A novel hexameric NADP+ -reducing [FeFe] hydrogenase from Moorella thermoacetica.

Acetogenic bacteria such as the thermophilic anaerobic model organism Moorella thermoacetica reduce CO2 with H2 as a reductant via the Wood-Ljungdahl pathway (WLP). The enzymes of the WLP of M. thermoacetica require NADH, NADPH, and reduced ferredoxin as reductants. Whereas an electron-bifurcating ferredoxin- and NAD+ -reducing hydrogenase HydABC had been described, the enzyme that reduces NADP+ remained to be identified. A likely candidate is the HydABCDEF hydrogenase from M. thermoacetica. Genes encoding for the HydABCDEF hydrogenase are expressed during growth on glucose and dimethyl sulfoxide (DMSO), an alternative electron acceptor in M. thermoacetica, whereas expression of the genes hydABC encoding for the electron-bifurcating hydrogenase is downregulated. Therefore, we have purified the hydrogenase from cells grown on glucose and DMSO to apparent homogeneity. The enzyme had six subunits encoded by hydABCDEF and contained 58 mol of iron and 1 mol of FMN. The enzyme reduced methyl viologen with H2 as reductant and of the physiological acceptors tested, only NADP+ was reduced. Electron bifurcation with pyridine nucleotides and ferredoxin was not observed. H2 -dependent NADP+ reduction was optimal at pH 8 and 60 °C; the specific activity was 8.5 U·mg-1 and the Km for NADP+ was 0.086 mm. Cell suspensions catalyzed H2 -dependent DMSO reduction, which is in line with the hypothesis that the NADP+ -reducing hydrogenase HydABCDEF is involved in electron transfer from H2 to DMSO.

Moorella caeni sp. nov., isolated from thermophilic anaerobic sludge from a methanol-fed reactor.

Strain AMPT has been previously suggested as a strain of the species Moorella thermoacetica Jiang et al. 2009 (based on the high 16S rRNA gene identity, 98.3 %). However, genome-based phylogenetic analysis of strain AMPT reveals that this bacterium is in fact a novel species of the genus Moorella. Genome relatedness indices between strain AMPT and Moorella thermoacetica DSM 521T were below the minimum threshold values required to consider them members of the same species (digital DNA-DNA hybridization, 52.2 % (<70%); average nucleotide identity, 93.2 % (<95%)). Based on phylogenetic and phenotypic results we recommend that strain AMPT (DSM 21394T=JCM 35360T) should be classified as representing new species, for which we propose the name Moorella caeni sp. nov.

Enhancing acetone production from H2 and CO2 using supplemental electron acceptors in an engineered Moorella thermoacetica.

Acetogens grow autotrophically and use hydrogen (H2) as the energy source to fix carbon dioxide (CO2). This feature can be applied to gas fermentation, contributing to a circular economy. A challenge is the gain of cellular energy from H2 oxidation, which is substantially low, especially when acetate formation coupled with ATP production is diverted to other chemicals in engineered strains. Indeed, an engineered strain of the thermophilic acetogen Moorella thermoacetica that produces acetone lost autotrophic growth on H2 and CO2. We aimed to recover autotrophic growth and enhance acetone production, in which ATP production was assumed to be a limiting factor, by supplementing with electron acceptors. Among the four selected electron acceptors, thiosulfate and dimethyl sulfoxide (DMSO) enhanced both bacterial growth and acetone titers. DMSO was the most effective and was further analyzed. We showed that DMSO supplementation enhanced intracellular ATP levels, leading to increased acetone production. Although DMSO is an organic compound, it functions as an electron acceptor, not a carbon source. Thus, supplying electron acceptors is a potential strategy to complement the low ATP production caused by metabolic engineering and to improve chemical production from H2 and CO2.

Moorella sulfitireducens sp. nov., a thermophilic anaerobic bacterium isolated from a terrestrial thermal spring.

