Engineering the cellulolytic bacterium, Clostridium thermocellum, to co-utilize hemicellulose.

Consolidated bioprocessing (CBP) of lignocellulosic biomass holds promise to realize economic production of second-generation biofuels/chemicals, and Clostridium thermocellum is a leading candidate for CBP due to it being one of the fastest degraders of crystalline cellulose and lignocellulosic biomass. However, CBP by C. thermocellum is approached with co-cultures, because C. thermocellum does not utilize hemicellulose. When compared with a single-species fermentation, the co-culture system introduces unnecessary process complexity that may compromise process robustness. In this study, we engineered C. thermocellum to co-utilize hemicellulose without the need for co-culture. By evolving our previously engineered xylose-utilizing strain in xylose, an evolved clonal isolate (KJC19-9) was obtained and showed improved specific growth rate on xylose by ∼3-fold and displayed comparable growth to a minimally engineered strain grown on the bacteria's naturally preferred substrate, cellobiose. To enable full xylan deconstruction to xylose, we recombinantly expressed three different ß-xylosidase enzymes originating from Thermoanaerobacterium saccharolyticum into KJC19-9 and demonstrated growth on xylan with one of the enzymes. This recombinant strain was capable of co-utilizing cellulose and xylan simultaneously, and we integrated the ß-xylosidase gene into the KJC19-9 genome, creating the KJCBXint strain. The strain, KJC19-9, consumed monomeric xylose but accumulated xylobiose when grown on pretreated corn stover, whereas the final KJCBXint strain showed significantly greater deconstruction of xylan and xylobiose. This is the first reported C. thermocellum strain capable of degrading and assimilating hemicellulose polysaccharide while retaining its cellulolytic capabilities, unlocking significant potential for CBP in advancing the bioeconomy.

Establishing Butyribacterium methylotrophicum as a Platform Organism for the Production of Biocommodities from Liquid C1 Metabolites.

Using the Wood-Ljungdahl pathway, acetogens can nonphotosynthetically fix gaseous C1 molecules, preventing them from entering the atmosphere. Many acetogens can also grow on liquid C1 compounds such as formate and methanol, which avoid the storage and mass transfer issues associated with gaseous C1 compounds. Substrate redox state also plays an important role in acetogen metabolism and can modulate products formed by these organisms. Butyribacterium methylotrophicum is an acetogen known for its ability to synthesize longer-chained molecules such as butyrate and butanol, which have significantly higher values than acetate or ethanol, from one-carbon (C1) compounds. We explored B. methylotrophicum's C1 metabolism by varying substrates, substrate concentrations, and substrate feeding strategies to improve four-carbon product titers. Our results showed that formate utilization by B. methylotrophicum favored acetate production and methanol utilization favored butyrate production. Cofeeding of both substrates produced a high butyrate titer of 4 g/liter when methanol was supplied in excess to formate. Testing of formate feeding strategies, in the presence of methanol, led to further increases in the butyrate to acetate ratio. Mixotrophic growth of liquid and gaseous C1 substrates expanded the B. methylotrophicum product profile, as ethanol, butanol, and lactate were produced under these conditions. We also showed that B. methylotrophicum is capable of producing caproate, a six-carbon product, presumably through chain elongation cycles of the reverse ß-oxidation pathway. Furthermore, we demonstrated butanol production via heterologous gene expression. Our results indicate that both selection of appropriate substrates and genetic engineering play important roles in determining titers of desired products. IMPORTANCE Acetogenic bacteria can fix single-carbon (C1) molecules. However, improvements are needed to overcome poor product titers. Butyribacterium methylotrophicum can naturally ferment C1 compounds into longer-chained molecules such as butyrate alongside traditional acetate. Here, we show that B. methylotrophicum can effectively grow on formate and methanol to produce high titers of butyrate. We improved ratios of butyrate to acetate through adjusted formate feeding strategies and produced higher-value six-carbon molecules. We also expanded the B. methylotrophicum product profile with the addition of C1 gases, as the organism produced ethanol, butanol, and lactate. Furthermore, we developed a transformation protocol for B. methylotrophicum to facilitate genetic engineering of this organism for the circular bioeconomy.

CO2-fixing one-carbon metabolism in a cellulose-degrading bacterium Clostridium thermocellum.

