Authors: Dong Liu, Zhengjiao Yang, Yong Chen...

BACKGROUND

Biofilms are cell communities wherein cells are embedded in a self-produced extracellular polymeric substances (EPS). The biofilm of confers the cells superior phenotypes and has been extensively exploited to produce a variety of liquid biofuels and bulk chemicals. However, little has been known about the physiology of in biofilm as well as the composition and biosynthesis of the EPS. Thus, this study is focused on revealing the cell physiology and EPS composition of biofilm.

RESULTS

Here, we revealed a novel lifestyle of in biofilm: elimination of sporulation and vegetative growth. Extracellular polymeric substances and wire-like structures were also observed in the biofilm. Furthermore, for the first time, the biofilm polysaccharides and proteins were isolated and characterized. The biofilm contained three heteropolysaccharides. The major fraction consisted of predominantly glucose, mannose and aminoglucose. Also, a great variety of proteins including many non-classically secreted proteins moonlighting as adhesins were found considerably present in the biofilm, with GroEL, a S-layer protein and rubrerythrin being the most abundant ones.

CONCLUSIONS

This study evidenced that vegetative cells rather than commonly assumed spore-forming cells were essentially the solvent-forming cells. The abundant non-classically secreted moonlighting proteins might be important for the biofilm formation. This study provides the first physiological and molecular insights into biofilm which should be valuable for understanding and development of the biofilm-based processes.

PubMed: 30479660
DOI: 10.1186/s13068-018-1316-4

Review

Authors: Huifang Zhang, Pengpeng Yang, Zhenyu Wang...

is an important industrial platform capable of producing a variety of biofuels and bulk chemicals. Biofilm of renders many production advantages and has been long and extensively applied in fermentation. However, molecular and genetic mechanisms underlying the biofilm have been much less studied and remain largely unknown. Here, we review studies to date focusing on biofilms, especially on its physiological and molecular aspects, summarizing the production advantages, cell physiological changes, extracellular matrix components and regulatory genes of the biofilm. This represents the first review dedicated to the biofilm of . Hopefully, it will deepen our understanding toward biofilm and inspire more research to learn and develop more efficient biofilm processes in this industrially important bacterium.

PubMed: 34150727
DOI: 10.3389/fbioe.2021.658568

Authors: Sangwoo Kim, Kyung-Jin Kim

Acetone-butanol-ethanol (ABE) fermentation by the anaerobic bacterium has been considered a promising process of industrial biofuel production. Phosphotransbutyrylase (phosphate butyryltransferase, PTB) plays a crucial role in butyrate metabolism by catalyzing the reversible conversion of butyryl-CoA into butyryl phosphate. Here, we report the crystal structure of PTB from the host for ABE fermentation, , (PTB) at a 2.9 Å resolution. The overall structure of the PTB monomer is quite similar to those of other acyltransferases, with some regional structural differences. The monomeric structure of PTB consists of two distinct domains, the N- and C-terminal domains. The active site cleft was formed at the interface between the two domains. Interestingly, the crystal structure of PTB contained eight molecules per asymmetric unit, forming an octamer, and the size-exclusion chromatography experiment also suggested that the enzyme exists as an octamer in solution. The structural analysis of PTB identifies the substrate binding mode of the enzyme and comparisons with other acyltransferase structures lead us to speculate that the enzyme undergoes a conformational change upon binding of its substrate.

Topics: Acetone; Acyl Coenzyme A; Amino Acid Sequence; Bacterial Proteins; Butanols; Catalytic Domain; Clostridium acetobutylicum; Ethanol; Fermentation; Phosphate Acetyltransferase; Protein Structure, Quaternary

PubMed: 34584034
DOI: 10.4014/jmb.2109.09036

Authors: Shunya Takahashi, Takenori Hama, Toshihiko Nogawa...

This paper considers the total synthesis of a cellular differentiation regulator of , clostrienose, which is a unique fatty-acid glycosyl ester consisting of clostrienoic acid, (3,5,8,10)-3-hydroxy-tetradeca-5,8,10-trienoic acid and α-d-galactofuranosyl-(1 → 2)-α-l-rhamnose. The key features of our synthesis include stereoselective construction of a skipped-triene system in clostrienoic acid and its esterification with a disaccharide residue. The partially protected clostrienoic acid employed for the coupling also served for the preparation of l-rhamnosyl clostrienoate, thus leading to confirmation of the proposed structure unambiguously.

PubMed: 37779990
DOI: 10.1021/acsomega.3c05277

Comparative Study

Authors: Jae Hyuk Chung, Jieun Lee, Sooah Kim...

