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[FeFe]-hydrogenases catalyze the reversible two-electron reduction of two protons to molecular hydrogen. Although these enzymes are among the most efficient H 2 -converting biocatalysts in nature, their catalytic cofactor (termed H-cluster) is irreversibly destroyed upon contact with dioxygen. The [FeFe]-hydrogenase CbA5H from Clostridium beijerinckii has a unique mechanism to protect the H-cluster from oxygen-induced degradation. The protective strategy of CbA5H was proposed based on a partial protein structure of CbA5H's oxygen-shielded form. Here, we present a cryo-EM structure of 2.2 Å resolution from the entire enzyme in its dimeric and active state and elucidate the structural parameters of the reversible cofactor protection mechanism. We found that both subunits of the homodimeric structure of CbA5H have a Zn 2+ -binding four-helix domain, which does not play a role in electron transport as described for other complex protein structures. Biochemical data instead confirm that two [4Fe-4S] clusters are responsible for electron transfer in CbA5H, while the identified zinc atom is critical for oligomerization and protein stability., Competing Interests: Competing interests statement:The authors declare no competing interest.

Methods such as gas stripping and vacuum-assisted gas stripping (VAGS) result in significant removal of water from the bioreactor, thus requiring continuous water replenishment in the bioreactor. In this study, we developed a hydrophobic stainless steel meshes capable of selectively recovering concentrated ABE stream from the bioreactor during VAGS. Three stainless steel meshes with pore sizes of 180 µm, 300 µm, and 425 µm were made hydrophobic and oleophilic with zinc oxide (ZnO) and polydimethylsiloxane (PDMS). Butanol concentrations in the model solutions range from 3 to 10 g/L which mimic concentrations typically produced during batch ABE fermentation. The meshes were integrated in a 5-L bioreactor containing 2.5 L of operational ABE model solution followed by the evaluation of selective extraction of ABE from both cell-free and Clostridium beijerinckii-rich ABE model solutions. The results show that the 180-µm ZnO/PDMS-coated mesh retained 54-64% more water in the bioreactor without C. beijerinckii cells and 61-65% more water with cells compared to the uncoated mesh. Furthermore, the butanol concentration of condensates recovered with 180-µm ZnO-PDMS-coated mesh was up to 10.8-fold greater than that of uncoated counterpart. Our data demonstrate that the developed ZnO-PDMS mesh can recover high concentrations of ABE while selectively retaining water in the bioreactor. Additionally, this technology demonstrates the potential for real-time ABE recovery during the fermentation of lignocellulosic and colloidal materials, without the concern of clogging the separation system. KEY POINTS: • Hydrophobic mesh enhanced water retention in the bioreactor by up to 1.65-fold. • Butanol concentration in the collected condensate was increased by up to 10.8-fold. • Hydrophobic mesh is compatible with fermentation of lignocellulose., (© 2024. The Author(s).)

Bioconversion of abundant lactose-replete whey permeate to value-added chemicals holds promise for valorization of this expanding food processing waste. Efficient conversion of whey permeate-borne lactose requires adroit microbial engineering to direct carbon to the desired chemical. An engineered strain of Clostridium beijerinckii NCIMB 8052 ( C. beijerinckii _mgsA+mgR) that produces 87% more butanol on lactose than the control strain was assessed for global transcriptomic changes. The results revealed broadly contrasting gene expression patterns in C. beijerinckii _mgsA+mgR relative to the control strain. These were characterized by widespread decreases in the abundance of mRNAs of Fe-S proteins in C. beijerinckii _mgsA+mgR, coupled with increased differential expression of lactose uptake and catabolic genes, iron uptake genes, two-component signal transduction and motility genes, and genes involved in the biosynthesis of vitamins B 5 and B 12 , aromatic amino acids (particularly tryptophan), arginine, and pyrimidines. Conversely, the mRNA patterns suggest that the L-aspartate-dependent de novo biosynthesis of NAD as well as biosynthesis of lysine and asparagine and metabolism of glycine and threonine were likely down-regulated. Furthermore, genes involved in cysteine and methionine biosynthesis and metabolism, including cysteine desulfurase-a central player in Fe-S cluster biosynthesis-equally showed reductions in mRNA abundance. Genes involved in biosynthesis of capsular polysaccharides and stress response also showed reduced mRNA abundance in C. beijerinckii _mgsA+mgR. The results suggest that remodeling of cellular and metabolic networks in C. beijerinckii _mgsA+mgR to counter anticipated effects of methylglyoxal production from heterologous expression of methylglyoxal synthase led to enhanced growth and butanol production in C. beijerinckii _mgsA+mgR., Importance: Biological production of commodity chemicals from abundant waste streams such as whey permeate represents an opportunity for decarbonizing chemical production. Whey permeate remains a vastly underutilized feedstock for bioproduction purposes. Thus, enhanced understanding of the cellular and metabolic repertoires of lactose-mediated production of chemicals such as butanol promises to identify new targets that can be fine tuned in recombinant and native microbial strains to engender stronger coupling of whey permeate-borne lactose to value-added chemicals. Our results highlight new genetic targets for future engineering of C. beijerinckii for improved butanol production on lactose and ultimately in whey permeate., Competing Interests: The authors declare no conflict of interest.

Furfural-tolerant and hydrogen-producing microbial consortia were enriched from soil, with hydrogen production of 259.84 mL/g-xylose under 1 g/L furfural stress. The consortia could degrade 2.5 g/L furfural within 24 h in the xylose system, more efficient than in the sugar-free system. Despite degradation of furfural to furfuryl alcohol, the release of reactive oxygen species and lactate dehydrogenase was also detected, suggesting that furfuryl alcohol is also a potential inhibitor of hydrogen production. The butyrate/acetate ratio was observed to decrease with increasing furfural concentration, leading to decreased hydrogen production. Furthermore, microbial community analysis suggested that dominated Clostridium butyricum was responsible for furfural degradation, while Clostridium beijerinckii reduction led to hydrogen production decrease. Overall, the enriched consortia in this study could efficiently degrade furfural and produce hydrogen, providing new insights into hydrogen-producing microbial consortia with furfural tolerance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The fusion of hydrogenases and photosynthetic reaction centers (RCs) has proven to be a promising strategy for the production of sustainable biofuels. Type I (iron-sulfur-containing) RCs, acting as photosensitizers, are capable of promoting electrons to a redox state that can be exploited by hydrogenases for the reduction of protons to dihydrogen (H 2 ). While both [FeFe] and [NiFe] hydrogenases have been used successfully, they tend to be limited due to either O 2 sensitivity, binding specificity, or H 2 production rates. In this study, we fuse a peripheral (stromal) subunit of Photosystem I (PS I), PsaE, to an O 2 -tolerant [FeFe] hydrogenase from Clostridium beijerinckii using a flexible [GGS] 4 linker group ( Cb HydA1-PsaE). We demonstrate that the Cb HydA1 chimera can be synthetically activated in vitro to show bidirectional activity and that it can be quantitatively bound to a PS I variant lacking the PsaE subunit. When illuminated in an anaerobic environment, the nanoconstruct generates H 2 at a rate of 84.9 ± 3.1 µmol H 2 mg chl -1 h -1 . Further, when prepared and illuminated in the presence of O 2 , the nanoconstruct retains the ability to generate H 2 , though at a diminished rate of 2.2 ± 0.5 µmol H 2 mg chl -1 h -1 . This demonstrates not only that PsaE is a promising scaffold for PS I-based nanoconstructs, but the use of an O 2 -tolerant [FeFe] hydrogenase opens the possibility for an in vivo H 2 generating system that can function in the presence of O 2 ., Competing Interests: Competing interests statement:The authors declare no competing interest.

