Modulation of sol mRNA expression by the long non-coding RNA Assolrna in Clostridium saccharoperbutylacetonicum affects solvent formation.

Solvents such as butanol are important platform chemicals and are often produced from petrochemical sources. Production of butanol and other compounds from renewable and sustainable resources can be achieved by solventogenic bacteria, such as the hyper-butanol producer Clostridium saccharoperbutylacetonicum. Its sol operon consists of the genes encoding butyraldehyde dehydrogenase, CoA transferase, and acetoacetate decarboxylase (bld, ctfA, ctfB, adc) and the gene products are involved in butanol and acetone formation. It is important to understand its regulation to further optimize the solvent production. In this study, a new long non-coding antisense transcript complementary to the complete sol operon, now called Assolrna, was identified by transcriptomic analysis and the regulatory mechanism of Assolrna was investigated. For this purpose, the promoter-exchange strain C. saccharoperbutylacetonicum ΔP asr ::P asr ** was constructed. Additionally, Assolrna was expressed plasmid-based under control of the native P asr promoter and the lactose-inducible P bgaL promoter in both the wild type and the promoter-exchange strain. Solvent formation was strongly decreased for all strains based on C. saccharoperbutylacetonicum ΔP asr ::P asr ** and growth could not be restored by plasmid-based complementation of the exchanged promoter. Interestingly, very little sol mRNA expression was detected in the strain C. saccharoperbutylacetonicum ΔP asr ::P asr ** lacking Assolrna expression. Butanol titers were further increased for the overexpression strain C. saccharoperbutylacetonicum [pMTL83151_asr_P bgaL ] compared to the wild type. These results suggest that Assolrna has a positive effect on sol operon expression. Therefore, a possible stabilization mechanism of the sol mRNA by Assolrna under physiological concentrations is proposed.

Anaerobic Production of Poly(3-hydroxybutyrate) and Its Precursor 3-Hydroxybutyrate from Synthesis Gas by Autotrophic Clostridia.

Anaerobic production of the biopolymer poly(3-hydroxybutyrate) (PHB) and the monomer 3-hydroxybutyrate (3-HB) was achieved using recombinant clostridial acetogens supplied with syn(thesis) gas as the sole carbon and energy source. 3-HB production was successfully accomplished by a new synthetic pathway containing the genes thlA (encoding thiolase A), ctfA/B (encoding CoA-transferase A/B), and bdhA (encoding (R)-3-hydroxybutyrate dehydrogenase). The respective recombinant Clostridium coskatii [p83_tcb] strain produced autotrophically 0.98 ± 0.12 mM and heterotrophically 21.7 ± 0.27 mM 3-HB. As a proof of concept, production of PHB was achieved using recombinant C. coskatii and Clostridium ljungdahlii strains expressing a novel synthetic PHB pathway containing the genes thlA (encoding thiolase A), hbd (encoding 3-hydroxybutyryl-CoA dehydrogenase), crt (encoding crotonase), phaJ (encoding (R)-enoyl-CoA hydratase), and phaEC (encoding PHA synthase). The strain C. coskatii [p83_PHB_Scaceti] synthesized heterotrophically 3.4 ± 0.29% PHB per cell dry weight (CDW) and autotrophically 1.12 ± 0.12% PHB per CDW.

Genome Sequence of the Acetogenic Bacterium Acetobacterium wieringae DSM 1911T.

Here, we report the draft genome sequence of Acetobacterium wieringae DSM 1911T, an anaerobic, autotrophic, acetogenic, d,l-lactate-utilizing bacterium. The genome consists of a chromosome (3.88 Mb) and 3,620 predicted protein-encoding genes.

Genome Sequence of the Poly-3-Hydroxybutyrate Producer Clostridium acetireducens DSM 10703.

Here, we report the genome sequence of Clostridium acetireducens (DSM 10703T), a strictly anaerobic bacterium capable of fermenting acetate and leucine to butyrate, isovalerate, and poly-3-hydroxybutyrate. The draft genome consists of a circular chromosome with a size of 2.4 Mb and harbors 2,239 predicted protein-encoding genes.

Genome Sequence of the Acetogenic Bacterium Butyribacterium methylotrophicum DSM 3468T.

Butyribacterium methylotrophicum DSM 3468T is an acetogenic methylotrophic, anaerobic, carbon monoxide-oxidizing bacterium that produces acetate, butyrate, and butanol. The genome consists of a single chromosome (4.3 Mb) and harbors 3,989 predicted protein-encoding genes.