In hydrothermal ecosystems, the dissolution of sulfur dioxide in water results in the formation of sulfite, which can be used in microbial metabolism. A limited number of thermophiles have been isolated using sulfite as an electron acceptor. From a terrestrial thermal spring, Sakhalin Island, Russia, we isolated a thermophilic anaerobic bacterium (strain SLA38T). Cells of strain SLA38T were spore-forming straight rods. Growth was observed at temperatures 45-65 °C (optimum at 60 °C) and pH 5.5-9.0 (optimum at pH 6.5-7.0). The novel isolate was capable of anaerobic respiration with sulfite, thiosulfate, fumarate and perchlorate or fermentative growth. Strain SLA38T utilized glycerol, lactate, pyruvate and yeast extract. It grew lithoautotrophically on carbon monoxide with thiosulfate as electron acceptor, producing acetate. The genome size of the isolate was 2.9 Mbp and genomic DNA G + C content was 53.6 mol%. Analysis of the 16S rRNA gene sequences revealed that strain SLA38T belongs to the genus Moorella. Based on the physiological features and phylogenetic analysis, we propose to assign strain SLA38T to a new species of the genus Moorella, as Moorella sulfitireducens sp. nov. The type strain is SLA38T (= DSM 111068T = VKM B-3584T).

Energy-conserving dimethyl sulfoxide reduction in the acetogenic bacterium Moorella thermoacetica.

Moorella thermoacetica is one of the well-studied thermophilic acetogenic bacteria. It grows by oxidation of organic substrates, CO or H2 coupled to CO2 reduction to acetate. Here, we describe that M. thermoacetica can also use dimethyl sulfoxide as terminal electron acceptor. Growth of M. thermoacetica on glucose or H2  + CO2 was stimulated by dimethyl sulfoxide (DMSO). Membranes showed a DMSO reductase activity, that was induced by growing cells in presence of DMSO. The enzyme used reduced anthraquinone-2,6-disulfonate, benzyl- and methyl viologen as electron donor, but not NAD(P)H. Activity was highest at pH 5 and 60°C, the Km for DMSO was 2.4 mM. Potential DMSO reductase subunits were identified by peptide mass fingerprinting; they are encoded in a genomic region that contains three potential dmsA genes, three dmsB genes and one dmsC gene. Transcriptome analysis revealed that two different dmsAB gene clusters were induced in the presence of DMSO. The function of these two and their predicted biochemical features are discussed. In addition, the data are in line with the hypothesis that M. thermoacetica can use DMSO alongside CO2 as electron acceptor and DMSO reduction is catalysed by an energy-conserving, membrane-bound electron transport chain with DMSO as final electron acceptor.

The Critical Role of Environmental Synergies in the Creation of Bionanohybrid Microbes.