, a strict gram-positive anaerobe, plays a pivotal role in biotechnological applications, particularly in the biosynthesis of 1,3-propanediol, a critical biofuel component and monomer for bioplastic production. This study introduces DG1, a metabolically engineered strain designed to enhance the 1,3-propanediol pathway. Despite its development, comprehensive metabolic comparisons between the parent and modified strains remain unexplored. Our research addresses this gap by employing gas chromatography coupled with time-of-flight mass spectrometry to delineate the global metabolite landscapes of both strains. Through multivariate statistical analysis such as principal component analysis and hierarchical clustering analysis, we discovered pronounced disparities in their metabolite profiles across the acidogenic and solventogenic phases. Detailed metabolomics investigations underscored significant divergences in amino acid metabolism, fatty acid metabolism, and the tricarboxylic acid cycle. These findings shed light on the metabolic alterations induced by genetic engineering in , offering novel insights into microbial metabolism that could guide future biotechnological innovations.

Topics: Clostridium acetobutylicum; Metabolic Engineering; Metabolomics; Propylene Glycols; Fatty Acids; Metabolic Networks and Pathways; Amino Acids; Citric Acid Cycle; Gas Chromatography-Mass Spectrometry; Biofuels; Metabolome; Fermentation

PubMed: 40016151
DOI: 10.4014/jmb.2407.07028

Authors: Alexandre Delarouzée, Nicolas Lopes Ferreira, François Wasels...

Efficient bioconversion processes of lignocellulose-derived carbohydrates into chemicals have received increasing interest in the last decades since they represent a promising alternative to petro-based processes. Despite efforts to adapt microorganisms to the use of such substrates, one of their major limitations remains their inability to consume multiple sugars simultaneously. In particular, the solventogenic model organism Clostridium acetobutylicum struggles to efficiently use second generation (2G) substrates because of carbon catabolite repression mechanisms that prevent the assimilation of xylose and arabinose in the presence of glucose. In this study, we addressed this issue by inactivating genes encoding transcriptional repressors involved in such mechanisms in the C. acetobutylicum strain DSM 792. Our results showed that the deletion of the two putative copies of (CA_C2613 and CA_C3673) had little or no effect on the ability of the strain to consume xylose. Unlikely, the deletion of (CA_C1340) led to a 2.5-fold growth rate increase on xylose. The deletion of both and genes resulted in the coassimilation of arabinose together with glucose, while xylose consumption remained inefficient. Transcriptional analyses of the wild-type strain and mutants grown on glucose, arabinose, xylose, and combinations of them provided a crucial, global overview of regulations triggered by the products of both and in C. acetobutylicum. As suggested by these data, overexpression of and led to further improvement of pentose assimilation. Those results represent a step forward in the development of genetically modified strains of C. acetobutylicum able to coassimilate lignocellulosic-derived sugars. C. acetobutylicum is a strong candidate to produce chemicals of interest such as C3 and C4 alcohols. Used for more than a century for its capacity to produce a mixture of acetone, butanol, and ethanol from first generation (1G) substrates, its natural ability to assimilate a wide variety of monoosides also predisposes it as an auspicious organism for the valorization of lignocellulose-derived sugar mixtures. To achieve this purpose, a better understanding of carbon catabolite repression mechanisms is essential. The work done here provides critical knowledge on how these mechanisms occur during growth on glucose, arabinose, and xylose mixtures, as well as strategies to tackle them.

Topics: Catabolite Repression; Xylose; Clostridium acetobutylicum; Arabinose; Sugars; Glucose; Fermentation

PubMed: 36779716
DOI: 10.1128/aem.02135-22

Authors: Ann-Kathrin Kotte, Oliver Severn, Zak Bean...

The strictly anaerobic bacterium is well known for its ability to convert sugars into organic acids and solvents, most notably the potential biofuel butanol. However, the regulation of its fermentation metabolism, in particular the shift from acid to solvent production, remains poorly understood. The aim of this study was to investigate whether cell-cell communication plays a role in controlling the timing of this shift or the extent of solvent formation. Analysis of the available genome sequences revealed the presence of eight putative RRNPP-type quorum-sensing systems, here designated to , each consisting of an RRNPP-type regulator gene followed by a small open reading frame encoding a putative signalling peptide precursor. The identified regulator and signal peptide precursor genes were designated to and to , respectively. Triplicate regulator mutants were generated in strain ATCC 824 for each of the eight systems and screened for phenotypic changes. The mutants showed increased solvent formation during early solventogenesis and hence the QssB system was selected for further characterization. Overexpression of severely reduced solvent and endospore formation and this effect could be overcome by adding short synthetic peptides to the culture medium representing a specific region of the QspB signalling peptide precursor. In addition, overexpression of increased the production of acetone and butanol and the initial (48 h) titre of heat-resistant endospores. Together, these findings establish a role for QssB quorum sensing in the regulation of early solventogenesis and sporulation in .

Topics: Bacterial Proteins; Base Composition; Base Sequence; Clostridium acetobutylicum; Gene Expression Regulation, Bacterial; Multigene Family; Quorum Sensing; Sequence Analysis, DNA; Spores, Bacterial

PubMed: 32375981
DOI: 10.1099/mic.0.000916

Design and validation of a multiplex PCR method for the simultaneous quantification of Clostridium acetobutylicum, Clostridium carboxidivorans and Clostridium cellulovorans.