The current study proposes RBBR biosorption by Clostridium beijerinckii DSMZ 6422 biomass remaining after biobutanol production from pumpkin peel (PP) by a zero-waste approach. Efficient biobutanol production was achieved by investigating initial PP concentrations (5-20% without or with enzymatic hydrolysis) and fermentation time. According to this, the highest concentrations of biobutanol and total ABE were obtained as 4.87 g/L and 8.13 g/L in the presence of 10% PP without enzymatic hydrolysis at 96 h. Furthermore, based on the zero-waste approach, C. beijerinckii DSMZ 6422 biomass obtained after biofuel production was used as a biosorbent for the removal of RBBR dye. Response surface methodology (RSM), commonly utilized for the experimental design, was used to specify the optimized biosorption conditions of RBBR, including initial dye concentration (50-200 mg/L), initial pH (2-6), biosorbent concentration (1-3 g/L), and contact time (0-240 min). The highest biosorption under optimized conditions with RSM was 98% in the presence of 194.36 mg/L RBBR and 2.65 g/L biosorbent at pH 2 and 15 min. This is the first report in the literature about the biosorption of RBBR dye by anaerobic C. beijerinckii biomass after the biobutanol production process. This study also shows the efficient usage of agricultural and microbial wastes in different areas based on zero-waste applications., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Clostridium species are re-emerging as biotechnological workhorses for industrial acetone-butanol-ethanol production. This re-emergence is largely due to advances in fermentation technologies but also due to advances in genome engineering and re-programming of the native metabolism. Several genome engineering techniques have been developed including the development of numerous CRISPR-Cas tools. Here, we expanded the CRISPR-Cas toolbox and developed a CRISPR-Cas12a genome engineering tool in Clostridium beijerinckii NCIMB 8052. By controlling the expression of FnCas12a with the xylose-inducible promoter, we achieved efficient (25-100%) single-gene knockout of five C. beijerinckii NCIMB 8052 genes (spo0A, upp, Cbei_1291, Cbei_3238, Cbei_3832). Moreover, we achieved multiplex genome engineering by simultaneously knocking out the spo0A and upp genes in a single step with an efficiency of 18%. Finally, we showed that the spacer sequence and position in the CRISPR array can affect the editing efficiency outcome., (© 2023. The Author(s).)

Serine/threonine protein kinases (STKs) are important for signal transduction and involved in multiple physiological processes, including cell growth, central metabolism, and sporulation in bacteria. However, the role of STKs in solventogenic clostridia remains unclear. Here, we identified and comprehensively investigated six STK candidates in Clostridium beijerinckii. These STKs were classified into four groups with distinct characteristics via analysis of genetic organizations, prediction of protein domains, and multiple sequence alignment. Cbei0566 is a member of the PrkA family with 41% identity to PrkA from Bacillus subtilis, and both Cbei0666 and Cbei0813 are two-component-like STKs. Cbei1151 and Cbei1929 belong to the Hanks family STKs and consist of a cytoplasmic catalytic domain, a transmembrane region, and extracellular sensor domains. In-frame deletion mutants of cbei0566, cbei0666, cbei1929, and cbei2661 displayed similar cell growth with wild type. Both Δcbei0666 and Δcbei2661 improved acetone-butanol-ethanol (ABE) production by 14.3% (19.2 g/L vs. 16.8 g/L), and the sporulation frequencies of Δcbei0566, Δcbei1929, and Δcbei2661 significantly decreased to 35.5%, 55.1% and 44.8%, respectively. The restored phenotypes after genetic complementation demonstrated their direct link to STKs deletion. Remarkably, overexpressing cbei0566 contributed to 41.5% more spore formation and cbei1929 overexpression enhanced ABE production from 19.3 to 24.2 g/L, along with 25% less acids. These results revealed that Cbei0566 and Cbei1929 had prominent regulatory functions. This study expands the current knowledge of the existence and functions of STKs in prokaryotes and highlights the importance of STK-mediated signaling networks in developing superior strains. KEY POINTS: • First reported serine/threonine protein kinases in solventogenic clostridia • Six STKs with distinct properties possessed diverse functions in C. beijerinckii • Cbei1929 and Cbei0566 remarkably regulated solventogenesis and sporulation., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Solventogenic clostridia are not a strictly defined group within the genus Clostridium but its representatives share some common features, i.e. they are anaerobic, non-pathogenic, non-toxinogenic and endospore forming bacteria. Their main metabolite is typically 1-butanol but depending on species and culture conditions, they can form other metabolites such as acetone, isopropanol, ethanol, butyric, lactic and acetic acids, and hydrogen. Although these organisms were previously used for the industrial production of solvents, they later fell into disuse, being replaced by more efficient chemical production. A return to a more biological production of solvents therefore requires a thorough understanding of clostridial metabolism. Transcriptome analysis, which reflects the involvement of individual genes in all cellular processes within a population, at any given (sampling) moment, is a valuable tool for gaining a deeper insight into clostridial life. In this review, we describe techniques to study transcription, summarize the evolution of these techniques and compare methods for data processing and visualization of solventogenic clostridia, particularly the species Clostridium acetobutylicum and Clostridium beijerinckii. Individual approaches for evaluating transcriptomic data are compared and their contributions to advancements in the field are assessed. Moreover, utilization of transcriptomic data for reconstruction of computational clostridial metabolic models is considered and particular models are described. Transcriptional changes in glucose transport, central carbon metabolism, the sporulation cycle, butanol and butyrate stress responses, the influence of lignocellulose-derived inhibitors on growth and solvent production, and other respective topics, are addressed and common trends are highlighted., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A new aerotolerant strain of Clostridium beijerinckii LY-5 was isolated from the pit mud of the Chinese Baijiu-making process for butanol production. Plackett-Burman design and artificial neural network were used to optimize the fermentation medium and a total of 13.54 ± 0.22 g/L butanol and 19.91 ± 0.52 g/L ABE were attained under aerotolerant condition. Moreover, distillers' grain waste (DGW), the main by-product in the Baijiu production process, was utilized as potential substrate for butanol production. DGW was hydrolyzed by α-amylase and glucoamylase and then fermented after a detoxifying process of overliming. Butanol and ABE concentrations were 9.02 ± 0.18 and 9.57 ± 0.19 g/L with the yield of 0.21 and 0.23 g/g sugar, respectively. The higher ratio of butanol to ABE might be caused by the inhibitors in DGW medium affecting the metabolic pathways of C. beijerinckii LY-5 and approximately 1.48 ± 0.04 g/L isopropanol was found at the end of fermentation. This work highlights the feasibility of using DGW as a promising feedstock for butanol production by a new aerotolerant strain of C. beijerinckii LY-5, with benefit to the environment., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The main bottleneck in the return of industrial butanol production from renewable feedstock through acetone-butanol-ethanol (ABE) fermentation by clostridia, such as Clostridium beijerinckii, is the low final butanol concentration. The problem is caused by the high toxicity of butanol to the production cells, and therefore, understanding the mechanisms by which clostridia react to butanol shock is of key importance. Detailed analyses of transcriptome data that were obtained after butanol shock and their comparison with data from standard ABE fermentation have resulted in new findings, while confirmed expected population responses. Although butanol shock resulted in upregulation of heat shock protein genes, their regulation is different than was assumed based on standard ABE fermentation transcriptome data. While glucose uptake, glycolysis, and acidogenesis genes were downregulated after butanol shock, solventogenesis genes were upregulated. Cyclopropanation of fatty acids and formation of plasmalogens seem to be significant processes involved in cell membrane stabilization in the presence of butanol. Surprisingly, one of the three identified Agr quorum-sensing system genes was upregulated. Upregulation of several putative butanol efflux pumps was described after butanol addition and a large putative polyketide gene cluster was found, the transcription of which seemed to depend on the concentration of butanol., (© 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Enzymatic hydrolysis of lignocellulose under industrial conditions is prone to contamination by lactic acid bacteria, and in this study, a cellulose hydrolysate produced from dilute-acid pretreatedsugarcane bagasse contained 13 g/L lactic acid and was used for IBE production by Clostridium beijerinckii DSM 6423. In fermentation of the cellulose hydrolysate supplemented with sugarcane molasses for nutrients and buffering of the medium (40 g/L total sugar), 92% of the lactic acid was consumed, and the butanol yield was as high as 0.28 (7.9 g/L butanol), suggesting that lactic acid was preferentially metabolized to butanol. When the hydrolysate was mixed with a detoxified bagasse hemicellulose hydrolysate and supplemented with molasses (35 g/L total sugar), the culture was able to exhaust glucose and utilized sucrose (by 38%), xylose (31%), and lactic acid (70%). Overall, this study shows that C. beijerinckii DSM 6423 can co-ferment first- and second-generation sugars while consuming lactic acid., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Palm oil mill effluent (POME) was tested as a substrate to produce hydrogen by dark fermentation. Two microbial consortia and a pure culture of Clostridium beijerinckii (ATCC 8260) were cultured anaerobically in raw, diluted and hydrolyzed POME to compare biohydrogen production yields in all three media. Experiments were done in 15 mL Hungate tubes containing 5 mL of medium and 1 mL of inoculum. When Clostridium beijerinckii was cultivated at 30 °C in the hydrolyzed POME (P003), containing 7.5 g/L of sucrose, during 8 days of fermentation and 20 % of the inoculum, the maximum biohydrogen production yield was 4.62 LH 2 /L med . Consortium C3 also showed the best production in hydrolyzed POME while consortium C6 achieved its maximum production in raw POME. This effluent is a potential substrate for biohydrogen production., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Sago hampas is a starch-based biomass from sago processing industries consisted of 58% remaining starch. This study has demonstrated the bioconversion of sago hampas to volatile fatty acids (VFAs) by Clostridium beijerinckii SR1 via anaerobic digestion. Higher total VFAs were obtained from sago hampas (5.04 g/L and 0.287 g/g) as compared to commercial starch (5.94 g/L and 0.318 g/g). The physical factors have been investigated for the enhancement of VFAs production using one-factor-at-a-time (OFAT). The optimum condition; 3% substrate concentration, 3 g/L of yeast extract concentration and 2 g/L of ammonium nitrate enhanced the production of VFAs by 52.6%, resulted the total VFAs produced is 7.69 g/L with the VFAs yield of 0.451 g/g. VFAs hydrolysate produced successfully generated 273.4 mV of open voltage circuit and 61.5 mW/m 2 of power density in microbial fuel cells. It was suggested that sago hampas provide as an alternative carbon feedstock for bioelectricity generation.