Cap0037, a Novel Global Regulator of Clostridium acetobutylicum Metabolism.

An operon comprising two genes, CA_P0037 and CA_P0036, that encode proteins of unknown function that were previously shown to be highly expressed in acidogenic cells and repressed in solventogenic and alcohologenic cells is located on the pSOL1 megaplasmid of Clostridium acetobutylicum upstream of adhE2 A CA_P0037::int (189/190s) mutant in which an intron was inserted at position 189/190 in the sense strand of CA_P0037 was successfully generated by the Targetron technique. The resultant mutant showed significantly different metabolic flux patterns in acidogenic (producing mainly lactate, butyrate, and butanol) and alcohologenic (producing mainly butyrate, acetate, and lactate) chemostat cultures but not in solventogenic or batch cultures. Transcriptomic investigation of the CA_P0037::int (189/190s) mutant showed that inactivation of CA_P0037 significantly affected the expression of more than 258 genes under acidogenic conditions. Surprisingly, genes belonging to the Fur regulon, involved in iron transport (CA_C1029-CA_C1032), or coding for the main flavodoxin (CA_C0587) were the most significantly expressed genes under all conditions, whereas fur (coding for the ferric uptake regulator) gene expression remained unchanged. Furthermore, most of the genes of the Rex regulon, such as the adhE2 and ldhA genes, and of the PerR regulon, such as rbr3A-rbr3B and dfx, were overexpressed in the mutant. In addition, the whole CA_P0037-CA_P0036 operon was highly expressed under all conditions in the CA_P0037::int (189/190s) mutant, suggesting a self-regulated expression mechanism. Cap0037 was shown to bind to the CA_P0037-CA_P0036 operon, sol operon, and adc promoters, and the binding sites were determined by DNA footprinting. Finally, a putative Cap0037 regulon was generated using a bioinformatic approach. IMPORTANCE: Clostridium acetobutylicum is well-known for its ability to produce solvents, especially n-butanol. Understanding the regulatory network of C. acetobutylicum will be crucial for further engineering to obtain a strain capable of producing n-butanol at high yield and selectivity. This study has discovered that the Cap0037 protein is a novel regulator of C. acetobutylicum that drastically affects metabolism under both acidogenic and alcohologenic fermentation conditions. This is pioneering work for further determining the regulatory mechanism of Cap0037 in C. acetobutylicum and studying the role of proteins homologous to Cap0037 in other members of the phylum Firmicutes.

Draft Genome Sequence of the Strict Anaerobe Clostridium neopropionicum X4 (DSM 3847T).

Here, we report the draft genome sequence ofClostridium neopropionicumX4 (DSM 3847(T)), a strictly anaerobic bacterium capable of fermenting ethanol and CO2to propionate, acetate, and propanol. The genome consists of a single chromosome (3.19 Mb).

Genome Sequence of the Acetogenic Bacterium Oxobacter pfennigii DSM 3222T.

Here, we report the draft genome sequence of Oxobacter pfennigii DSM 3222(T), an anaerobic, acetogenic, carbon monoxide-oxidizing, and butyrate-producing bacterium. The genome consists of a chromosome with a size of 4.49 Mbp.

Complete Genome Sequence of the Acetogenic Bacterium Moorella thermoacetica DSM 2955T.

Here, we report the complete genome sequence of Moorella thermoacetica DSM 2955(T), an acetogenic bacterium, which uses the Wood-Ljungdahl pathway for reduction of H2 + CO2 or CO. The genome consists of a single circular chromosome (2.62 Mb).

Complete Genome Sequence of the Type Strain of the Acetogenic Bacterium Moorella thermoacetica DSM 521T.

Here we report the closed genome sequence of the type strain Moorella thermoacetica DSM 521(T), an acetogenic bacterium, which is able to grow autotrophically on H2 + CO2 and/or CO, using the Wood-Ljungdahl pathway. The genome consists of a circular chromosome (2.53 Mb).

Draft Genome Sequence of the Strict Anaerobe Clostridium homopropionicum LuHBu1 (DSM 5847).

Here, we report the draft genome sequence of Clostridium homopropionicum LuHBu1 (DSM 5847(T)), a strictly anaerobic bacterium, which performs propionate fermentation and is capable of growing with 2-, 3-, or 4-hydroxybutyrate as its sole substrate. The genome consists of a single chromosome of 3.65 Mb.

Draft Genome Sequence of Purine-Degrading Gottschalkia purinilyticum (Formerly Clostridium purinilyticum) WA1 (DSM 1384).

