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Isotope-Assisted Metabolite Analysis Sheds Light on Central Carbon Metabolism of a Model Cellulolytic Bacterium Clostridium Thermocellum

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Isotope-Assisted Metabolite Analysis Sheds Light on Central Carbon Metabolism of a Model Cellulolytic Bacterium Clostridium Thermocellum ORIGINAL RESEARCH published: 23 August 2018 doi: 10.3389/fmicb.2018.01947 Isotope-Assisted Metabolite Analysis Sheds Light on Central Carbon Metabolism of a Model Cellulolytic Bacterium Clostridium thermocellum Wei Xiong*, Jonathan Lo, Katherine J. Chou, Chao Wu, Lauren Magnusson, Tao Dong and PinChing Maness* National Renewable Energy Laboratory, Golden, CO, United States Cellulolytic bacteria have the potential to perform lignocellulose hydrolysis and fermentation simultaneously. The metabolic pathways of these bacteria, therefore, require more comprehensive and quantitative understanding. Using isotope tracer, gas chromatography-mass spectrometry, and metabolic flux modeling, we decipher the metabolic network of Clostridium thermocellum, a model cellulolytic bacterium which represents as an attractive platform for conversion of lignocellulose to dedicated Edited by: Ivan A. Berg, products. We uncover that the Embden–Meyerhof–Parnas (EMP) pathway is the Universität Münster, Germany predominant glycolytic route whereas the Entner–Doudoroff (ED) pathway and oxidative Reviewed by: pentose phosphate pathway are inactive. We also observe that C. thermocellum’s TCA Wolfgang Buckel, Philipps-Universität Marburg, cycle is initiated by both Si- and Re-citrate synthase, and it is disconnected between Germany 2-oxoglutarate and oxaloacetate in the oxidative direction; C. thermocellum uses a Wolfgang Eisenreich, citramalate shunt to synthesize isoleucine; and both the one-carbon pathway and the Technische Universität München, Germany malate shunt are highly active in this bacterium. To gain a quantitative understanding, *Correspondence: we further formulate a fluxome map to quantify the metabolic fluxes through central Wei Xiong metabolic pathways. This work represents the first global in vivo investigation of the [email protected] PinChing Maness principal carbon metabolism of C. thermocellum. Our results elucidate the unique [email protected] structure of metabolic network in this cellulolytic bacterium and demonstrate the capability of isotope-assisted metabolite studies in understanding microbial metabolism Specialty section: This article was submitted to of industrial interests. Microbial Physiology and Metabolism, Keywords: 13C-isotope tracer, cellulolytic bacteria, citrate synthase, glycolytic pathways, isoleucine biosynthesis, a section of the journal metabolic flux analysis, tricarboxylic acid cycle Frontiers in Microbiology Received: 23 May 2018 Accepted: 31 July 2018 INTRODUCTION Published: 23 August 2018 Citation: Lignocellulosic biomass is the most abundant bio-renewable resource on earth. It has the potential Xiong W, Lo J, Chou KJ, Wu C, to substitute the current sugar feedstocks for sustainable biofuels and biochemicals production. The Magnusson L, Dong T and Maness P use of lignocellulosic biomass has significant merits including improving net carbon and energy (2018) Isotope-Assisted Metabolite balances, reducing production cost, and avoiding the competition between food vs. fuel (Lynd Analysis Sheds Light on Central et al., 2005, 2008). However, effective application of lignocellulose in bio-refineries is currently Carbon Metabolism of a Model Cellulolytic Bacterium Clostridium impeded by biomass recalcitrance: its inherent resistance to depolymerization. A potential solution thermocellum. is consolidated bioprocessing (CBP), in which microbes of outstanding cellulolytic ability are Front. Microbiol. 9:1947. recruited for combined cellulose hydrolysis and fermentation without extra addition of cellulases doi: 10.3389/fmicb.2018.01947 (Lynd et al., 2005). Clostridium thermocellum is among the most attractive CBP host microbes. Frontiers in Microbiology | www.frontiersin.org 1 August 2018 | Volume 9 | Article 1947 Xiong et al. Central Carbon Metabolism in Clostridium thermocellum C. thermocellum is a Gram-positive, thermophilic, and strict It also showcases the capability of isotope-assisted metabolite anaerobic bacterium. By taking advantage of an extracellular analysis in complementing genome annotation with a more in- cellulase system called the cellulosome (Gold and Martin, depth understanding of microbial metabolism. 