Symposium 13 Regulation of Photosynthetic Gene Expression 315 Unique central carbon metabolic pathways and novel enzymes in phototrophic bacteria revealed by integrative genomics, 13C-based metabolomics and fluxomics Kuo-Hsiang Tanga,b, Xueyang Fengc, Anindita Bandyopadhyaya, Himadri B. Pakrasia,c, Yinjie J. Tangc, Robert E. Blankenshipa,b,* Departments of aBiology, bChemistry, cEnergy, Environment, and Chemical Engineering, Washington University in St. Louis, St. Louis, Missouri 63130, USA *Corresponding author. Tel. No. 1-314-935-7971; Fax No. 1-314-935-4432; E-mail: [email protected]. Abstract: Photosynthesis is the process to convert solar energy to biomass and biofuels, which are the only major solar energy storage means on Earth. To satisfy the increased demand for sustainable energy sources, it is essential to understand the process of solar energy storage, that is, the carbon metabolism in photosynthetic organisms. It has been well-recognized that one bottleneck of photosynthesis is carbon assimilation. In this report, we summarize our recent studies on the carbon metabolism pathways of several types of photosynthetic bacteria, including aerobic anoxygenic phototrophic proteobacteria, green sulfur bacteria, heliobacteria and cyanobacteria, using physiological studies, transcriptomics, enzyme assays, 13C-based metabolomics and fluxomics. Our studies have revealed several unique and/or significant central carbon metabolic pathways and novel enzymes that operate in these phototrophs, quantified CO2 assimilation pathways operative during mixotrophic cultivation conditions, and also suggested evolutionary links between photosynthetic and non-photosynthetic organisms. Keywords: CO2 fixation; Citramalate pathway; Entner-Doudoroff pathway; Metabolomics; Reductive TCA cycle; (Re)-citrate synthase Introduction microorganisms is of substantial interest. In this review, we briefly summarize our recent studies on Solar energy is the most abundant, albeit dilute, the carbon metabolism mechanisms of aerobic sustainable energy source on Earth, and anoxygenic phototrophic proteobacteria, green sulfur photosynthesis is a process that converts solar energy bacteria, heliobacteria and cyanobacteria using into biofuels and biomass (Blankenship, 2002). physiological studies, transcriptomics, enzyme assays, However, the photosynthetic efficiency for bimass 13C-based metabolomics and fluxomics. Our studies production in higher plants is at maximum only 4-6% have revealed several unique carbon metabolic (Zhu et al., 2010). As the demand for sustainable pathways and enzymes that operate in these energy has expanded significantly, it is important to phototrophs. Possible evolutionary links between improve the efficiency of photosynthesis. It has been photosynthetic and non-photosynthetic organisms are determined that carbon assimilation is one important suggested. bottleneck of photosynthesis, so it is necessary to understand carbon metabolism pathways in photosynthetic organisms. In contrast to the rich Materials and Methods information reported for biomedically relevant microbes, much less knowledge has been obtained for Physiological studies and integrative genomics. the metabolism of photosynthetic bacteria. Many The methods for bacterial growth, physiological photosynthetic microorgnisms live in environments studies, gene expression profiles via quantitative real- that higher plants cannot survive in, so an time PCR (QRT-PCR) (Tang et al., 2009b) and understanding of the metabolism of these enzyme assays of Roseobacter denitrificans (Tang et 316 Photosynthesis: Research for Food, Fuel and Future—15th International Conference on Photosynthesis al., 2009a), Chlorobaculum tepidum (Tang & Results and Discussion Blankenship, 2010; Tang et al., 2010a), Heliobacterium modesticaldum (Tang et al., 2010b) Using multiple approaches, we have identified and Cyanothece sp. ATCC 51142 (Feng et al., 2010a; several unique pathways and novel enzymes in several Wu et al., 2010) are described elsewhere. photosynthetic bacteria. Here we report some unique 13C-isotopic labeling and flux analysis. 