n -Butyl acetate is an important food additive commonly produced via concentrated sulfuric acid catalysis or immobilized lipase catalysis of butanol and acetic acid. Compared with chemical methods, an enzymatic approach is more environmentally friendly; however, it incurs a higher cost due to lipase production. In vivo biosynthesis via metabolic engineering offers an alternative to produce n -butyl acetate. This alternative combines substrate production (butanol and acetyl-coenzyme A (acetyl-CoA)), alcohol acyltransferase expression, and esterification reaction in one reactor. The alcohol acyltransferase gene ATF1 from Saccharomyces cerevisiae was introduced into Clostridium beijerinckii NCIMB 8052, enabling it to directly produce n -butyl acetate from glucose without lipase addition. Extractants were compared and adapted to realize glucose fermentation with in situ n -butyl acetate extraction. Finally, 5.57 g/L of butyl acetate was produced from 38.2 g/L of glucose within 48 h, which is 665-fold higher than that reported previously. This demonstrated the potential of such a metabolic approach to produce n -butyl acetate from biomass.

Acetone-butanol-ethanol (ABE) fermentation was performed with sugarcane bagasse (SCB) hydrolysate using Clostridium beijerinckii strains. A cost-effective SCB medium was developed with no enzymatic hydrolysis and no supplementation of extra carbon source or expensive nitrogen source. One of the C. beijerinckii strains studied was able to produce butanol with butanol productivity of 1.23 g/L/day with butanol yield of 0.18 g/g of sugars from the developed medium. High utilization rate of both glucose and xylose was observed in SCB medium during ABE fermentation. This study shows that SCB is a promising substrate for cellulosic biobutanol production., (© 2019 International Union of Biochemistry and Molecular Biology, Inc.)

This study addressed the functionality of genetic circuits carrying natural regulatory elements of Clostridium acetobutylicum ATCC 824 in the presence of the respective inducer molecules. Specifically, promoters and their regulators involved in diverse carbon source utilization were characterized using mCherryOpt or beta-galactosidase as a reporter. Consequently, most of the genetic circuits tested in this study were functional in Clostridium acetobutylicum ATCC 824 in the presence of an inducer, leading to the expression of reporter proteins. These genetic sensor-regulators were found to be transferable to another Clostridium species, such as Clostridium beijerinckii NCIMB 8052. The gradual expression of reporter protein was observed as a function of the carbohydrates of interest. A xylose-inducible promoter allows a titratable and robust expression of a reporter protein with stringency and efficacy. This xylose-inducible circuit was seen to enable induction of the expression of reporter proteins in the presence of actual sugar mixtures incorporated in woody hydrolysate wherein glucose and xylose are present as predominant carbon sources.

The production of acetone-butanol-ethanol by solventogenic Clostridium using lignocellulosic biomass can be a potential alternative to petroleum-based butanol. However, previous studies on nondetoxified lignocellulose hydrolysate could not provide better results when compared to those in synthetic medium. In this study, we engineered the pentose pathway of Clostridium beijerinckii NCIMB 8052, which was then subjected to adaptive laboratory evolution in the gradient mixture of synthetic medium and pretreated corn stover enzymatic hydrolysate (CSH) prepared according to the National Renewable Energy Laboratory (NREL) standard. The final resultant strain CIBTS1274A produced 20.7 g/L of total solvents in NREL CSH diluted to 6% initial total sugars, supplemented with ammonium acetate. This performance was comparable with that of corn-based butanol. In addition, this strain was successfully used in the scale-up operation using nondetoxified corn stover and corncob hydrolysate at Lignicell Refining Biotechnologies Ltd., which once was the only commercial biobutanol industry in the world.

Synthetic microbial communities have become a focus of biotechnological research since they can overcome several of the limitations of single-specie cultures. A paradigmatic example is Clostridium cellulovorans DSM 743B, which can decompose lignocellulose but cannot produce butanol. Clostridium beijerinckii NCIMB 8052 however, is unable to use lignocellulose but can produce high amounts of butanol from simple sugars. In our previous studies, both organisms were cocultured to produce butanol by consolidated bioprocessing. However, such consolidated bioprocessing implementation strongly depends on pH regulation. Since low pH (pH 4.5-5.5) is required for butanol fermentation, C. cellulovorans cannot grow well and saccharify sufficient lignocellulose to feed both strains at a pH below 6.4. To overcome this bottleneck, this study engineered C. cellulovorans by adaptive laboratory evolution, inactivating cell wall lyases genes (Clocel_0798 and Clocel_2169), and overexpressing agmatine deiminase genes (augA, encoded by Cbei_1922) from C. beijerinckii NCIMB 8052. The generated strain WZQ36: 743B*6.0*3△lyt0798△lyt2169-(pXY1-P thl -augA) can tolerate a pH of 5.5. Finally, the alcohol aldehyde dehydrogenase gene adhE1 from Clostridium acetobutylicum ATCC 824 was introduced into the strain to enable butanol production at low pH, in coordination with solvent fermentation of C. beijerinckii in consortium. The engineered consortium produced 3.94 g/L butanol without pH control within 83 hr, which is more than 5-fold of the level achieved by wild consortia under the same conditions. This exploration represents a proof of concept on how to combine metabolic and evolutionary engineering to coordinate coculture of a synthetic microbial community., (© 2020 Wiley Periodicals, Inc.)

Production of esters from the acetone-butanol-ethanol (ABE) fermentation by Clostridium often focuses on butyl butyrate, leaving acetone as an undesired product. Addition of butyrate is also often needed because ABE fermentation does not produce enough butyrate. Here we addressed the problems using Clostridium beijerinckii BGS1 that preferred to produce isopropanol instead of acetone, and co-culturing it with Clostridium tyrobutyricum ATCC 25,755 that produced butyrate. Unlike acetone, isopropanol could be converted into ester using lipase and acids. C. tyrobutyricum ATCC 25,755 produced acids at pH 6, while C. beijerinckii BGS1 produced mainly solvents at the same pH. When the two strains were co-cultured, more butyrate was produced, leading to a higher titer of esters than the mono-culture of C. beijerinckii BGS1. As the first study reporting the production of isopropyl butyrate from the Clostridium fermentation, this study highlighted the potential use of lipase and co-culture strategy in ester production.