A wide range of bacteria can synthesize surface-associated nanoparticles (SANs) through exogenous metal ions reacting with sulfide produced via cysteine metabolism, resulting in the emergence of a biological-nanoparticle hybrid (bionanohybrid). The attached nanoparticles may couple to extracellular electron transfer, facilitating de novo photoelectrochemical processes. While SAN-cell coupling in hybrid organisms is opening a range of biotechnological possibilities, observation of bionanohybrids in nature is not commonly reported and their lab-based behavior remains difficult to control. We describe the critical role environmental synergy (microbial growth stage, cell densities, cysteine, and exogenous metal concentrations) plays in controlling the form and occurrence of Escherichia coli and Moorella thermoacetica bionanohybrids. SAN development depends on an appropriate cell density to metal ratio, with too few cells resulting in nanoparticle suppression through cytotoxicity or inhibition of cysteine conversion, and with too many cells diluting the number and size of particles produced. This cell number is governed by the concentration of cysteine present, which acts to protect the cells from metal ion toxicity. Exposing cells to metal and cysteine during the lag phase leads to SAN development, whereas cells in the exponential growth phase predominantly produce dispersed nanoparticles. Applying these principles more broadly, E. coli is shown to biosynthesize composite Bi/Cu sulfide SANs, and Clostridioides difficile can be coaxed into a bionanohybrid lifestyle by fine-tuning the cysteine dosage. Bionanohybrids maintain a remarkable ability for binary fission and sustained growth, opening doors to the production of SANs tailored to specific technological functions. IMPORTANCE Some bacteria can produce nanoscale-sized particles, which remain attached to the surface of the organism. The surface association of these nanoparticles creates a new mode of interaction between the microbe's environment and its internal cellular function, giving rise to a new hybrid lifeform, a biological nanoparticle hybrid (bionanohybrid). These hybrid organisms gain new or enhanced biological functions, and thus their creation opens a wide range of biotechnological possibilities. Despite this potential, the fundamental controls on bionanohybrid formation and occurrence remain poorly constrained. In this study, Escherichia coli K-12, Moorella thermoacetica, and Clostridioides difficile were used to test the combined influences of the growth phase, cell density, cysteine dose, and metal concentration in determining single and composite metal sulfide surface-associated nanoparticle production. The significance of this study is that it defined the critical synergies controlling nanoparticle formation on bacterial cell surfaces, unlocking the potential for bionanohybrid applications in a range of organisms.

Characterization of the nickel-inserting cyclometallase LarC from Moorella thermoacetica and identification of a cytidinylylated reaction intermediate.

LarC catalyzes the CTP-dependent insertion of nickel ion into pyridinium-3,5-bisthiocarboxylic acid mononucleotide (P2TMN), the final biosynthetic step for generating the nickel-pincer nucleotide (NPN) enzyme cofactor. In this study, we characterized a LarC homolog from Moorella thermoacetica (LarCMt) and characterized selected properties of the protein. We ruled out the hypothesis that enzyme inhibition by its product pyrophosphate accounts for its apparent single-turnover activity. Most notably, we identified a cytidinylylated-substrate intermediate that is formed during the reaction of LarCMt. Selected LarCMt variants with substitutions at the predicted CTP-binding site retained substantial amounts of activity, but exhibited greatly reduced levels of the CMP-P2TMN intermediate. In contrast, enhanced amounts of the CMP-P2TMN intermediate were generated when using LarCMt from cells grown on medium without supplemental nickel. On the basis of these results, we propose a functional role for CTP in the unprecedented nickel-insertase reaction during NPN biosynthesis.

Insights into the Structure and Protein Composition of Moorella thermoacetica Spores Formed at Different Temperatures.

The bacterium Moorella thermoacetica produces the most heat-resistant spores of any spoilage-causing microorganism known in the food industry. Previous work by our group revealed that the resistance of these spores to wet heat and biocides was lower when spores were produced at a lower temperature than the optimal temperature. Here, we used electron microcopy to characterize the ultrastructure of the coat of the spores formed at different sporulation temperatures; we found that spores produced at 55 °C mainly exhibited a lamellar inner coat tightly associated with a diffuse outer coat, while spores produced at 45 °C showed an inner and an outer coat separated by a less electron-dense zone. Moreover, misarranged coat structures were more frequently observed when spores were produced at the lower temperature. We then analyzed the proteome of the spores obtained at either 45 °C or 55 °C with respect to proteins putatively involved in the spore coat, exosporium, or in spore resistance. Some putative spore coat proteins, such as CotSA, were only identified in spores produced at 55 °C; other putative exosporium and coat proteins were significantly less abundant in spores produced at 45 °C. Altogether, our results suggest that sporulation temperature affects the structure and protein composition of M. thermoacetica spores.

Autotrophic growth and ethanol production enabled by diverting acetate flux in the metabolically engineered Moorella thermoacetica.