Clostridium thermocellum can ferment cellulosic biomass to formate and other end products, including CO2 This organism lacks formate dehydrogenase (Fdh), which catalyzes the reduction of CO2 to formate. However, feeding the bacterium 13C-bicarbonate and cellobiose followed by NMR analysis showed the production of 13C-formate in C. thermocellum culture, indicating the presence of an uncharacterized pathway capable of converting CO2 to formate. Combining genomic and experimental data, we demonstrated that the conversion of CO2 to formate serves as a CO2 entry point into the reductive one-carbon (C1) metabolism, and internalizes CO2 via two biochemical reactions: the reversed pyruvate:ferredoxin oxidoreductase (rPFOR), which incorporates CO2 using acetyl-CoA as a substrate and generates pyruvate, and pyruvate-formate lyase (PFL) converting pyruvate to formate and acetyl-CoA. We analyzed the labeling patterns of proteinogenic amino acids in individual deletions of all five putative PFOR mutants and in a PFL deletion mutant. We identified two enzymes acting as rPFOR, confirmed the dual activities of rPFOR and PFL crucial for CO2 uptake, and provided physical evidence of a distinct in vivo "rPFOR-PFL shunt" to reduce CO2 to formate while circumventing the lack of Fdh. Such a pathway precedes CO2 fixation via the reductive C1 metabolic pathway in C. thermocellum These findings demonstrated the metabolic versatility of C. thermocellum, which is thought of as primarily a cellulosic heterotroph but is shown here to be endowed with the ability to fix CO2 as well.

Acetogenic production of 3-Hydroxybutyrate using a native 3-Hydroxybutyryl-CoA Dehydrogenase.

3-Hydroxybutyrate (3HB) is a product of interest as it is a precursor to the commercially produced bioplastic polyhydroxybutyrate. It can also serve as a platform for fine chemicals, medicines, and biofuels, making it a value-added product and feedstock. Acetogens non-photosynthetically fix CO2 into acetyl-CoA and have been previously engineered to convert acetyl-CoA into 3HB. However, as acetogen metabolism is poorly understood, those engineering efforts have had varying levels of success. 3HB, using acetyl-CoA as a precursor, can be synthesized by a variety of different pathways. Here we systematically compare various pathways to produce 3HB in acetogens and discover a native (S)-3-hydroxybutyryl-CoA dehydrogenase, hbd2, responsible for endogenous 3HB production. In conjunction with the heterologous thiolase atoB and CoA transferase ctfAB, hbd2 overexpression improves yields of 3HB on both sugar and syngas (CO/H2/CO2), outperforming the other tested pathways. These results uncovered a previously unknown 3HB production pathway, inform data from prior metabolic engineering efforts, and have implications for future physiological and biotechnological anaerobic research.

The Metabolism of Clostridium ljungdahlii in Phosphotransacetylase Negative Strains and Development of an Ethanologenic Strain.

The sustainable production of chemicals from non-petrochemical sources is one of the greatest challenges of our time. CO2 release from industrial activity is not environmentally friendly yet provides an inexpensive feedstock for chemical production. One means of addressing this problem is using acetogenic bacteria to produce chemicals from CO2, waste streams, or renewable resources. Acetogens are attractive hosts for chemical production for many reasons: they can utilize a variety of feedstocks that are renewable or currently waste streams, can capture waste carbon sources and covert them to products, and can produce a variety of chemicals with greater carbon efficiency over traditional fermentation technologies. Here we investigated the metabolism of Clostridium ljungdahlii, a model acetogen, to probe carbon and electron partitioning and understand what mechanisms drive product formation in this organism. We utilized CRISPR/Cas9 and an inducible riboswitch to target enzymes involved in fermentation product formation. We focused on the genes encoding phosphotransacetylase (pta), aldehyde ferredoxin oxidoreductases (aor1 and aor2), and bifunctional alcohol/aldehyde dehydrogenases (adhE1 and adhE2) and performed growth studies under a variety of conditions to probe the role of those enzymes in the metabolism. Finally, we demonstrated a switch from acetogenic to ethanologenic metabolism by these manipulations, providing an engineered bacterium with greater application potential in biorefinery industry.