The AdhR regulatory protein is an activator of σ 54 -dependent transcription of adhA1 and adhA2 genes, which are required for alcohol synthesis in Clostridium beijerinckii Here, we identified the signal perceived by AdhR and determined the regulatory mechanism of AdhR activity. By assaying the activity of AdhR in N-terminally truncated forms, a negative control mechanism of AdhR activity was identified in which the central AAA + domain is subject to repression by the N-terminal GAF and PAS domains. Binding of Fe 2+ to the GAF domain was found to relieve intramolecular repression and stimulate the ATPase activity of AdhR, allowing the AdhR to activate transcription. This control mechanism enables AdhR to regulate transcription of adhA1 and adhA2 in response to cellular redox status. The mutants deficient in AdhR or σ 54 showed large shifts in intracellular redox state indicated by the NADH/NAD + ratio under conditions of increased electron availability or oxidative stress. We demonstrated that the Fe 2+ -activated transcriptional regulator AdhR and σ 54 control alcohol synthesis to maintain redox homeostasis in clostridial cells. Expression of N-terminally truncated forms of AdhR resulted in improved solvent production by C. beijerinckii IMPORTANCE Solventogenic clostridia are anaerobic bacteria that can produce butanol, ethanol, and acetone, which can be used as biofuels or building block chemicals. Here, we show that AdhR, a σ 54 -dependent transcriptional activator, senses the intracellular redox status and controls alcohol synthesis in Clostridium beijerinckii AdhR provides a new example of a GAF domain coordinating a mononuclear non-heme iron to sense and transduce the redox signal. Our study reveals a previously unrecognized functional role of σ 54 in control of cellular redox balance and provides new insights into redox signaling and regulation in clostridia. Our results reveal AdhR as a novel engineering target for improving solvent production by C. beijerinckii and other solventogenic clostridia., (Copyright © 2020 American Society for Microbiology.)

Recent developments in CRISPR technologies have opened new possibilities for improving genome editing tools dedicated to the Clostridium genus. In this study we adapted a two-plasmid tool based on this technology to enable scarless modification of the genome of two reference strains of Clostridium beijerinckii producing an Acetone/Butanol/Ethanol (ABE) or an Isopropanol/Butanol/Ethanol (IBE) mix of solvents. In the NCIMB 8052 ABE-producing strain, inactivation of the SpoIIE sporulation factor encoding gene resulted in sporulation-deficient mutants, and this phenotype was reverted by complementing the mutant strain with a functional spoIIE gene. Furthermore, the fungal cellulase-encoding celA gene was inserted into the C. beijerinckii NCIMB 8052 chromosome, resulting in mutants with endoglucanase activity. A similar two-plasmid approach was next used to edit the genome of the natural IBE-producing strain C. beijerinckii DSM 6423, which has never been genetically engineered before. Firstly, the catB gene conferring thiamphenicol resistance was deleted to make this strain compatible with our dual-plasmid editing system. As a proof of concept, our dual-plasmid system was then used in C. beijerinckii DSM 6423 ΔcatB to remove the endogenous pNF2 plasmid, which led to a sharp increase of transformation efficiencies., Competing Interests: Declaration of Competing Interest The authors declare no financial or commercial conflict of interest., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

This study aimed to produce acetone-butanol-ethanol (ABE) using lignocellulosic crop residues as renewable bioresources. Butanol production from banana crop residue (BCR) was studied using a newly isolated solventogenic Clostridium beijerinckii YVU1. BCR is one of the abundant lignocellulosic substrates available in tropical countries containing 4·3 ± 3·5% cellulose, 28·5 ± 3·0% hemicellulose and 20·3 ± 2·6% lignin. The sequential dilute alkali and acid pretreatments solubilized 69% of lignin and 73% of hemicellulose. Ten percent (w/v) of pretreated substrate was subjected to enzymatic saccharification with cellulase, and it was found to release 0·481 ± 0·035 g glucose per g pretreated biomass. In the batch fermentation process, 20·5 g l -1 ABE (14·0 g l -1 of butanol, 5·4 g l -1 of acetone and 1·1 g l -1 of ethanol) was obtained. The executed fermentation process yielded 0·39 g ABE per g hydrolysate with 0·14 g l -1  h -1 of volumetric productivity. On the basis of the results, we believe that sequential alkali and acid pretreatment on the enzymatic hydrolysis for butanol production is indeed a technology with the potential to be applied and newly isolated. C. beijerinckii YVU1 is also a potential candidate organism for butanol production agricultural residues. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that a banana crop residue (BCR) can be successfully utilized as an inexpensive and alternative bioresource for the production of acetone-butanol-ethanol (ABE). The sequential pretreatment of BCR with alkali and acid solubilized lignin and hemicellulose leading to high glucose release during enzymatic hydrolysis. A newly isolated Clostridium beijerinckii YVU1 was able to produce comparable amount of ABE with previous reports. Therefore, we can state that the utilization of BCR as substrate for C. beijerinckii YVU1 leads to an economical bioprocess for the microbial production of ABE., (© 2019 The Society for Applied Microbiology.)

In-depth knowledge of cell metabolism and nutrient uptake mechanisms can lead to the development of a tool for improving acetone-butanol-ethanol (ABE) fermentation performance and help to overcome bottlenecks in the process, such as the high cost of substrates and low production rates. Over 300 genes potentially encoding transport of amino acids, metal ions, vitamins and carbohydrates were identified in the genome of the butanol-producing strain Clostridium beijerinckii NRRL B-598, based on similarity searches in protein function databases. Transcriptomic data of the genes were obtained during ABE fermentation by RNA-Seq experiments and covered acidogenesis, solventogenesis and sporulation. The physiological roles of the selected 81 actively expressed transport genes were established on the basis of their expression profiles at particular stages of ABE fermentation. This article describes how genes encoding the uptake of glucose, iron, riboflavin, glutamine, methionine and other nutrients take part in growth, production and stress responses of C. beijerinckii NRRL B-598. These data increase our knowledge of transport mechanisms in solventogenic Clostridium and may be used in the selection of individual genes for further research., Competing Interests: The authors have declared that no competing interests exist.

In situ detoxification of lignocellulose-derived microbial inhibitory compounds is an economical strategy for the fermentation of lignocellulose-derived sugars to fuels and chemicals. In this study, we investigated homologous integration and constitutive expression of Cbei_3974 and Cbei_3904, which encode aldo-keto reductase and previously annotated short chain dehydrogenase/reductase, respectively, in Clostridium beijerinckii NCIMB 8052 (Cb), resulting in two strains: Cb_3974 and Cb_3904. Expression of Cbei_3974 led to 2-fold increase in furfural detoxification relative to Cb_3904 and Cb_wild type. Correspondingly, butanol production was up to 1.2-fold greater in furfural-challenged cultures of Cb_3974 relative to Cb_3904 and Cb_wild type. With 4-hydroxybezaldehyde and syringaldehyde supplementation, Cb_3974 showed up to 2.4-fold increase in butanol concentration when compared to Cb_3904 and Cb_wild type. Syringic and vanillic acids were considerably less deleterious to all three strains of Cb tested. Overall, Cb_3974 showed greater tolerance to furfural, 4-hydroxybezaldehyde, and syringaldehyde with improved capacity for butanol production. Hence, development of Cb_3974 represents a significant progress towards engineering solventogenic Clostridium species that are tolerant to lignocellulosic biomass hydrolysates as substrates for ABE fermentation.

The solventogenic C. beijerinckii DSM 6423, a microorganism that naturally produces isopropanol and butanol, was previously modified by random mutagenesis. In this work, one of the resulting mutants was characterized. This strain, selected with allyl alcohol and designated as the AA mutant, shows a dominant production of acids, a severely diminished butanol synthesis capacity, and produces acetone instead of isopropanol. Interestingly, this solvent-deficient strain was also found to have a limited consumption of two carbohydrates and to be still able to form spores, highlighting its particular phenotype. Sequencing of the AA mutant revealed point mutations in several genes including CIBE_0767 (sigL), which encodes the σ 54 sigma factor. Complementation with wild-type sigL fully restored solvent production and sugar assimilation and RT-qPCR analyses revealed its transcriptional control of several genes related to solventogensis, demonstrating the central role of σ 54 in C. beijerinckii DSM 6423. Comparative genomics analysis suggested that this function is conserved at the species level, and this hypothesis was further confirmed through the deletion of sigL in the model strain C. beijerinckii NCIMB 8052.