Gas fermentation is a promising biological process for the conversion of CO2 or syngas into valuable chemicals. Homoacetogens are microorganisms growing autotrophically using CO2 and H2 or CO and metabolizing them to form acetate coupled with energy conservation. The challenge in the metabolic engineering of the homoacetogens is divergence of the acetate formation, whose intermediate is acetyl-CoA, to a targeted chemical with sufficient production of adenosine triphosphate (ATP). In this study, we report that an engineered strain of the thermophilic homoacetogen Moorella thermoacetica, in which a pool of acetyl-CoA is diverted to ethanol without ATP production, can maintain autotrophic growth on syngas. We estimated the ATP production in the engineered strains under different gaseous compositions by considering redox-balanced metabolism for ethanol and acetate formation. The culture test showed that the combination of retaining a level of acetate production and supplying the energy-rich CO allowed maintenance of the autotrophic growth during ethanol production. In contrast, autotrophy was collapsed by complete elimination of the acetate pathway or supplementation of H2-CO2. We showed that the intracellular level of ATP was significantly lowered on H2-CO2 in consistent with the incompetence. In the meantime, the complete disruption of the acetate pathway resulted in the redox imbalance to produce ethanol from CO, albeit a small loss in the ATP production. Thus, preservation of a fraction of acetate formation is required to maintain sufficient ATP and balanced redox in CO-containing gases for ethanol production.

Anaerobic fermentation metabolism of Moorella thermoacetica inhibited by copper nanoparticles: Comprehensive analyses of transcriptional response and enzyme activity.

Engineered nanoparticles are observed to be released into the environment and ended up in wastewater treatment plants. It has been reported that these nanoparticles in sewage might have a toxic effect on microorganisms, and thus affect anaerobic microbial fermentation. However, the mechanisms involved in nanoparticles-induced effects on the anaerobic acidification process and its related bacterial metabolism are still unclear. This work indicated that copper nanoparticles (Cu NPs) were able to cause cell membrane oxidative damage and inhibit the growth and metabolism of Moorella thermoacetica (a model acetogen). The OD600 and acetic acid production of M. thermoacetica in the presence of 1 mg/L of Cu NPs were decreased to 29.2% and 40.7% of the control, respectively. The key mechanism of the inhibitory effect was governed by the fact that Cu NPs significantly reduced the glucose consumption, and led to the decreased pyruvate metabolism levels. Additionally, Cu NPs inhibited the gene expressions and catalytic activities of the key enzymes related to acetic acid production. It was identified that the relative activities of phosphofructokinase, pyruvate kinase, phosphotransacetylase, and acetate kinase of M. thermoacetica in the presence of 1 mg/L of Cu NPs decreased to only 70.1%, 69.3%, 50.1%, and 65.2% of the control, respectively. These results demonstrated that the release of Cu NPs in the environment could pose risks to anaerobic fermentation processes via regulating microbial transcriptional response and enzyme activity.

Compost Samples from Different Temperature Zones as a Model to Study Co-occurrence of Thermophilic and Psychrophilic Bacterial Population: a Metagenomics Approach.

In this study, using a metagenomic approach, we explore the bacterial diversity of compost sites categorized based on their ambient temperatures. The two sites were Reckong Peo in the lower Himalayas and Tambaram in the southern region of the country, namely, CPR and CT. Following assembly of the raw reads from shotgun metagenomics, similarity hits were generated using NCBI BLAST + and SILVA database. A total of 1463 and 1483 species were annotated from CPR and CT. A species-level annotation was performed using a python-based literature search pipeline revealing their growth characteristics. Thermophiles Thermomonospora curvata and Thermus scotoductus were among the prominent species in CT. CPR too was seen abundant with Acidothermus cellulolyticus and Moorella thermoacetica, constituting 10% of the population. Nearly 3% of the identified species in the site CPR were psychrophilic. Although found higher in CPR, psychrophilic species were identified in CT too. Flavobacterium and Psychrobacter spp. were present in both sites without any significant changes in their relative distribution contrary to the thermophilic species abundance (z = - 4.3). Akin to the sequenced samples, database-derived metagenomes also showed similar distribution of thermophiles and psychrophiles. Identifying such peculiar prevalence of extremophiles can be central to understanding extended growth temperatures.

