Recombinant Production of Pseudomonas aeruginosa Rhamnolipids in P. putida KT2440 on Acetobacterium woodii Cultures Grown Chemo-Autotrophically with Carbon Dioxide and Hydrogen.

The establishment of sustainable processes for the production of commodity chemicals is one of today's central challenges for biotechnological industries. The chemo-autotrophic fixation of CO2 and the subsequent production of acetate by acetogenic bacteria via anaerobic gas fermentation represents a promising platform for the ecologically sustainable production of high-value biocommodities via sequential fermentation processes. In this study, the applicability of acetate-containing cell-free spent medium of the gas-fermenting acetogenic bacterium A. woodii WP1 as the feeder strain for growth and the recombinant production of P. aeruginosa PAO1 mono-rhamnolipids in the well-established nonpathogenic producer strain P. putida KT2440 were investigated. Additionally, the potential possibility of a simplified production process without the necessary separation of feeder strain cells was elucidated via the cultivation of P. putida in cell-containing A. woodii culture broth. For these cultures, the content of both strains was investigated by examining the relative quantification of strain-exclusive genes via qPCR. The recombinant production of mono-rhamnolipids was successfully achieved with maximum titers of approximately 360-400 mg/L for both cell-free and cell-containing A. woodii spent medium. The reported processes therefore represent a successful proof of principle for gas fermentation-derived acetate as a potential sustainable carbon source for future recombinant rhamnolipid production processes by P. putida KT2440.

New Insights into the Physiology of the Propionate Producers Anaerotignum propionicum and Anaerotignum neopropionicum (Formerly Clostridium propionicum and Clostridium neopropionicum).

Propionate is an important platform chemical that is available through petrochemical synthesis. Bacterial propionate formation is considered an alternative, as bacteria can convert waste substrates into valuable products. In this regard, research primarily focused on propionibacteria due to high propionate titers achieved from different substrates. Whether other bacteria could also be attractive producers is unclear, mostly because little is known about these strains. Therefore, two thus far less researched strains, Anaerotignum propionicum and Anaerotignum neopropionicum, were investigated with regard to their morphologic and metabolic features. Microscopic analyses revealed a negative Gram reaction despite a Gram-positive cell wall as well as surface layers for both strains. Furthermore, growth, product profiles, and the potential for propionate formation from sustainable substrates, i.e., ethanol or lignocellulosic sugars, were assessed. Results showed that both strains can oxidize ethanol to different extents. While A. propionicum only partially used ethanol, A. neopropionicum converted 28.3 mM ethanol to 16.4 mM propionate. Additionally, the ability of A. neopropionicum to produce propionate from lignocellulose-derived substrates was analyzed, leading to propionate concentrations of up to 14.5 mM. Overall, this work provides new insights into the physiology of the Anaerotignum strains, which can be used to develop effective propionate producer strains.

Heterologous 1,3-Propanediol Production Using Different Recombinant Clostridium beijerinckii DSM 6423 Strains.

1,3-propanediol (1,3-PDO) is a valuable basic chemical, especially in the polymer industry to produce polytrimethylene terephthalate. Unfortunately, the production of 1,3-PDO mainly depends on petroleum products as precursors. Furthermore, the chemical routes have significant disadvantages, such as environmental issues. An alternative is the biobased fermentation of 1,3-PDO from cheap glycerol. Clostridium beijerinckii DSM 6423 was originally reported to produce 1,3-PDO. However, this could not be confirmed, and a genome analysis revealed the loss of an essential gene. Thus, 1,3-PDO production was genetically reinstalled. Genes for 1,3-PDO production from Clostridium pasteurianum DSM 525 and Clostridium beijerinckii DSM 15410 (formerly Clostridium diolis) were introduced into C. beijerinckii DSM 6423 to enable 1,3-PDO production from glycerol. 1,3-PDO production by recombinant C. beijerinckii strains were investigated under different growth conditions. 1,3-PDO production was only observed for C. beijerinckii [pMTL83251_Ppta-ack_1,3-PDO.diolis], which harbors the genes of C. beijerinckii DSM 15410. By buffering the growth medium, production could be increased by 74%. Furthermore, the effect of four different promoters was analyzed. The use of the constitutive thlA promoter from Clostridium acetobutylicum led to a 167% increase in 1,3-PDO production compared to the initial recombinant approach.

Production of propionate using metabolically engineered strains of Clostridium saccharoperbutylacetonicum.

