Abstract
The cellulolytic bacterium Clostridium thermocellum is a promising candidate for lignocellulosic biofuel production, however ethanol titer needs to be improved for commercialization. To understand the factors limiting ethanol titer in C. thermocellum, we developed a cell-free extract reaction (CFER) system. We demonstrated that 15 mM cellobiose could be converted, in vitro, to 25 mM ethanol, and that this reaction functions both at thermophilic (55C) and mesophilic (37C) temperatures. Although the yield was similar to that produced by whole cells, the rate was much slower (~0.5 vs 12 mM/h). In order to reliably quantify metabolites, rapid CFER quenching is necessary. Among the methods tested, filtration with a 3 kDa molecular weight cutoff filter proved to be the most effective. Metabolomic analysis revealed high levels of glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) in the CFER, identifying potential rate limiting enzymes downstream of F6P. NADH was also found to accumulate in the CFER, suggesting NADH recycling rate-limiting. We used two complementary strategies to identify enzymes that limit metabolic flux, including feeding different substrates and supplementing with exogenous enzymes. In the enzyme addition experiment, the largest improvement was observed with the addition of yeast alcohol dehydrogenase (ADH), indicating a limitation at that reaction. The development of a CFER system for C. thermocellum, combined with detailed measurements of intermediate metabolites, allowed us to directly observe the metabolism of this organism, and suggested several potential metabolic engineering interventions for increasing ethanol titer. This demonstrates a technique that may be of general use for metabolic engineering in non-model organisms.
Highlights
Clostridium thermocellum is a cellulolytic thermophilic bacterium that is a good candidate organism for conversion of lignocellulosic biomass to biofuels, such as ethanol, through consolidated bioprocessing (CBP)
The strain used for the cell-free extract reaction (CFER) is an engineered strain of Clostridium thermocellum DSM1313: LL1570, which was engineered for increased ethanol production by introducing genes from Thermoanaerobacterium saccharolyticum, including adhA, nfnAB, adhE, ferredoxin, and pfor; and deletion of native pfor genes
Metabolite Measurement by Liquid chromatography-mass spectrometry (LC-MS) To prepare for Liquid chromatography (LC)-MS analysis, samples were thawed on ice, diluted 1:10 in Solvent A [97:3 water:methanol with 10 mM tributylamine (TBA), adjusted to pH 8.2 with ∼10 mM acetic acid] and injected to the LC-MS system, consisting of a Thermo Scientific Vanquish UHPLC coupled by heated electrospray ionization (HESI; negative mode) to a hybrid quadrupole-highresolution mass spectrometer (Q Exactive Orbitrap, Thermo Scientific) for detection of targeted compounds based on their accurate masses and retention times
Summary
Clostridium thermocellum is a cellulolytic thermophilic bacterium that is a good candidate organism for conversion of lignocellulosic biomass to biofuels, such as ethanol, through consolidated bioprocessing (CBP). Three key enzymes of the pathway: hexokinase (HK), phosphofructokinase (PFK), and phosphoglycerate kinase (PGK) use different cofactors (PPi/Pi or GTP/GDP) instead of the (ATP/ADP) pair usually associated with these reactions. Many genetic modifications are slow, in non-model organisms These challenges motivate the search for other tools, including the use of cell-free extract reaction systems. The use of cell-free systems to characterize physiology and prototype metabolic engineering strategies is impactful in non-model organisms, like thermophilic bacteria or obligate anaerobes, where genetic tools would otherwise limit progress. For this reason, we set out to develop a cell-free system in a thermophilic obligate anaerobe, C. thermocellum
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