Cysteine: an overlooked energy and carbon source.

Biohybrids composed of microorganisms and nanoparticles have emerged as potential systems for bioenergy and high-value compound production from CO2 and light energy, yet the cellular and metabolic processes within the biological component of this system are still elusive. Here we dissect the biohybrid composed of the anaerobic acetogenic bacterium Moorella thermoacetica and cadmium sulphide nanoparticles (CdS) in terms of physiology, metabolism, enzymatics and transcriptomic profiling. Our analyses show that while the organism does not grow on L-cysteine, it is metabolized to acetate in the biohybrid system and this metabolism is independent of CdS or light. CdS cells have higher metabolic activity, despite an inhibitory effect of Cd2+ on key enzymes, because of an intracellular storage compound linked to arginine metabolism. We identify different routes how cysteine and its oxidized form can be innately metabolized by the model acetogen and what intracellular mechanisms are triggered by cysteine, cadmium or blue light.

Organic waste streams as feedstock for the production of high volume-low value products.

Valorisation of organic wastes to produce industrially relevant commodity products is a sustainable, cost-effective and viable alternative providing a green platform for chemical production while simultaneously leading to waste disposal management. In the present study, organic wastes such as agricultural residue-derived sugars, oilseed meals, poultry waste and molasses were used for substituting expensive organic fermentation medium components. Moorella thermoacetica and Aurantiochytrium limacinum were adapted on these waste-derived hydrolysates to produce high volume-low value products such as bio-acetic acid (80% theoretical yields) and oil-rich fish/animal feed (more than 85% dry cell weight as compared with conventional nutrient sources) respectively. Use of these waste-derived nutrients led to ~ 75% and ~ 90% reduction in media cost for acetic acid and oil-rich biomass production respectively as compared with that of traditionally used high-priced medium components. The strategy will assist in the cost reduction for high volume-low value products while also ensuring waste recovery.

Gas channel rerouting in a primordial enzyme: Structural insights of the carbon-monoxide dehydrogenase/acetyl-CoA synthase complex from the acetogen Clostridium autoethanogenum.

Clostridium autoethanogenum, the bacterial model for biological conversion of waste gases into biofuels, grows under extreme carbon-monoxide (CO) concentrations. The strictly anaerobic bacterium derives its entire cellular energy and carbon from this poisonous gas, therefore requiring efficient molecular machineries for CO-conversion. Here, we structurally and biochemically characterized the key enzyme of the CO-converting metabolism: the CO-dehydrogenase/Acetyl-CoA synthase (CODH/ACS). We obtained crystal structures of natively isolated complexes from fructose-grown and CO-grown C. autoethanogenum cultures. Both contain the same isoforms and if the overall structure adopts the classic α2ß2 architecture, comparable to the model enzyme from Moorella thermoacetica, the ACS binds a different position on the CODH core. The structural characterization of a proteolyzed complex and the conservation of the binding interface in close homologs rejected the possibility of a crystallization artefact. Therefore, the internal CO-channeling system, critical to transfer CO generated at the C-cluster to the ACS active site, drastically differs in the complex from C. autoethanogenum. The 1.9-Å structure of the CODH alone provides an accurate picture of the new CO-routes, leading to the ACS core and reaching the surface. Increased gas accessibility would allow the simultaneous CO-oxidation and acetyl-CoA production. Biochemical experiments showed higher flexibility of the ACS subunit from C. autoethanogenum compared to M. thermoacetica, albeit monitoring similar CO-oxidation and formation rates. These results show a reshuffling of internal CO-tunnels during evolution of these Firmicutes, putatively leading to a bidirectional complex that ensure a high flux of CO-conversion toward energy conservation, acting as the main cellular powerplant.

Negative-Stain Electron Microscopy Reveals Dramatic Structural Rearrangements in Ni-Fe-S-Dependent Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase.