Yellow Top (Physaria fendleri) is a plant that belongs to the mustard family. This plant is used to produce seeds that are rich in hydroxy oil. After extraction of oil, the presscake is land filled. The seedcake is rich in polymeric sugars and can be used for various bioconversions. For the present case, the seedcake or presscake was hydrolyzed with dilute (0.50% [v/v]) H 2 SO 4 and enzymes to release sugars including glucose, xylose, galactose, arabinose, and mannose. Then, the hydrolyzate was used to produce acetone-butanol-ethanol (ABE). Using 100 gL -1 presscake (prior to pretreatment), 19.22 gL -1 of ABE was successfully produced of which butanol was the major product. In this process, an ABE productivity of 0.48 gL -1 h -1 was obtained. These results are superior to glucose fermentation to produce ABE in which an ABE productivity of 0.42 gL -1 h -1 was obtained. Use of Yellow Top to produce butanol has the following advantages: (i) it is an economic feedstock and is expected to produce butanol economically; (ii) it avoids pollution concerns when not land filled; and (iii) rate of ABE production is not inhibited when fermented this substrate. It is suggested that the potential of this feedstock be further explored by optimizing process parameters for this valuable fermentation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2767, 2019., (© 2018 American Institute of Chemical Engineers.)

Macroalgae (or seaweeds) are considered potential biomass feedstocks for the production of renewable fuels and chemicals. Their sugar composition is different from that of lignocellulosic biomasses, and in green species, including Ulva lactuca , the major sugars are l-rhamnose and d-glucose. C. beijerinckii DSM 6423 utilized these sugars in a U. lactuca hydrolysate to produce acetic acid, butyric acid, isopropanol, butanol, and ethanol (IBE), and 1,2-propanediol. d-Glucose was almost completely consumed in diluted hydrolysates, while l-rhamnose or d-xylose was only partially utilized. In this study, the metabolism of l-rhamnose by C. beijerinckii DSM 6423 was investigated to improve its utilization from natural resources. Fermentations on d-glucose, l-rhamnose, and a mixture of d-glucose and l-rhamnose were performed. On l-rhamnose, the cultures showed low growth and sugar consumption and produced 1,2-propanediol, propionic acid, and n -propanol in addition to acetic and butyric acids, whereas on d-glucose, IBE was the major product. On a d-glucose-l-rhamnose mixture, both sugars were converted simultaneously and l-rhamnose consumption was higher, leading to high levels of 1,2-propanediol (78.4 mM), in addition to 59.4 mM butanol and 31.9 mM isopropanol. Genome and transcriptomics analysis of d-glucose- and l-rhamnose-grown cells revealed the presence and transcription of genes involved in l-rhamnose utilization and in bacterial microcompartment (BMC) formation. These data provide useful insights into the metabolic pathways involved in l-rhamnose utilization and the effects on the general metabolism (glycolysis, early sporulation, and stress response) induced by growth on l-rhamnose. IMPORTANCE A prerequisite for a successful biobased economy is the efficient conversion of biomass resources into useful products, such as biofuels and bulk and specialty chemicals. In contrast to other industrial microorganisms, natural solvent-producing clostridia utilize a wide range of sugars, including C 5 , C 6 , and deoxy-sugars, for production of long-chain alcohols (butanol and 2,3-butanediol), isopropanol, acetone, n -propanol, and organic acids. Butanol production by clostridia from first-generation sugars is already a commercial process, but for the expansion and diversification of the acetone, butanol, and ethanol (ABE)/IBE process to other substrates, more knowledge is needed on the regulation and physiology of fermentation of sugar mixtures. Green macroalgae, produced in aquaculture systems, harvested from the sea or from tides, can be processed into hydrolysates containing mixtures of d-glucose and l-rhamnose, which can be fermented. The knowledge generated in this study will contribute to the development of more efficient processes for macroalga fermentation and of mixed-sugar fermentation in general., (Copyright © 2019 Diallo et al.)

The solventogenic anaerobe Clostridium beijerinckii has potential for use in the sustainable bioconversion of plant-derived carbohydrates into solvents, such as butanol or acetone. However, relatively few strains have been extensively characterised either at the genomic level or through exemplification of a complete genetic toolkit. To remedy this situation, a new strain of C. beijerinckii, NCIMB 14988, is selected from among a total of 55 new clostridial isolates capable of growth on hexose and pentose sugars. Chosen on the basis of its favorable properties, the complete genome sequence of NCIMB 14988 is determined and a high-efficiency plasmid transformation protocol devised. The developed DNA transfer procedure allowed demonstration in NCIMB 14988 of the forward and reverse genetic techniques of transposon mutagenesis and gene knockout, respectively. The latter is accomplished through the successful deployment of both group II intron retargeting (ClosTron) and allelic exchange. In addition to gene inactivation, the developed allelic exchange procedure is used to create point mutations in the chromosome, allowing for the effect of amino acid changes in enzymes involved in primary metabolism to be characterized. ClosTron mediated disruption of the currently unannotated non-coding region between genes LF65_05915 and LF65_05920 is found to result in a non-sporulating phenotype., (© 2018 The Authors. Biotechnology Journal Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Background: Thinning supplies of natural resources increase attention to sustainable microbial production of bio-based fuels. The strain Clostridium beijerinckii NRRL B-598 is a relatively well-described butanol producer regarding its genotype and phenotype under various conditions. However, a link between these two levels, lying in the description of the gene regulation mechanisms, is missing for this strain, due to the lack of transcriptomic data., Results: In this paper, we present a transcription profile of the strain over the whole fermentation using an RNA-Seq dataset covering six time-points with the current highest dynamic range among solventogenic clostridia. We investigated the accuracy of the genome sequence and particular genome elements, including pseudogenes and prophages. While some pseudogenes were highly expressed, all three identified prophages remained silent. Furthermore, we identified major changes in the transcriptional activity of genes using differential expression analysis between adjacent time-points. We identified functional groups of these significantly regulated genes and together with fermentation and cultivation kinetics captured using liquid chromatography and flow cytometry, we identified basic changes in the metabolism of the strain during fermentation. Interestingly, C. beijerinckii NRRL B-598 demonstrated different behavior in comparison with the closely related strain C. beijerinckii NCIMB 8052 in the latter phases of cultivation., Conclusions: We provided a complex analysis of the C. beijerinckii NRRL B-598 fermentation profile using several technologies, including RNA-Seq. We described the changes in the global metabolism of the strain and confirmed the uniqueness of its behavior. The whole experiment demonstrated a good reproducibility. Therefore, we will be able to repeat the experiment under selected conditions in order to investigate particular metabolic changes and signaling pathways suitable for following targeted engineering.

Background: There is a worldwide interest for sustainable and environmentally-friendly ways to produce fuels and chemicals from renewable resources. Among them, the production of acetone, butanol and ethanol (ABE) or Isopropanol, Butanol and Ethanol (IBE) by anaerobic fermentation has already a long industrial history. Isopropanol has recently received a specific interest and the best studied natural isopropanol producer is C. beijerinckii DSM 6423 (NRRL B-593). This strain metabolizes sugars into a mix of IBE with only low concentrations of ethanol produced (< 1 g/L). However, despite its relative ancient discovery, few genomic details have been described for this strain. Research efforts including omics and genetic engineering approaches are therefore needed to enable the use of C. beijerinckii as a microbial cell factory for production of isopropanol., Results: The complete genome sequence and a first transcriptome analysis of C. beijerinckii DSM 6423 are described in this manuscript. The combination of MiSeq and de novo PacBio sequencing revealed a 6.38 Mbp chromosome containing 6254 genomic objects. Three Mobile Genetic Elements (MGE) were also detected: a linear double stranded DNA bacteriophage (ϕ6423) and two plasmids (pNF1 and pNF2) highlighting the genomic complexity of this strain. A first RNA-seq transcriptomic study was then performed on 3 independent glucose fermentations. Clustering analysis allowed us to detect some key gene clusters involved in the main life cycle steps (acidogenesis, solvantogenesis and sporulation) and differentially regulated among the fermentation. These putative clusters included some putative metabolic operons comparable to those found in other reference strains such as C. beijerinckii NCIMB 8052 or C. acetobutylicum ATCC 824. Interestingly, only one gene was encoding for an alcohol dehydrogenase converting acetone into isopropanol, suggesting a single genomic event occurred on this strain to produce isopropanol., Conclusions: We present the full genome sequence of Clostridium beijerinckii DSM 6423, providing a complete genetic background of this strain. This offer a great opportunity for the development of dedicated genetic tools currently lacking for this strain. Moreover, a first RNA-seq analysis allow us to better understand the global metabolism of this natural isopropanol producer, opening the door to future targeted engineering approaches.