Authors: Laura Feliu-Paradeda, Sebastià Puig, Lluis Bañeras...

Co-cultures of clostridia with distinct physiological properties have emerged as an alternative to increase the production of butanol and other added-value compounds from biomass. The optimal performance of mixed tandem cultures may depend on the stability and fitness of each species in the consortium, making the development of specific quantification methods to separate their members crucial. In this study, we developed and tested a multiplex qPCR method targeting the 16S rRNA gene for the simultaneous quantification of Clostridium acetobutylicum, Clostridium carboxidivorans and Clostridium cellulovorans in co-cultures. Designed primer pairs and probes could specifically quantify the three Clostridium species with no cross-reactions thus allowing significant changes in their growth kinetics in the consortia to be detected and correlated with productivity. The method was used to test a suitable medium composition for simultaneous growth of the three species. We show that higher alcohol productions were obtained when combining C. carboxidivorans and C. acetobutylicum compared to individual cultures, and further improved (> 90%) in the triplet consortium. Altogether, the methodology could be applied to fermentation processes targeting butanol productions from lignocellulosic feedstocks with a higher substrate conversion efficiency.

Topics: Clostridium acetobutylicum; Clostridium cellulovorans; Multiplex Polymerase Chain Reaction; RNA, Ribosomal, 16S; Clostridium; Butanols; 1-Butanol; Fermentation

PubMed: 37973932
DOI: 10.1038/s41598-023-47007-w

Authors: Wei Zhuang, Xiaojing Liu, Jing Yang...

Immobilized fermentation has several advantages over traditional suspended fermentation, including simple and continuous operation, improved fermentation performance and reduced cost. Carrier is the most adjustable element among three elements of immobilized fermentation, including carrier, bacteria and environment. In this study, we characterized carrier roughness and surface properties of four types of natural fibres, including linen, cotton, bamboo fibre and silk, to assess their effects on cell immobilization, fermentation performance and stability. Linen with higher specific surface area and roughness could adsorb more bacteria during immobilized fermentation, thereby improving fermentation performance; thus, linen was selected as a suitable carrier and was applied for acetone-butanol-ethanol (ABE) fermentation. To further improve fermentation performance, we also found that microbes of Clostridium acetobutylicum were negatively charged surfaces during fermentation. Therefore, we then modified linen with polyetherimide (PEI) and steric acid (SA) to increase surface positive charge and improve surface property. During ABE fermentation, the adhesion between modified linen and bacteria was increased, adsorption was increased about twofold compared with that of unmodified linen, and butanol productivity was increased 8.16% and 6.80% with PEI- and SA-modified linen as carriers respectively.

Topics: Acetone; Bacterial Adhesion; Butanols; Cells, Immobilized; Chemical Phenomena; Clostridium acetobutylicum; Ethanol; Fermentation; Surface Properties; Textiles

PubMed: 28112488
DOI: 10.1111/1751-7915.12557

Review

Authors: Mohab A Al-Hinai, Shawn W Jones, Eleftherios T Papoutsakis...

Bacillus and Clostridium organisms initiate the sporulation process when unfavorable conditions are detected. The sporulation process is a carefully orchestrated cascade of events at both the transcriptional and posttranslational levels involving a multitude of sigma factors, transcription factors, proteases, and phosphatases. Like Bacillus genomes, sequenced Clostridium genomes contain genes for all major sporulation-specific transcription and sigma factors (spo0A, sigH, sigF, sigE, sigG, and sigK) that orchestrate the sporulation program. However, recent studies have shown that there are substantial differences in the sporulation programs between the two genera as well as among different Clostridium species. First, in the absence of a Bacillus-like phosphorelay system, activation of Spo0A in Clostridium organisms is carried out by a number of orphan histidine kinases. Second, downstream of Spo0A, the transcriptional and posttranslational regulation of the canonical set of four sporulation-specific sigma factors (σ(F), σ(E), σ(G), and σ(K)) display different patterns, not only compared to Bacillus but also among Clostridium organisms. Finally, recent studies demonstrated that σ(K), the last sigma factor to be activated according to the Bacillus subtilis model, is involved in the very early stages of sporulation in Clostridium acetobutylicum, C. perfringens, and C. botulinum as well as in the very late stages of spore maturation in C. acetobutylicum. Despite profound differences in initiation, propagation, and orchestration of expression of spore morphogenetic components, these findings demonstrate not only the robustness of the endospore sporulation program but also the plasticity of the program to generate different complex phenotypes, some apparently regulated at the epigenetic level.

Topics: Bacillus; Bacillus subtilis; Clostridium; Clostridium acetobutylicum; Clostridium botulinum; Clostridium perfringens; Gene Expression Regulation, Bacterial; Histidine Kinase; Phenotype; Protein Kinases; Sigma Factor; Spores, Bacterial; Transcription Factors

PubMed: 25631287
DOI: 10.1128/MMBR.00025-14