13C- and/or significant central carbon metabolic pathways labeled glucose, pyruvate, acetate or bicarbonate was and novel enzymes; details can be found in our recent used as the sole carbon source in cell cultures, and the published studies (Feng et al., 2010a; Feng et al., labeled protein-based amino acids were extracted 2010b; Tang & Blankenship, 2010; Tang et al., 2009a; from cell pellets, hydrolyzed, derivitized and analyzed Tang et al., 2010a; Tang et al., 2010b; Wu et al., by GC/MS (Figure 1). For flux analysis, fluxes cannot 2010). be calculated directly from labeling data, but their accurate determination was achieved through a (1) The Entner-Doudoroff pathway. heuristic recursive procedure: the known metabolic The Embden-Meyerhof-Parnas (EMP) pathway reactions, atomic transitions, metabolite labeling, and (glycolysis), Entner-Doudoroff (ED) pathway and extracellular fluxes are combined to produce an error pentose phosphate pathway are three major function ε (the difference between the measured carbohydrate metabolic pathways. While the majority isotopomer data and the predicted isotopomer data of organisms use the EMP pathway for digesting from assumed fluxes), and then a search algorithm is glucose, some microorganisms, most of which are applied to determine the actual fluxes by minimizing aerobes, have been reported to use the ED pathway the error function (equation 1): (Conway, 1992; Fuhrer et al., 2005). Our studies indicate that the aerobic anoxygenic phototrophic k 2 (AAP) proteobacterium Roseobacter denitrificans j M N (v ) i i n (eq. 1) (vn ) predominantly uses the ED pathway for metabolizing i1 i sugars (Figure 2), and that their EMP pathway is Subject to: S • vn = 0 inactive (Tang et al., 2009a). Similar results were also reported in two other AAP bacteria using 13C- where S is the stoichiometry matrix for all unknown metabolomics and fluxomics (Furch et al., 2009) as fluxes, v are the unknown fluxes to be optimized in n well as some purple bacteria (such as Rhodobacter the program, M is the measured isotopomer data, N is i i capsulatus and Rhodobacter sphaeroides) under the corresponding model-predicted isotopomer data, certain growth conditions (Conrad & Schlegel, 1977; and δ is the corresponding standard deviation of the i Fuhrer et al., 2005). All of the cultured AAPs are measured isotopomer data. The unknown metabolic photoheterotrophs and require organic carbon for fluxes are calculated to minimize ε (Feng et al., 2010b; growth. It has not been established if the ED pathway Pingitore et al., 2007; Wahl et al., 2004). is active in all of the purple bacteria, and is also not entirely clear why those bacteria use the ED pathway, which has a lower thermodynamic efficiency compared to the EMP pathway. It is possible that those bacteria may use a high glucose metabolic rate to compensate the thermodynamic inefficiency of the ED pathway (Molenaar et al., 2009). (2) (Re)-citrate synthase. The majority of studied organisms synthesize citrate from acetyl-CoA and oxaloacetate by (Si)- citrate synthase ((Si)-CS) for initiating the TCA cycle. Our studies showed that the anoxygenic heliobacterium Heliobacterium modesticaldum uses (Re)-citrate synthase ((Re)-CS), an alternative Fig. 1. 13C-based metabolomics and fluxomics biosynthetic pathway, to produce citrate (Tang et al., Symposium 13 Regulation of Photosynthetic Gene Expression 317 2010a). Activity of (Si)-CS has not been detected oxidative TCA cycle, in which the reducing (Tang et al., 2010b) and the gene encoding (Si)-CS equivalents (i.e. NADH) are generated (Figure 4), has not been annotated in the H. modesticaldum although all of the genes, except one, in the reductive genome (Sattley & Blankenship, 2009; Sattley et al., TCA cycle have been annotated in H. modesticaldum 2008). As indicated in Figure 3, the acetyl group from genome (Sattley et al., 2008) and the enzymatic acetyl-CoA is attached to the pro-S or pro-R arm of activity of encoded proteins in the TCA cycle have citrate by (Si)-CS or (Re)-CS, respectively. been detected in H. modesticaldum (Tang et al., 2010b). Our studies may shed light on the proposed photosynthetic cyclic electron transport pathway in the heliobacteria (Kramer et al., 1997). Fig. 4. The proposed central carbon flow in heliobacteria. Fig. 2. The EMP pathway versus the ED pathway. (3) Citramalate pathway. Many organisms synthesize isoleucine through the threonine-deaminase dependent pathway, whereas we discovered that several photosynthetic bacteria, including R. denitrificans (Tang et al., 2009a), Chlorobaculum tepidum (Feng et al., 2010b), H. modesticaldum (Tang et al., 2010a) and Cyanothece sp. ATCC 51142 (Wu et al., 2010), use the citramalate-dependent pathway to synthesize Fig. 3. The reactions catalyzed by (Si)- versus (Re)-CS. isoleucine (Figure 5). Together with an active citramalate pathway reported in several non- Previous studies indicate that several Clostridia photosynthetic bacteria (Feng et al., 2009; Risso et al., species exclusively use (Re)-CS to synthesize citrate 2008), it is suggested that the citramalate-dependent (Li et al., 2007). These reports, together with our pathway is much more widespread than previously work on heliobacteria, suggest a possible evolutionary recognized (Howell et al., 1999). Compared to the link between heliobacteria and Clostridia, both of threonine-dependent pathway, it is more efficient to which are