Background: Phenolic compounds generated in hydrolysis of lignocellulosic materials are major limiting factors for biological production of solvents by Clostridia, but it lacks the attention on the study of adaptation or resistance mechanisms in response to phenolic compounds., Results: Gene Cbei&#95;3304, encoding a hypothetical membrane transport protein, was analyzed by bioinformatic method. After insertional inactivation of the functionally uncertain gene Cbei&#95;3304 in Clostridium beijerinckii NCIMB 8052, resulted in enhanced phenolic compounds tolerance. Compared to the parent strain C. beijerinckii NCIMB 8052, evaluation of toxicity showed the recombination stain C. beijerinckii 3304::int had a higher level of tolerance to four model phenolic compounds of lignocellulose-derived microbial inhibitory compounds. A comparative transcriptome analysis showed that the genes were involved in membrane transport proteins (ABC and MFS family) and were up-regulated expression after disrupting gene Cbei&#95;3304. Additionally, the adaptation of C. beijerinckii NCIMB 8052 in response to non-detoxified hemicellulosic hydrolysate was improved by disrupting gene Cbei&#95;3304., Conclusion: Toxicity evaluation of lignocellulose-derived phenolic compounds shows that Cbei&#95;3304 plays a significant role in regulating toxicities tolerance for ABE fermentation by C. beijerinckii, and the adaptation of non-detoxified hemicellulosic hydrolysate is significantly improved after inactivation of Cbei&#95;3304 in wild-type strain C. beijerinckii NCIMB 8052. It provided a potential strategy for generating high inhibitor tolerance strains for using lignocellulosic materials to produce solvents by clostridia in this study.

A wild type solventogenic Clostridium beijerinckii NJP7 capable of converting polysaccharides, such as hemicellulose, into butanol and isopropanol via a unique acetone-isopropanol-butanol (AIB) pathway was isolated and characterized. This represents the first wild type isopropanol-butanol generating bacterium which could achieve butanol production directly from lignocellulose through consolidated bioprocessing (CBP). Strain NJP7 was isolated from decomposite soil from Laoshan Nature Park, China, and its genome shows 98.6% identical to 89.5% of the Clostridium diolis submitted genome sequence. The assembled draft genome contains 5.76 Mb and 5101 predicted encoding proteins with a GC content of 29.73%. Among these annotated proteins, hemicellulase and the secondary alcohol dehydrogenase play key roles in achievement of AIB production from hemicellulose through CBP.

Clostridium beijerinckii is a predominant solventogenic clostridia with great attraction for renewable liquid biofuel and biochemical production. Metabolic engineering and synthetic biology can be employed to engineer the strain toward desirable phenotypes. However, current limited information such as promoter strength and gene regulation may hinder the efficient engineering of the strain. To investigate genetic information and complex cellular bioprocesses of C. beijerinckii, an in vivo fluorescence reporter system can be employed. In general, green fluorescence protein (GFP) and relative analogs have been widely used as real-time reporters. However, GFP-family proteins require molecular oxygen for fluorescence maturation. Considering the strict anaerobic growth requirement of the clostridia, an oxygen-independent fluorescence reporter such as a flavin mononucleotide-based fluorescent protein (FbFP) can be used as an alternative fluorescence reporter. In this study, we synthesized and expressed the codon-optimized FbFP gene for C. beijerinckii (CbFbFP) based on the nucleotide sequence of Bacillus subtilis YtvA variant EcFbFP in C. beijerinckii NCIMB 8052 wild-type. Protein expression and in vivo fluorescence of CbFbFP in C. beijeirnckii were confirmed under anaerobic growth conditions. Through fluorescence-activated cell sorting (FACS), we isolated the bright cells from the heterogenous population of C. beijerinckii cells expressing CbFbFP. Several mutations were found in the isolated plasmid which may be responsible for the high-level expression of CbFbFP in C. beijerinckii. The mutant plasmid and CbFbFP reporter were further utilized for strain selection, real-time fluorescence measurement, population analysis, and metabolic engineering in this study., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Apple pomace was studied as a possible raw material for biobutanol production. Five different soft physicochemical pretreatments (autohydrolysis, acids, alkalis, organic solvents and surfactants) were compared in a high-pressure reactor, whose working parameters (temperature, time and reagent concentration) were optimised to maximise the amount of simple sugars released and to minimise inhibitor generation. The pretreated biomass was subsequently subjected to a conventional enzymatic treatment to complete the hydrolysis. A thermal analysis (DSC) of the solid biomass indicated that lignin was mainly degraded during the enzymatic treatment. The hydrolysate obtained with the surfactant polyethylene glycol 6000 (PEG 6000) (1.96% w/w) contained less inhibitors than any other pretreatment, yet providing 42 g/L sugars at relatively mild conditions (100 °C, 5 min), and was readily fermented by Clostridium beijerinckii CECT 508 in 96 h (3.55 g/L acetone, 9.11 g/L butanol, 0.26 g/L ethanol; 0.276 g B /g S yield; 91% sugar consumption). Therefore, it is possible to optimise pretreatment conditions of lignocellulosic apple pomace to reduce inhibitor concentrations in the final hydrolysate and perform successful ABE fermentations without the need of a detoxification stage.

Xylose is the second most abundant sugar derived from lignocellulose; it is considered less desirable than glucose for fermentation, and strategies that specifically increase xylose utilization in wild-type cells are goals for biofuel production. Xylose consumption, butanol production, and hydrogen production increased in both Clostridium beijerinckii and a novel solventogenic bacterium (strain DC-1) when anthraquinone-2,6,-disulfonate (AQDS) or riboflavin were used as redox mediators to transfer electrons to poorly crystalline Fe(OH) 3 as an extracellular electron sink. Strain DC-1 was most closely related to Rhizobiales bacterium Mfc52 based on 95% 16S rRNA gene sequence similarity, which demonstrates that this response is not limited to a single genus of xylose-fermenting bacteria. Xylose utilization and butanol production were negligible in control incubations containing cells plus 3% (w/v) xylose alone during a 10-day batch fermentation, for both strains tested (n-butanol titers of 0.05 g L -1 ). Micromolar concentrations of AQDS and riboflavin were added as electron shuttling compounds with poorly crystalline Fe(OH) 3 as an insoluble electron acceptor, and respective n-butanol titers increased to 6.35 and 7.46 g L -1 . Increases in xylose consumption for the iron treatments were relatively high, from less than 0.49 g L -1 (xylose alone, no iron or electron shuttling molecules) to 25.98 and 29.15 g L -1 for the AQDS and riboflavin treatments, respectively. Hydrogen production was also 3.68 times greater for the AQDS treatment and 5.27 greater for the riboflavin treatment relative to controls. Strain DC-1 data were similar, again indicating that the effects are not specific to the genus Clostridium.

Clostridium beijerinckii is a predominant solventogenic bacterium that is used for the ABE fermentation. Various C. beijerinckii mutants are constructed for desirable phenotypes. The C. beijerinckii mutant BA105 harboring a glucose derepression phenotype was previously isolated and demonstrated the enhanced amylolytic activity in the presence of glucose. Despite its potential use, BA105 is not further characterized and utilized. Therefore, the authors investigate fermentation phenotypes of BA105 in this study. Under the typical batch fermentation conditions, BA105 consistently exhibits acid crash phenotype resulting in limited glucose uptake and cell growth. However, when the culture pH is maintained above 5.5, BA105 exhibits the increased glucose uptake and butanol production than did the wild-type. To further analyze BA105, the authors perform genome sequencing and RNA sequencing. Genome analysis identifies two SNPs unique to BA105, in the upstream region of AbrB regulator (Cbei&#95;4885) and the ROK family glucokinase (Cbei&#95;4895) which are involved in catabolite repression and regulation of sugar metabolism. Transcriptional analysis of BA105 reveals significant differential expression of the genes associated with the PTS sugar transport system and acid production. This study improves understanding of the acid crash phenomenon and provides the genetic basis underlying the catabolite derepression phenotype of C. beijericnkii., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The production of acetone, butanol and ethanol by fermentation of renewable biomass has potential to become a valuable industrial process. Mechanisms of solvent production and sporulation involve some common regulators in some ABE-producing clostridia, although details of the links between the pathways are not clear. In this study, we compare a wild-type (WT) Clostridium beijerinckii NRRL B-598 with its mutant strain OESpo0A, in which the gene encoding Spo0A, an important regulator of both sporulation and solventogenesis, is overexpressed in terms of solvent and acid production. We also compare morphologies during growth on two different media: TYA broth, where the WT culture sporulates, and RCM, where the WT culture does not. In addition, RT-qPCR-based analysis of expression profiles of spo0A, spoIIE, sigG, spoVD, ald and buk1 genes involved in sporulation or solvent production in these strains, were compared. The OESpo0A mutant did not produce spores and butanol titre was lower compared to the WT, but increased amounts of butyric acid and ethanol were produced. The gene spo0A had high levels of expression in the WT under non-sporulating culture conditions while other selected genes for sporulation factors were downregulated significantly. Similar observations were obtained for OESpo0A where spo0A overexpression and downregulation of other sporulation genes were demonstrated. Higher expression of spo0A led to higher expression of buk1 and ald, which could confirm the role of spo0A in activation of the solventogenic pathway, although solvent production was not affected significantly in the WT and was weakened in the OESpo0A mutant.