Continuous hydrogen production during fermentation of alpha-cellulose by the thermophillic bacterium Clostridium thermocellum.

Continuous hydrogen (H2) production during fermentation of alpha-cellulose was established using the thermophillic, anaerobic bacterium Clostridium thermocellum ATCC 27405. The objectives of this work were to characterize growth of C. thermocellum, quantify H2 production and determine soluble end-product synthesis patterns during fermentation of a cellulosic substrate under continuous culture conditions. A 5 L working volume fermentor was established and growth experiments were maintained for over 3,000 h. Substrate concentrations were varied from 1 to 4 g/L and the feed was introduced with continuous nitrogen gas sparging to prevent clogging of the feed-line. The pH and temperature of the reactor were maintained at 7.0 and 600 degrees C, respectively, throughout the study. At concentrations above 4 g/L, the delivery of alpha-cellulose was impaired due to feed-line clogging and it became difficult to maintain a homogenous suspension. The highest total gas (H2 plus CO2) production rate, 56.6 mL L(-1) h(-1), was observed at a dilution rate of 0.042 h(-1) and substrate concentration of 4 g/L. Under these conditions, the H2 production rate was 5.06 mmol h(-1). Acetate and ethanol were the major soluble end-products, while lactate and formate were greatly reduced compared to production in batch cultures. Concentrations of all metabolites increased with increasing substrate concentration, with the exception of lactate. Despite a number of short-term electrical and mechanical failures during the testing period, the system recovered quickly, exhibiting substantial robustness. A carbon balance was completed to ensure that all end-products were accounted for, with final results indicating near 100% carbon recovery. This study shows that long-term, stable H2 production can be achieved during direct fermentation of an insoluble cellulosic substrate under continuous culture conditions.

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

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 products. We uncover that the Embden-Meyerhof-Parnas (EMP) pathway is the predominant glycolytic route whereas the Entner-Doudoroff (ED) pathway and oxidative pentose phosphate pathway are inactive. We also observe that C. thermocellum's TCA cycle is initiated by both Si- and Re-citrate synthase, and it is disconnected between 2-oxoglutarate and oxaloacetate in the oxidative direction; C. thermocellum uses a citramalate shunt to synthesize isoleucine; and both the one-carbon pathway and the malate shunt are highly active in this bacterium. To gain a quantitative understanding, we further formulate a fluxome map to quantify the metabolic fluxes through central metabolic pathways. This work represents the first global in vivo investigation of the principal carbon metabolism of C. thermocellum. Our results elucidate the unique structure of metabolic network in this cellulolytic bacterium and demonstrate the capability of isotope-assisted metabolite studies in understanding microbial metabolism of industrial interests.

Comparison of transcriptional profiles of Clostridium thermocellum grown on cellobiose and pretreated yellow poplar using RNA-Seq.

The anaerobic, thermophilic bacterium, Clostridium thermocellum, secretes multi-protein enzyme complexes, termed cellulosomes, which synergistically interact with the microbial cell surface and efficiently disassemble plant cell wall biomass. C. thermocellum has also been considered a potential consolidated bioprocessing (CBP) organism due to its ability to produce the biofuel products, hydrogen, and ethanol. We found that C. thermocellum fermentation of pretreated yellow poplar (PYP) produced 30 and 39% of ethanol and hydrogen product concentrations, respectively, compared to fermentation of cellobiose. RNA-seq was used to analyze the transcriptional profiles of these cells. The PYP-grown cells taken for analysis at the late stationary phase showed 1211 genes up-regulated and 314 down-regulated by more than two-fold compared to the cellobiose-grown cells. These affected genes cover a broad spectrum of specific functional categories. The transcriptional analysis was further validated by sub-proteomics data taken from the literature; as well as by quantitative reverse transcription-PCR (qRT-PCR) analyses of selected genes. Specifically, 47 cellulosomal protein-encoding genes, genes for 4 pairs of SigI-RsgI for polysaccharide sensing, 7 cellodextrin ABC transporter genes, and a set of NAD(P)H hydogenase and alcohol dehydrogenase genes were up-regulated for cells growing on PYP compared to cellobiose. These genes could be potential candidates for future studies aimed at gaining insight into the regulatory mechanism of this organism as well as for improvement of C. thermocellum in its role as a CBP organism.