Catenovulum adriaticum sp. nov., isolated from algae in the harbour of Susak, Croatia.

The use of algae as feedstock for industrial purposes, such as in bioethanol production, is desirable. During a search for new agarolytic marine bacteria, a novel Gram-stain-negative, strictly aerobic, and agarolytic bacterium, designated as TS8T, was isolated from algae in the harbour of the island of Susak, Croatia. The cells were rod-shaped and motile. The G+C content of the sequenced genome was 38.6 mol%. Growth was observed at 11-37 °C, with 0.5-13 % (w/v) NaCl, and at pH 6.0-9.0. The main fatty acids were summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c), summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), and C16 : 0. The main respiratory quinone was ubiquinone-8. The major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. Analysis of 16S rRNA gene sequences indicated that the newly isolated strain belongs to the genus Catenovulum. Based on 16S rRNA gene sequence data, strain TS8T is closely related to Catenovulum sediminis D2T (95.7 %), Catenovulum agarivorans YM01T (95.0 %), and Catenovulum maritimum Q1T (93.2 %). Digital DNA-DNA hybridization values between TS8T and the other Catenovulum strains were below 25 %. Based on genotypic, phenotypic, and phylogenetic data, strain TS8T represents a new species of the genus Catenovulum, for which the name Catenovulum adriaticum sp. nov. is proposed. The type strain is TS8T (=DSM 114830T=NCIMB 15451T).

Refining and illuminating acetogenic Eubacterium strains for reclassification and metabolic engineering.

BACKGROUND: The genus Eubacterium is quite diverse and includes several acetogenic strains capable of fermenting C1-substrates into valuable products. Especially, Eubacterium limosum and closely related strains attract attention not only for their capability to ferment C1 gases and liquids, but also due to their ability to produce butyrate. Apart from its well-elucidated metabolism, E. limosum is also genetically accessible, which makes it an interesting candidate to be an industrial biocatalyst. RESULTS: In this study, we examined genomic, phylogenetic, and physiologic features of E. limosum and the closest related species E. callanderi as well as E. maltosivorans. We sequenced the genomes of the six Eubacterium strains 'FD' (DSM 3662T), 'Marburg' (DSM 3468), '2A' (DSM 2593), '11A' (DSM 2594), 'G14' (DSM 107592), and '32' (DSM 20517) and subsequently compared these with previously available genomes of the E. limosum type strain (DSM 20543T) as well as the strains 'B2', 'KIST612', 'YI' (DSM 105863T), and 'SA11'. This comparison revealed a close relationship between all eleven Eubacterium strains, forming three distinct clades: E. limosum, E. callanderi, and E. maltosivorans. Moreover, we identified the gene clusters responsible for methanol utilization as well as genes mediating chain elongation in all analyzed strains. Subsequent growth experiments revealed that strains of all three clades can convert methanol and produce acetate, butyrate, and hexanoate via reverse ß-oxidation. Additionally, we used a harmonized electroporation protocol and successfully transformed eight of these Eubacterium strains to enable recombinant plasmid-based expression of the gene encoding the fluorescence-activating and absorption shifting tag (FAST). Engineered Eubacterium strains were verified regarding their FAST-mediated fluorescence at a single-cell level using a flow cytometry approach. Eventually, strains 'FD' (DSM 3662T), '2A' (DSM 2593), '11A' (DSM 2594), and '32' (DSM 20517) were genetically engineered for the first time. CONCLUSION: Strains of E. limosum, E. callanderi, and E. maltosivorans are outstanding candidates as biocatalysts for anaerobic C1-substrate conversion into valuable biocommodities. A large variety of strains is genetically accessible using a harmonized electroporation protocol, and FAST can serve as a reliable fluorescent reporter protein to characterize genetically engineered cells. In total eleven strains have been assigned to distinct clades, providing a clear and updated classification. Thus, the description of respective Eubacterium species has been emended, improved, aligned, and is requested to be implemented in respective databases.

Establishment of Green- and Red-Fluorescent Reporter Proteins Based on the Fluorescence-Activating and Absorption-Shifting Tag for Use in Acetogenic and Solventogenic Anaerobes.

Anaerobic bacteria are promising biocatalysts to produce industrially relevant products from nonfood feedstocks. Several anaerobes are genetically accessible, and various molecular tools for metabolic engineering are available. Still, the use of bright fluorescent reporters, which are commonly used in molecular biological approaches is limited under anaerobic conditions. Therefore, the establishment of different anaerobic fluorescent reporter proteins is of great interest. Here, we present the establishment of the green- and red-fluorescent reporter proteins greenFAST and redFAST for use in different solventogenic and acetogenic bacteria. Green fluorescence of greenFAST was bright in Clostridium saccharoperbutylacetonicum, Clostridium acetobutylicum, Acetobacterium woodii, and Eubacterium limosum, while only C. saccharoperbutylacetonicum showed bright red fluorescence when producing redFAST. We used both reporter proteins in C. saccharoperbutylacetonicum for multicolor approaches. These include the investigation of the co-culture dynamics of metabolically engineered strains. Moreover, we established a tightly regulated inducible two-plasmid system and used greenFAST and redFAST to track the coexistence and interaction of both plasmids under anaerobic conditions in C. saccharoperbutylacetonicum. The establishment of greenFAST and redFAST as fluorescent reporters opens the door for further multicolor approaches to investigate cell dynamics, gene expression, or protein localization under anaerobic conditions.