The Ni-Fe-S-containing A-cluster of acetyl-coenzyme A (CoA) synthase (ACS) assembles acetyl-CoA from carbon monoxide (CO), a methyl group (CH3+), and CoA. To accomplish this feat, ACS must bind CoA and interact with two other proteins that contribute the CO and CH3+, respectively: CO dehydrogenase (CODH) and corrinoid Fe-S protein (CFeSP). Previous structural data show that, in the model acetogen Moorella thermoacetica, domain 1 of ACS binds to CODH such that a 70-Å-long internal channel is created that allows CO to travel from CODH to the A-cluster. The A-cluster is largely buried and is inaccessible to CFeSP for methylation. Here we use electron microscopy to capture multiple snapshots of ACS that reveal previously uncharacterized domain motion, forming extended and hyperextended structural states. In these structural states, the A-cluster is accessible for methylation by CFeSP.

Insights into the Chemical Reactivity in Acetyl-CoA Synthase.

The biological synthesis of acetyl-coenzyme A (acetyl-CoA), catalyzed by acetyl-CoA synthase (ACS), is of biological significance and chemical interest acting as a source of energy and carbon. The catalyst contains an unusual hexa-metal cluster with two nickel ions and a [Fe4S4] cluster. DFT calculations have been performed to investigate the ACS reaction mechanism starting from three different oxidation states (+2, +1, and 0) of Nip, the nickel proximal to [Fe4S4]. The results indicate that the ACS reaction proceeds first through a methyl radical transfer from cobalamin (Cbl) to Nip randomly accompanying with the CO binding. After that, C-C bond formation occurs between the Nip-bound methyl and CO, forming Nip-acetyl. The substrate CoA-S- then binds to Nip, allowing C-S bond formation between the Nip-bound acetyl and CoA-S-. Methyl transfer is rate-limiting with a barrier of ∼14 kcal/mol, which does not depend on the presence or absence of CO. Both the Nip2+ and Nip1+ states are chemically capable of catalyzing the ACS reaction independent of the state (+2 or +1) of the [Fe4S4] cluster. The [Fe4S4] cluster is not found to affect the steps of methyl transfer and C-C bond formation but may be involved in the C-S bond formation depending on the detailed mechanism chosen. An ACS active site containing a Nip(0) state could not be obtained. Optimizations always led to a Nip1+ state coupled with [Fe4S4]1+. The calculations show a comparable activity for Nip1+/[Fe4S4]1+, Nip1+/[Fe4S4]2+, and Nip2+/[Fe4S4]2+. The results here give significant insights into the chemistry of the important ACS reaction.

CobT and BzaC catalyze the regiospecific activation and methylation of the 5-hydroxybenzimidazole lower ligand in anaerobic cobamide biosynthesis.

Vitamin B12 and other cobamides are essential cofactors required by many organisms and are synthesized by a subset of prokaryotes via distinct aerobic and anaerobic routes. The anaerobic biosynthesis of 5,6-dimethylbenzimidazole (DMB), the lower ligand of vitamin B12, involves five reactions catalyzed by the bza operon gene products, namely the hydroxybenzimidazole synthase BzaAB/BzaF, phosphoribosyltransferase CobT, and three methyltransferases, BzaC, BzaD, and BzaE, that conduct three distinct methylation steps. Of these, the methyltransferases that contribute to benzimidazole lower ligand diversity in cobamides remain to be characterized, and the precise role of the bza operon protein CobT is unclear. In this study, we used the bza operon from the anaerobic bacterium Moorella thermoacetica (comprising bzaA-bzaB-cobT-bzaC) to examine the role of CobT and investigate the activity of the first methyltransferase, BzaC. We studied the phosphoribosylation catalyzed by MtCobT and found that it regiospecifically activates 5-hydroxybenzimidazole (5-OHBza) to form the 5-OHBza-ribotide (5-OHBza-RP) isomer as the sole product. Next, we characterized the domains of MtBzaC and reconstituted its methyltransferase activity with the predicted substrate 5-OHBza and with two alternative substrates, the MtCobT product 5-OHBza-RP and its riboside derivative 5-OHBza-R. Unexpectedly, we found that 5-OHBza-R is the most favored MtBzaC substrate. Our results collectively explain the long-standing observation that the attachment of the lower ligand in anaerobic cobamide biosynthesis is regiospecific. In conclusion, we validate MtBzaC as a SAM:hydroxybenzimidazole-riboside methyltransferase (HBIR-OMT). Finally, we propose a new pathway for the synthesis and activation of the benzimidazolyl lower ligand in anaerobic cobamide biosynthesis.