Recent efforts to combat increasing greenhouse gas emissions include their capture into advanced biofuels, such as butanol. Traditionally, biobutanol research has been centered solely on its generation from sugars. Our results show partial re-assimilation of CO 2 and H 2 by n-butanol-producer C. beijerinckii. This was detected as synchronous CO 2 /H 2 oscillations by direct (real-time) monitoring of their fermentation gasses. Additional functional analysis demonstrated increased total carbon recovery above heterotrophic values associated to mixotrophic assimilation of synthesis gas (H 2 , CO 2 and CO). This was further confirmed using 13 C-Tracer experiments feeding 13 CO 2 and measuring the resulting labeled products. Genome- and transcriptome-wide analysis revealed transcription of key C-1 capture and additional energy conservation genes, including partial Wood-Ljungdahl and complete reversed pyruvate ferredoxin oxidoreductase / pyruvate-formate-lyase-dependent (rPFOR/Pfl) pathways. Therefore, this report provides direct genetic and physiological evidences of mixotrophic inorganic carbon-capture by C. beijerinckii.

The biosynthesis of plasmalogens in anaerobic bacteria differs fundamentally from that in animal cells. Firstly, the formation of the alk-1'-enyl ether bond in animal cells is oxygen dependent. Secondly, the first step in plasmalogen formation in animal cells is an acylation of dihydroxyacetone phosphate, which has been ruled out as a precursor in anaerobes. In bacteria the alk-1'-enyl ether bond is formed after the fully formed acyl glycerolipids are synthesized. Evidence will be presented for the conversion of the sn-1 acyl-linked chain to an O-alk-1'-enyl ether by an as yet unknown mechanism., (© 2017 Federation of European Biochemical Societies.)

Low oxygen tolerance and substrate restriction continues to hamper the process of biobutanol industrialization. In this work, butanol fermentation with cocultures of Bacillus cereus China General Microbiological Culture Collection Center (CGMCC) 1.895 and Clostridium beijerinckii NCIMB 8052 under nonanaerobic conditions was investigated, and the interactions between these two strains were examined. The addition of B. cereus CGMCC 1.895 resulted in higher oxygen tolerance and a wider range of substrate utilization, compared with the pure culture of C. beijerinckii NCIMB 8052. Butanol concentration reached 10.49 g/L with an optimized inoculation size of 90% under nonanaerobic conditions, and this concentration was close to that of pure C. beijerinckii NCIMB 8052 culture under anaerobic conditions. Dynamic relative abundance analysis demonstrated that the ratio of C. beijerinckii NCIMB 8052 accounted for nearly 99% of the cocultured cells. Furthermore, the substrate utilization range was expanded, allowing the use of corn mash for butanol production. The final concentration of butanol and total solvents was 6.78 and 10.52 g/L, respectively. Coculture also was performed successfully in a 5-L fermenter and 8.75 g/L butanol was obtained. Dynamic dissolved oxygen analysis demonstrated that B. cereus consumed the dissolved oxygen in the broth and resulted in the anaerobic condition for C. beijerinckii., (© 2016 International Union of Biochemistry and Molecular Biology, Inc.)

d-xylose, the main building block of plant biomass, is a pentose sugar that can be used by bacteria as a carbon source for bio-based fuel and chemical production through fermentation. In bacteria, the first step for d-xylose metabolism is signal perception at the membrane. We previously identified a three-component system in Firmicutes bacteria comprising a membrane-associated sensor protein (XylFII), a transmembrane histidine kinase (LytS) for periplasmic d-xylose sensing, and a cytoplasmic response regulator (YesN) that activates the transcription of the target ABC transporter xylFGH genes to promote the uptake of d-xylose. The molecular mechanism underlying signal perception and integration of these processes remains elusive, however. Here we purified the N-terminal periplasmic domain of LytS (LytSN) in a complex with XylFII and determined the conformational structures of the complex in its d-xylose-free and d-xylose-bound forms. LytSN contains a four-helix bundle, and XylFII contains two Rossmann fold-like globular domains with a xylose-binding cleft between them. In the absence of d-xylose, LytSN and XylFII formed a heterodimer. Specific binding of d-xylose to the cleft of XylFII induced a large conformational change that closed the cleft and brought the globular domains closer together. This conformational change led to the formation of an active XylFII-LytSN heterotetramer. Mutations at the d-xylose binding site and the heterotetramer interface diminished heterotetramer formation and impaired the d-xylose-sensing function of XylFII-LytS. Based on these data, we propose a working model of XylFII-LytS that provides a molecular basis for d-xylose utilization and metabolic modification in bacteria., Competing Interests: The authors declare no conflict of interest.

Clostridium beijerinckii 4693:int with high ferulic acid (FA) tolerance was engineered and characterized in our lab. In this study, the minimum inhibition concentrations of FA against C. beijerinckii NCIMB 8052 (wild-type) and 4693:int were 1.0 and 1.5g/l, respectively; cell viability was 18.5% and 106.7%, respectively, in the presence of 0.5g/l FA. A comparative transcriptome analysis was carried out at two different growth stages to evaluate sensitivity to FA. Genes that were differentially expressed included those related to redox and associated cofactors, riboflavin metabolism, two-component system, glycolysis and butanoate metabolism, and DNA replication as well as those encoding ATP-binding cassette transporters. Cbei&#95;2134 and Cbei&#95;2135 encoding alkyl hydroperoxide reductases are thought to be involved in antibacterial and adaptation mechanisms in C. beijerinckii in the presence of FA., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Clostridium saccharoperbutylacetonicum N1-4 is well known as a hyper-butanol-producing strain. However, the lack of genetic engineering tools hinders further elucidation of its solvent production mechanism and development of more robust strains. In this study, we set out to develop an efficient genome engineering system for this microorganism based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (CRISPR-Cas9) system. First, the functionality of the CRISPR-Cas9 system previously customized for Clostridium beijerinckii was evaluated in C. saccharoperbutylacetonicum by targeting pta and buk , two essential genes for acetate and butyrate production, respectively. pta and buk single and double deletion mutants were successfully obtained based on this system. However, the genome engineering efficiency was rather low (the mutation rate is <20%). Therefore, the efficiency was further optimized by evaluating various promoters for guide RNA (gRNA) expression. With promoter P J23119 , we achieved a mutation rate of 75% for pta deletion without serial subculturing as suggested previously for C. beijerinckii Thus, this developed CRISPR-Cas9 system is highly desirable for efficient genome editing in C. saccharoperbutylacetonicum Batch fermentation results revealed that both the acid and solvent production profiles were altered due to the disruption of acid production pathways; however, neither acetate nor butyrate production was eliminated with the deletion of the corresponding gene. The butanol production, yield, and selectivity were improved in mutants, depending on the fermentation medium. In the pta buk double deletion mutant, the butanol production in P2 medium reached 19.0 g/liter, which is one of the highest levels ever reported from batch fermentations. IMPORTANCE An efficient CRISPR-Cas9 genome engineering system was developed for C. saccharoperbutylacetonicum N1-4. This paves the way for elucidating the solvent production mechanism in this hyper-butanol-producing microorganism and developing strains with desirable butanol-producing features. This tool can be easily adapted for use in closely related microorganisms. As also reported by others, here we demonstrated with solid data that the highly efficient expression of gRNA is the key factor determining the efficiency of CRISPR-Cas9 for genome editing. The protocol developed in this study can provide essential references for other researchers who work in the areas of metabolic engineering and synthetic biology. The developed mutants can be used as excellent starting strains for development of more robust ones for desirable solvent production., (Copyright © 2017 American Society for Microbiology.)