2007), C. thermocellum can degrade cellulose into soluble oligosaccharides. The latter can be further utilized by the cells and MATERIALS AND METHODS fermented to products of industrial interest (e.g., H2, formate, lactate, acetate, ethanol) (Ellis et al., 2012; Holwerda et al., 2014). Strains, Culture Conditions, and Medium Recent advances in genetic modification tools (Tripathi et al., C. thermocellum DSM 1313 derived strain hpt (Xiong et al., 2010; Argyros et al., 2011) further enable heterologous expression 2016) was grown anaerobically at 55◦C on 5 g/L glucose. We used of new functional pathways (e.g., pentose sugar utilization the DSM122-defined medium referred to as CTFUD minimum Xiong et al., 2018, isobutanol production Lin et al., 2015) into medium (Olson and Lynd, 2012). The rich medium was added the bacterium, making this CBP bacterium an attractive and with 1 g/L yeast extract. The growth medium was deoxygenated engineerable platform for biofuels or biochemicals production. by gassing with argon for 20 min and autoclaved before use. Applications of C. thermocellum in CBP require not only knowledge in molecular and genetic details of its cellulolytic Quantitative Analysis of Fermentation system but also global understanding of its carbon metabolism. Products Detailing the carbon fluxes from metabolic pathways in C. Cell growth was measured as a function of optical density by thermocellum will guide design principles for efficient conversion spectrophotometry (DU800; Beckman Coulter) at OD . An of lignocellulosic sugars into dedicated products. To date, there 600nm OD of 1 correlated to 1.04 g/L cell dry weight (R2 = 0.9918). have been a few published works describing the fermentation 600 Hydrogen gas was measured using a gas chromatograph (7890A pathways in C. thermocellum (Zhang and Lynd, 2005; Lu et al., GC; Agilent Technologies) equipped with a thermal conductivity 2006; Nataf et al., 2010; Olson et al., 2010; Deng et al., 2013; Zhou detector and a stainless-steel Supelco 60/80 Mol Sieve column et al., 2013), whereas metabolic characteristics of this species (6 ft × 1/8 in) with argon as the carrier gas. Peak areas were have not been comprehensively and quantitatively understood. compared with a standard curve. Glucose, lactate, formate, Although the availability of genome sequence of C. thermocellum acetate, and ethanol were measured by HPLC (1200 series; strains (DSM 1313 and ATCC 27405) has provided essential Agilent Technologies) with a mobile phase of 4 mM H SO at information and blue prints in terms of metabolic pathways, 2 4 0.6 mL/min flow rate using an Aminex HPX-87H column with a elaborate experimental validation is still required to fully realize Micro Guard Cation H Cartridge. The column temperature was its biotechnological applications. set to 55◦C. Recently, 13C-tracer and metabolic flux analysis has emerged as a powerful tool for validating genome annotation and 13C-Tracer Experiment this technique has enabled us to identify a unique CO2- 13 fixing one-carbon metabolic pathway in C. thermocellum (Xiong We adopted a steady-state labeling strategy. The C-labeling et al., 2016). Complementing classic biochemical approaches experiment was performed with different nutrient combinations: 13 + in understanding metabolism such as enzyme activity assay, (1) 5.56mM [U- C6] glucose (20%) 22.22 mM unlabeled 13 + gene mutagenesis and the use of specific inhibitors to key glucose (80%); (2) 20 mM C-bicarbonate 27.78mM (5 13 + metabolic steps, it provides a uniquely non-invasive approach g/L) glucose; (3) 20 mM C-formate 27.78mM glucose. 13 + to delineate the cellular metabolism in vivo. Here we use 13C- (4) 20 mM [U- C5] glutamate 27.78mM glucose. These labeled substrates as isotopic tracers to follow the operation of nutrient combinations are supplemented into the CTFUD C. thermocellum’s central carbon metabolism and other primary defined medium, respectively. For cell growth, C. thermocellum metabolic pathways directly in living cells. As a verification strains were inoculated into these media with a starting OD600 of of genomic information, we find that the Embden–Meyerhof– 0.05. When late log phase was reached (OD600 above 0.6), 3mL Parnas (EMP) pathway is the predominant glycolytic route of cultures was sampled. whereas the Entner–Doudoroff (ED) pathway and oxidative pentose phosphate pathway are inactive. We also observe that GC-MS and Isotopomer Analysis for C. thermocellum’s TCA cycle is initiated with both Si- and Re- Proteinogenic Amino Acids citrate synthase and it is broken between 2-oxoglutarate and The sample treatment and GC-MS analysis were done as oxaloacetate in the oxidative direction. C. thermocellum uses a previously reported (5), with a few modifications. Briefly, citramalate shunt to synthesize isoleucine; and the one-carbon 3 mL of sampled cultures was centrifuged at 10,000 × g for metabolism and the malate shunt are active in this bacterium. 1min, and the cell pellets were digested with 500 µL of To gain a quantitative insight into the newly identified 6M HCl at 105◦C for 12 h. The hydrolysate was dried under metabolic activities, we constructed a fluxomic model that nitrogen gas flow at 65◦C and dissolved in 50 µL of water- enables us to quantify the metabolic fluxes through the EMP free dimethylformamide. For the GC/MS measurement, the pathway, the nonoxidative pentose phosphate
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