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.

Genome-Based Comparison of All Species of the Genus Moorella, and Status of the Species Moorella thermoacetica and Moorella thermoautotrophica.

Fermentation of gases provides a promising opportunity for the production of biochemicals from renewable resources, which has resulted in a growing interest in acetogenic bacteria. Thermophilic organisms provide potential advantages for the fermentation of, e.g., syngas into for example volatile compounds, and the thermophiles Moorella thermoacetica and Moorella thermoautotrophica have become model organisms of acetogenic metabolism. The justification for the recognition of the closely related species M. thermoautotrophica has, however, recently been disputed. In order to expand knowledge on the genus, we have here genome sequenced a total of 12 different M. thermoacetica and M. thermoautotrophica strains. From the sequencing results, it became clear that M. thermoautotrophica DSM 1974T consists of at least two different strains. Two different strains were isolated in Lyngby and Ulm from a DSM 1974T culture obtained from the DSMZ (Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Brunswick, Germany). Phylogenetic analysis revealed a close relationship between all the sequenced genomes, suggesting that the two strains detected in the type strain of the species M. thermoautotrophica could not be distinguished at the species level from M. thermoacetica. Despite genetic similarities, differences in genomic features were observed between the strains. Differences in compounds that can serve as carbon and energy sources for selected strains were also identified. On the contrary, strain DSM 21394, currently still named M. thermoacetica, obviously represents a new Moorella species. In addition, based on genome analysis and comparison M. glycerini NMP, M. stamsii DSM 26217T, and M. perchloratireducens An10 cannot be distinguished at the species level. Thus, this comprehensive analysis provides a significantly increased knowledge of the genetic diversity of Moorella strains.

Application of the thermostable ß-galactosidase, BgaB, from Geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions.

Use of thermophilic organisms has a range of advantages, but the significant lack of engineering tools limits their applications. Here we show that ß-galactosidase from Geobacillus stearothermophilus (BgaB) can be applicable in a range of conditions, including different temperatures and oxygen concentrations. This protein functions both as a marker, promoting colony color development in the presence of a lactose analogue S-gal, and as a reporter enabling quantitative measurement by a simple colorimetric assay. Optimal performance was observed at 70 °C and pH 6.4. The gene was introduced into G. thermoglucosidans. The combination of BgaB expressed from promoters of varying strength with S-gal produced distinct black colonies in aerobic and anaerobic conditions at temperatures ranging from 37 to 60 °C. It showed an important advantage over the conventional ß-galactosidase (LacZ) and substrate X-gal, which were inactive at high temperature and under anaerobic conditions. To demonstrate the versatility of the reporter, a promoter library was constructed by randomizing sequences around -35 and -10 regions in a wild type groES promoter from Geobacillus sp. GHH01. The library contained 28 promoter variants and encompassed fivefold variation. The experimental pipeline allowed construction and measurement of expression levels of the library in just 4 days. This ß-galactosidase provides a promising tool for engineering of aerobic, anaerobic, and thermophilic production organisms such as Geobacillus species.

Thermodynamics and economic feasibility of acetone production from syngas using the thermophilic production host Moorella thermoacetica.

BACKGROUND: Syngas fermentation is a promising option for the production of biocommodities due to its abundance and compatibility with anaerobic fermentation. Using thermophilic production strains in a syngas fermentation process allows recovery of products with low boiling point from the off-gas via condensation. RESULTS: In this study we analyzed the production of acetone from syngas with the hypothetical production host derived from Moorella thermoacetica in a bubble column reactor at 60 °C with respect to thermodynamic and economic feasibility. We determined the cost of syngas production from basic oxygen furnace (BOF) process gas, from natural gas, and from corn stover and identified BOF gas as an economically interesting source for syngas. Taking gas-liquid mass transfer limitations into account, we applied a thermodynamics approach to derive the CO to acetone conversion rate under the process conditions. We estimated variable costs of production of 389 $/t acetone for a representative production scenario from BOF gas with costs for syngas as the main contributor. In comparison, the variable costs of production from natural gas- and corn stover-derived syngas were determined to be higher due to the higher feedstock costs (1724 and 2878 $/t acetone, respectively). CONCLUSION: We applied an approach of combining thermodynamic and economic assessment to analyze a hypothetical bioprocess in which the volatile product acetone is produced from syngas with a thermophilic microorganism. Our model allowed us to identify process metrics and quantify the variable production costs for different scenarios. Economical production of bulk chemicals is challenging, making rigorous thermodynamic/economic modeling critical before undertaking an experimental program and as an ongoing guide during the program. We intend this study to give an incentive to apply the demonstrated approach to other bioproduction processes.

Targeted mutagenesis of the Clostridium acetobutylicum acetone-butanol-ethanol fermentation pathway.

The production of the chemical solvents acetone and butanol by the bacterium Clostridium acetobutylicum was one of the first large-scale industrial processes to be developed, and in the first part of the last century ranked second in importance only to ethanol production. After a steep decline in its industrial use, there has been a recent resurgence of interest in the acetone-butanol-ethanol (ABE) fermentation process, with a particular emphasis on butanol production. In order to generate strains suitable for efficient use on an industrial scale, metabolic engineering is required to alter the AB ratio in favour of butanol, and eradicate the production of unwanted products of fermentation. Using ClosTron technology, a large-scale targeted mutagenesis in C. acetobutylicum ATCC 824 was carried out, generating a set of 10 mutants, defective in alcohol/aldehyde dehydrogenases 1 and 2 (adhE1, adhE2), butanol dehydrogenases A and B (bdhA, bdhB), phosphotransbutyrylase (ptb), acetate kinase (ack), acetoacetate decarboxylase (adc), CoA transferase (ctfA/ctfB), and a previously uncharacterised putative alcohol dehydrogenase (CAP0059). However, inactivation of the main hydrogenase (hydA) and thiolase (thl) could not be achieved. Constructing such a series of mutants is paramount for the acquisition of information on the mechanism of solvent production in this organism, and the subsequent development of industrial solvent producing strains. Unexpectedly, bdhA and bdhB mutants did not affect solvent production, whereas inactivation of the previously uncharacterised gene CAP0059 resulted in increased acetone, butanol, and ethanol formation. Other mutants showed predicted phenotypes, including a lack of acetone formation (adc, ctfA, and ctfB mutants), an inability to take up acids (ctfA and ctfB mutants), and a much reduced acetate formation (ack mutant). The adhE1 mutant in particular produced very little solvents, demonstrating that this gene was indeed the main contributor to ethanol and butanol formation under the standard batch culture conditions employed in this study. All phenotypic changes observed could be reversed by genetic complementation, with exception of those seen for the ptb mutant. This mutant produced around 100 mM ethanol, no acetone and very little (7 mM) butanol. The genome of the ptb mutant was therefore re-sequenced, together with its parent strain (ATCC 824 wild type), and shown to possess a frameshift mutation in the thl gene, which perfectly explained the observed phenotype. This finding reinforces the need for mutant complementation and Southern Blot analysis (to confirm single ClosTron insertions), which should be obligatory in all further ClosTron applications.