Solar-Powered Organic Semiconductor-Bacteria Biohybrids for CO2 Reduction into Acetic Acid.

An organic semiconductor-bacteria biohybrid photosynthetic system is used to efficiently realize CO2 reduction to produce acetic acid with the non-photosynthetic bacteria Moorella thermoacetica. Perylene diimide derivative (PDI) and poly(fluorene-co-phenylene) (PFP) were coated on the bacteria surface as photosensitizers to form a p-n heterojunction (PFP/PDI) layer, affording higher hole/electron separation efficiency. The π-conjugated semiconductors possess excellent light-harvesting ability and biocompatibility, and the cationic side chains of organic semiconductors could intercalate into cell membranes, ensuring efficient electron transfer to bacteria. Moorella thermoacetica can thus harvest photoexcited electrons from the PFP/PDI heterojunction, driving the Wood-Ljungdahl pathway to synthesize acetic acid from CO2 under illumination. The efficiency of this organic biohybrid is about 1.6 %, which is comparable to those of reported inorganic biohybrid systems.

Ethanol yield and sugar usability in thermophilic ethanol production from lignocellulose hydrolysate by genetically engineered Moorella thermoacetica.

Bioconversion from inexpensive renewable resource, such as biomass, to liquid fuel is one of the promising technologies to reduce the use of petroleum. We previously reported the genetically engineered Moorella thermoacetica could produce ethanol from the lignocellulosic feedstock. However, it was still unclear which carbon source in the substrate was preferentially consumed to produce ethanol. To identify the hierarchy of the sugar utilization during ethanol fermentation of this strain, we analyzed the sugar composition of lignocellulosic feedstock, and consumption rate of sugars during the fermentation process. The hydrolysates after acid pretreatment and enzymatic saccharification contained glucose, xylose, galactose, arabinose, and mannose. Time course data suggested that xylose was the most preferred carbon source among those sugars during ethanol fermentation. Ethanol yield was 0.40 ± 0.06 and 0.40 ± 0.12 g/g-total sugar, from lignocellulosic hydrolysates of Japanese cedar (Cryptomeria japonica) and rice straw (Oryza sativa), respectively. The results demonstrated that the genetically engineered M. thermoacetica is a promising candidate for thermophilic ethanol fermentation of lignocellulosic feedstocks, especially hemicellulosic sugars.

Genome-wide systematic identification of methyltransferase recognition and modification patterns.

Genome-wide analysis of DNA methylation patterns using single molecule real-time DNA sequencing has boosted the number of publicly available methylomes. However, there is a lack of tools coupling methylation patterns and the corresponding methyltransferase genes. Here we demonstrate a high-throughput method for coupling methyltransferases with their respective motifs, using automated cloning and analysing the methyltransferases in vectors carrying a strain-specific cassette containing all potential target sites. To validate the method, we analyse the genomes of the thermophile Moorella thermoacetica and the mesophile Acetobacterium woodii, two acetogenic bacteria having substantially modified genomes with 12 methylation motifs and a total of 23 methyltransferase genes. Using our method, we characterize the 23 methyltransferases, assign motifs to the respective enzymes and verify activity for 11 of the 12 motifs.