Here, we report the draft genome sequence of Gottschalkia purinilyticum (formerly Clostridium purinilyticum) WA1, an anaerobic bacterium specialized on degradation of purines (including adenine) and glycine, which uses the selenoprotein glycine reductase for substrate degradation. The genome consists of a single chromosome (3.40 Mb).

The Complete Genome Sequence of Clostridium aceticum: a Missing Link between Rnf- and Cytochrome-Containing Autotrophic Acetogens.

UNLABELLED: Clostridium aceticum was the first isolated autotrophic acetogen, converting CO2 plus H2 or syngas to acetate. Its genome has now been completely sequenced and consists of a 4.2-Mbp chromosome and a small circular plasmid of 5.7 kbp. Sequence analysis revealed major differences from other autotrophic acetogens. C. aceticum contains an Rnf complex for energy conservation (via pumping protons or sodium ions). Such systems have also been found in C. ljungdahlii and Acetobacterium woodii. However, C. aceticum also contains a cytochrome, as does Moorella thermoacetica, which has been proposed to be involved in the generation of a proton gradient. Thus, C. aceticum seems to represent a link between Rnf- and cytochrome-containing autotrophic acetogens. In C. aceticum, however, the cytochrome is probably not involved in an electron transport chain that leads to proton translocation, as no genes for quinone biosynthesis are present in the genome. IMPORTANCE: Autotrophic acetogenic bacteria are receiving more and more industrial focus, as CO2 plus H2 as well as syngas are interesting new substrates for biotechnological processes. They are both cheap and abundant, and their use, if it results in sustainable products, also leads to reduction of greenhouse gases. Clostridium aceticum can use both gas mixtures, is phylogenetically not closely related to the commonly used species, and may thus become an even more attractive workhorse. In addition, its energy metabolism, which is characterized here, and the ability to synthesize cytochromes might offer new targets for improving the ATP yield by metabolic engineering and thus allow use of C. aceticum for production of compounds by pathways that currently present challenges for energy-limited acetogens.

Draft Genome Sequence of Purine-Degrading Clostridium cylindrosporum HC-1 (DSM 605).

Here, we report the draft genome sequence of Clostridium cylindrosporum HC-1, a purine- and glycine-fermenting anaerobe, which uses selenoprotein glycine reductase for substrate degradation. The genome consists of a single chromosome (2.72 Mb) and a circular plasmid (14.4 kb).

Complete Genome Sequence of Rnf- and Cytochrome-Containing Autotrophic Acetogen Clostridium aceticum DSM 1496.

Here, we report the closed genome sequence of Clostridium aceticum, an Rnf- and cytochrome-containing autotrophic acetogen that is able to convert CO2 and H2 to acetate using the Wood-Ljungdahl pathway. The genome consists of a circular chromosome (4.2 Mbp) and a small circular plasmid (5.7 kbp).

Butanol fermentation.

This review provides an overview on bacterial butanol production and recent developments concerning strain improvement, newly built butanol production plants, and the importance of alternative substrates, especially lignocellulosic hydrolysates. The butanol fermentation using solventogenic clostridial strains, particularly Clostridium acetobutylicum, is a very old industrial process (acetone-butanol-ethanol-ABE fermentation). The genome of this organism has been sequenced and analysed, leading to important improvements in rational strain construction. As the traditional ABE fermentation process is economically unfavourable, novel butanol production strains are being developed. In this review, some newly engineered solvent-producing Clostridium strains are described and strains of which sequences are available are compared with C. acetobutylicum. Furthermore, the past and present of commercial butanol fermentation are presented, including active plants and companies. Finally, the use of biomass as substrate for butanol production is discussed. Some advances concerning processing of biomass in a biorefinery are highlighted, which would allow lowering the price of the butanol fermentation process at industrial scale.

Pathway engineering and synthetic biology using acetogens.

Acetogenic anaerobic bacteria are defined as organisms employing the Wood-Ljungdahl pathway to synthesize acetyl-CoA from CO(2) or CO. Their autotrophic mode of metabolism offers the biotechnological chance to combine use of abundantly available substrates with reduction of greenhouse gases. Several companies have already established pilot and demonstration plants for converting waste gases into ethanol, an important biofuel and a natural product of many acetogens. Recombinant DNA approaches now opened the door to construct acetogens, synthesizing important industrial bulk chemicals and biofuels such as acetone and butanol. Thus, novel microbial production platforms are available that no longer compete with nutritional feedstocks.