The carboxylic acid propionate is a valuable platform chemical with applications in various fields. The biological production of this acid has become of great interest as it can be considered a sustainable alternative to petrochemical synthesis. In this work, Clostridium saccharoperbutylacetonicum was metabolically engineered to produce propionate via the acrylate pathway. In total, the established synthetic pathway comprised eight genes encoding the enzymes catalyzing the conversion of pyruvate to propionate. These included the propionate CoA-transferase, the lactoyl-CoA dehydratase, and the acryloyl-CoA reductase from Anaerotignum neopropionicum as well as a D-lactate dehydrogenase from Leuconostoc mesenteroides subsp. mesenteroides. Due to difficulties in assembling all genes on one plasmid under the control of standard promoters, the PtcdB-tcdR promoter system from Clostridium difficile was integrated into a two-plasmid system carrying the acrylate pathway genes. Several promoters were analyzed for their activity in C. saccharoperbutylacetonicum using the fluorescence-activating and absorption-shifting tag (FAST) as a fluorescent reporter to identify suitable candidates to drive tcdR expression. After selecting the lactose-inducible PbgaL promoter, engineered C. saccharoperbutylacetonicum strains produced 0.7 mM propionate upon induction of gene expression. The low productivity was suspected to be a consequence of a metabolic imbalance leading to acryloyl-CoA accumulation in the cells. To even out the proposed imbalance, the propionate-synthesis operons were rearranged, thereby increasing the propionate concentration by almost four-fold. This study is the first one to report recombinant propionate production using a clostridial host strain that has opened a new path towards bio-based propionate to be improved further in subsequent work. KEY POINTS: • Determination of promoter activities in C. saccharoperbutylacetonicum using FAST. • Implementation of propionate production in C. saccharoperbutylacetonicum. • Elevation of propionate production by 375% to a concentration of 3 mM.

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.

HspB5/αB-crystallin phosphorylation at S45 and S59 is essential for protection of the dendritic tree of rat hippocampal neurons.

Rarefaction of the dendritic tree leading to neuronal dysfunction is a hallmark of many neurodegenerative diseases and we have shown previously that heat shock protein B5 (HspB5)/αB-crystallin is able to increase dendritic complexity in vitro. The aim of this study was to investigate if this effect is also present in vivo, if HspB5 can counteract dendritic rarefaction under pathophysiological conditions and the impact of phosphorylation of HspB5 in this process. HspB5 and eight mutants inhibiting or mimicking phosphorylation at the three phosphorylation sites serine (S)19, S45, and S59 were over-expressed in cultured rat hippocampal neurons with subsequent investigation of the complexity of the dendritic tree. Sholl analysis revealed significant higher complexity of the dendritic tree after over-expression of wild-type HspB5 and the mutant HspB5-AEE. All other mutants showed no or minor effects. For in vivo investigation in utero electroporation of mouse embryos was applied. At embryonal day E15.5 the respective plasmids were injected, cornu ammonis 1 (CA1) pyramidal cells transfected by electroporation and their basal dendritic trees were analyzed at post-natal day P15. In vivo, HspB5 and HspB5-AEE led to an increase of total dendritic length as well as a higher complexity. Finally, the dendritic effect of HspB5 was investigated under a pathophysiological condition, that is, iron deficiency which reportedly results in dendritic rarefaction. HspB5 and HspB5-AEE but not the non-phosphorylatable mutant HspB5-AAA significantly counteracted the dendritic rarefaction. Thus, our data suggest that up-regulation and selective phosphorylation of HspB5 in neurodegenerative diseases may preserve dendritic morphology and counteract neuronal dysfunction.

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).

Cdc48/p97 promotes reformation of the nucleus by extracting the kinase Aurora B from chromatin.

During division of metazoan cells, the nucleus disassembles to allow chromosome segregation, and then reforms in each daughter cell. Reformation of the nucleus involves chromatin decondensation and assembly of the double-membrane nuclear envelope around the chromatin; however, regulation of the process is still poorly understood. In vitro, nucleus formation requires p97 (ref. 3), a hexameric ATPase implicated in membrane fusion and ubiquitin-dependent processes. However, the role and relevance of p97 in nucleus formation have remained controversial. Here we show that p97 stimulates nucleus reformation by inactivating the chromatin-associated kinase Aurora B. During mitosis, Aurora B inhibits nucleus reformation by preventing chromosome decondensation and formation of the nuclear envelope membrane. During exit from mitosis, p97 binds to Aurora B after its ubiquitylation and extracts it from chromatin. This leads to inactivation of Aurora B on chromatin, thus allowing chromatin decondensation and nuclear envelope formation. These data reveal an essential pathway that regulates reformation of the nucleus after mitosis and defines ubiquitin-dependent protein extraction as a common mechanism of Cdc48/p97 activity also during nucleus formation.

NSF- and SNARE-mediated membrane fusion is required for nuclear envelope formation and completion of nuclear pore complex assembly in Xenopus laevis egg extracts.

Despite the progress in understanding nuclear envelope (NE) reformation after mitosis, it has remained unclear what drives the required membrane fusion and how exactly this is coordinated with nuclear pore complex (NPC) assembly. Here, we show that, like other intracellular fusion reactions, NE fusion in Xenopus laevis egg extracts is mediated by SNARE proteins that require activation by NSF. Antibodies against Xenopus NSF, depletion of NSF or the dominant-negative NSF(E329Q) variant specifically inhibited NE formation. Staging experiments further revealed that NSF was required until sealing of the envelope was completed. Moreover, excess exogenous alpha-SNAP that blocks SNARE function prevented membrane fusion and caused accumulation of non-flattened vesicles on the chromatin surface. Under these conditions, the nucleoporins Nup107 and gp210 were fully recruited, whereas assembly of FxFG-repeat-containing nucleoporins was blocked. Together, we define NSF- and SNARE-mediated membrane fusion events as essential steps during NE formation downstream of Nup107 recruitment, and upstream of membrane flattening and completion of NPC assembly.