesis, degradation of the photosynthetic appara- sorokiniana UTEX1230, and Tetrachlorella alterns UTEX2453 tus, and the movement of primary carbon into long- (Wase et al., 2017). The two compounds induced both term storage molecules as starch and TAGs (Blaby similar and unique profiles regarding cellular physiology et al., 2013; Alboresi et al., 2016). This is correlated and metabolism. Briefly, both induced FA and triglyc- with increased expression of diglyceride acyltrans- eride accumulation without altering galactolipid levels ferase (DGTT1) and the major lipid droplet protein (Wase et al., 2017). WD30030 did not influence starch (MLDP). However, it is unclear if these factors and storage, whereas WD10784 increased starch 2.5-fold. processes are necessary to induce lipid accumulation Additionally, the WD10784 compound reduced chlo- or are merely expressed coincident with the nutrient rophyll (Chl) a and b levels and total carotenoids and deprivation response. the WD30030 compound did not affect these pigments. A novel approach to increase lipid yield is to select In this work, proteomics, transcriptomics, and metab- chemical inducers of TAG production during photo- olomics profiling were conducted to provide further trophic or mixotrophic growth conditions (for review, insight into pathway adjustments leading to lipid syn- see Wase et al., 2018). Franz et al. (2013) employed thesis and storage induced by these two compounds. microplate-based phenotypic screening of a small li- The goal of this work was to define similarities and brary of bioactive molecules (;50) in four strains of differences between each compound and to contrast oleaginous microalgae (Nannochloropsis salina, Nanno- the mechanistic impact of the compounds on cellular chloropsis

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