Production of the biocommodities butanol and acetone from methanol with fluorescent FAST-tagged proteins using metabolically engineered strains of Eubacterium limosum.

BACKGROUND: The interest in using methanol as a substrate to cultivate acetogens increased in recent years since it can be sustainably produced from syngas and has the additional benefit of reducing greenhouse gas emissions. Eubacterium limosum is one of the few acetogens that can utilize methanol, is genetically accessible and, therefore, a promising candidate for the recombinant production of biocommodities from this C1 carbon source. Although several genetic tools are already available for certain acetogens including E. limosum, the use of brightly fluorescent reporter proteins is still limited. RESULTS: In this study, we expanded the genetic toolbox of E. limosum by implementing the fluorescence-activating and absorption shifting tag (FAST) as a fluorescent reporter protein. Recombinant E. limosum strains that expressed the gene encoding FAST in an inducible and constitutive manner were constructed. Cultivation of these recombinant strains resulted in brightly fluorescent cells even under anaerobic conditions. Moreover, we produced the biocommodities butanol and acetone from methanol with recombinant E. limosum strains. Therefore, we used E. limosum cultures that produced FAST-tagged fusion proteins of the bifunctional acetaldehyde/alcohol dehydrogenase or the acetoacetate decarboxylase, respectively, and determined the fluorescence intensity and product concentrations during growth. CONCLUSIONS: The addition of FAST as an oxygen-independent fluorescent reporter protein expands the genetic toolbox of E. limosum. Moreover, our results show that FAST-tagged fusion proteins can be constructed without negatively impacting the stability, functionality, and productivity of the resulting enzyme. Finally, butanol and acetone can be produced from methanol using recombinant E. limosum strains expressing genes encoding fluorescent FAST-tagged fusion proteins.

Caproicibacter fermentans gen. nov., sp. nov., a new caproate-producing bacterium and emended description of the genus Caproiciproducens.

A strictly anaerobic bacterial strain designated EA1T was isolated from an enrichment culture inoculated with biogas reactor content. Cells of strain EA1T are spore-forming rods (1-3×0.4-0.8 µm) and stain Gram-negative, albeit they possess a Gram-positive type of cell-wall ultrastructure. Growth of strain EA1T was observed at 30 and 37 °C and within a pH range of pH 5-9. The major components recovered in the fatty acid fraction were C14:0, C16:0, C16:0 DMA (dimethyl acetal) and C16:1 ω7c. Strain EA1T fermented several mono- and disaccharides. Metabolic end products from fructose were acetate, butyrate, caproate and lactate. Furthermore, ethanol, CO2 and H2 were identified as products. The genome consists of a chromosome (3.9 Mbp) with 3797 predicted protein-encoding genes and a G+C content of 51.25 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain EA1T represents a novel taxon within the family Oscillospiraceae. The most closely related type strains of EA1T, based on 16S rRNA gene sequence identity, are Caproiciproducens galactitolivorans BS-1T (94.9 %), [Clostridium] leptum DSM 753T (93.8 %), [Clostridium] sporosphaeroides DSM 1294T (91.7 %) and Ruminococcus bromii ATCC 27255T (91.0 %). Further phenotypic characteristics of strain EA1T differentiate it from related, validly described bacterial species. Strain EA1T represents a novel genus and novel species within the family Oscillospiraceae. The proposed name is Caproicibacter fermentans gen. nov., sp. nov. The type strain is EA1T (DSM 107079T=JCM 33110T).

Induced heterologous expression of the arginine deiminase pathway promotes growth advantages in the strict anaerobe Acetobacterium woodii.

The advantage of using acetogens such as Acetobacterium woodii as biocatalysts converting the cheap substrate and greenhouse gas carbon dioxide (CO2) into value-added chemicals comes together with the disadvantage of a low overall ATP gain due to the bioenergetics associated with the Wood-Ljungdahl pathway. Expanding the product spectrum of recombinant A. woodii strains to compounds with high ATP-demanding biosynthesis is therefore challenging. As a least invasive strategy for improved ATP generation, the exploitation of the arginine deiminase pathway (ADI) was examined under native conditions and via using heterologously expressed genes in A. woodii. Several promoters were analyzed for application of different gene expression levels in A. woodii using ß-glucuronidase assays. Heterologous expression of the ADI pathway genes from Clostridium autoethanogenum was controlled using either the constitutive pta-ack promoter from Clostridium ljungdahlii or a tightly regulated tetracycline-inducible promoter Ptet. Unlike constitutive expression, only induced expression of the ADI pathway genes led to a 36% higher maximal OD600 when using arginine (OD600 3.4) as nitrogen source and a 52% lower acetate yield per biomass compared to cells growing with yeast extract as nitrogen source (OD600 2.5). In direct comparison, a 69% higher maximal OD600 and about 60% lower acetate yield per biomass in induced to non-induced recombinant A. woodii cells was noticed when using arginine. Our data suggests the application of the ADI pathway in A. woodii for expanding the product spectrum to compounds with high ATP-demanding biosynthesis.