Producing biobutanol from lignocellulosic biomass has shown promise to ultimately reduce greenhouse gases and alleviate the global energy crisis. However, because of the recalcitrance of a lignocellulosic biomass, a pretreatment of the substrate is needed which in many cases releases soluble lignin compounds (SLCs), which inhibit growth of butanol-producing clostridia. In this study, we found that SLCs changed the acetone/butanol ratio (A/B ratio) during butanol fermentation. The typical A/B molar ratio during Clostridium beijerinckii NCIMB 8052 batch fermentation with glucose as the carbon source is about 0.5. In the present study, the A/B molar ratio during batch fermentation with a lignocellulosic hydrolysate as the carbon source was 0.95 at the end of fermentation. Structural and redox potential changes of the SLCs were characterized before and after fermentation by using gas chromatography/mass spectrometry and electrochemical analyses, which indicated that some exogenous SLCs were involved in distributing electron flow to C. beijerinckii , leading to modulation of the redox balance. This was further demonstrated by the NADH/NAD + ratio and trxB gene expression profile assays at the onset of solventogenic growth. As a result, the A/B ratio of end products changed significantly during C. beijerinckii fermentation using corn stover-derived hydrolysate as the carbon source compared to glucose as the carbon source. These results revealed that SLCs not only inhibited cell growth but also modulated the A/B ratio during C. beijerinckii butanol fermentation. IMPORTANCE Bioconversion of lignocellulosic feedstocks to butanol involves pretreatment, during which hundreds of soluble lignin compounds (SLCs) form. Most of these SLCs inhibit growth of solvent-producing clostridia. However, the mechanism by which these compounds modulate electron flow in clostridia remains elusive. In this study, the results revealed that SLCs changed redox balance by producing oxidative stress and modulating electron flow as electron donors. Production of H 2 and acetone was stimulated, while butanol production remained unchanged, which led to a high A/B ratio during C. beijerinckii fermentation using corn stover-derived hydrolysate as the carbon source. These observations provide insight into utilizing C. beijerinckii to produce butanol from a lignocellulosic biomass., (Copyright © 2017 American Society for Microbiology.)

Spo0A is a master regulator that governs the metabolic shift of solventogenic Clostridium species such as Clostridium beijerinckii. Its disruption can thus potentially cause a significant alteration of cellular physiology as well as metabolic patterns. To investigate the specific effect of spo0A disruption in C. beijerinckii, a spo0A mutant of C. beijerinckii was characterized in this study. In a batch fermentation with pH control at 6.5, the spo0A mutant accumulated butyrate and butanol up to 8.96 g/L and 3.32 g/L, respectively from 60 g/L glucose. Noticing the unique phenotype of the spo0A mutant accumulating both butyrate and butanol at significant concentrations, we decided to use the spo0A mutant for the production of butyl butyrate that can be formed by the condensation of butyrate and butanol during the ABE fermentation in the presence of the enzyme lipase. Butyl butyrate is a value-added chemical that has numerous uses in the food and fragrance industry. Moreover, butyl butyrate as a biofuel is compatible with Jet A-1 aviation kerosene and used for biodiesel enrichment. In an initial trial of small-scale extractive batch fermentation using hexadecane as the extractant with supplementation of lipase CalB, the spo0A mutant was subjected to acid crash due to the butyrate accumulation, and thus produced only 98 mg/L butyl butyrate. To alleviate the butyrate toxicity, the biphasic medium was supplemented with 10 g/L CaCO 3 and 5 g/L butanol. The butyl butyrate production was then increased up to 2.73 g/L in the hexadecane layer. When continuous agitation was performed to enhance the esterification and extraction of butyl butyrate, 3.32 g/L butyl butyrate was obtained in the hexadecane layer. In this study, we successfully demonstrated the use of the C. beijerinckii spo0A mutant for the butyl butyrate production through the simultaneous ABE fermentation, condensation, and extraction. Biotechnol. Bioeng. 2017;114: 106-112. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Degenerate Clostridium beijerinckii strain (DG-8052) can be partially recovered by supplementing CaCO 3 to fermentation media. Genome resequencing of DG-8052 showed no general regulator mutated. This study focused on transcriptional analysis of DG-8052 and its response to CaCO 3 treatment via microarray. The expressions of 5168 genes capturing 98.6% of C. beijerinckii NCIMB 8052 genome were examed. The results revealed that with addition of CaCO 3 565 and 916 genes were significantly up-regulated, and 704 and 1044 genes significantly down-regulated at acidogenic and solventogenic phase of DG-8052, respectively. These genes are primarily responsible for glycolysis to solvent/acid production (poR, pfo), solventogensis (buk, ctf, aldh, adh, bcd) and sporulation (spo0A, sigE, sigma-70, bofA), cell motility and division (ftsA, ftsK, ftsY, ftsH, ftsE, mreB, mreC, mreD, rodA), and molecular chaperones (grpE, dnaK, dnaJ, hsp20, hsp90), etc. The functions of some altered genes in DG-8052, totalling 5.7% at acidogenisis and 8.0% at sovlentogenisis, remain unknown. The response of the degenerate strain to CaCO 3 was suggested significantly pleiotropic. This study reveals the multitude of regulatory function that CaCO 3 has in clostridia and provides detailed insights into degeneration mechanisms at gene regulation level. It also enables us to develop effective strategies to prevent strain degeneration in future.

Lignocellulose-derived microbial inhibitors (LDMICs) prevent efficient fermentation of Miscanthus giganteus (MG) hydrolysates to fuels and chemicals. To address this problem, we explored detoxification of pretreated MG biomass by Cupriavidus basilensis ATCC(®)BAA-699 prior to enzymatic saccharification. We document three key findings from our test of this strategy to alleviate LDMIC-mediated toxicity on Clostridium beijerinckii NCIMB 8052 during fermentation of MG hydrolysates. First, we demonstrate that growth of C. basilensis is possible on furfural, 5-hydroxymethyfurfural, cinnamaldehyde, 4-hydroxybenzaldehyde, syringaldehyde, vanillin, and ferulic, p-coumaric, syringic and vanillic acid, as sole carbon sources. Second, we report that C. basilensis detoxified and metabolized ~98 % LDMICs present in dilute acid-pretreated MG hydrolysates. Last, this bioabatement resulted in significant payoffs during acetone-butanol-ethanol (ABE) fermentation by C. beijerinckii: 70, 50 and 73 % improvement in ABE concentration, yield and productivity, respectively. Together, our results show that biological detoxification of acid-pretreated MG hydrolysates prior to fermentation is feasible and beneficial.

This article aims to validate the use of calorimetry to measure the growth of anaerobic microbes. It has been difficult to monitor the growth of strict anaerobes while maintaining optimal growth conditions. Traditionally, optical density and ATP concentration are usually used as measures of the growth of anaerobic microbes. However, to take these measurements it is necessary to extract an aliquot of the culture, which can be difficult while maintaining anaerobic conditions. In this study, calorimetry was used to continuously and nondestructively measure the heat generated by the growth of anaerobic microbes as a function of time. Clostridium acetobutylicum, Clostridium beijerinckii, and Clostridium cellulovorans were used as representative anaerobic microbes. Using a multiplex isothermal calorimeter, we observed that peak time (tp) of C. acetobutylicum heat evolution increased as the inoculation rate decreased. This strong correlation between the inoculation rate and tp showed that it was possible to measure the growth rate of anaerobic microbes by calorimetry. Overall, our results showed that there is a very good correlation between heat evolution and optical density/ATP concentration, validating the use of the method., (Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

A mutant strain of Clostridium beijerinckii, with high tolerance to ferulic acid, was generated using atmospheric pressure glow discharge and high-throughput screening of C. beijerinckii NCIMB 8052. The mutant strain M11 produced 7.24 g/L of butanol when grown in P2 medium containing 30 g/L of glucose and 0.5 g/L of ferulic acid, which is comparable to the production from non-ferulic acid cultures (8.11 g/L of butanol). When 0.8 g/L of ferulic acid was introduced into the P2 medium, C. beijerinckii M11 grew well and produced 4.91 g/L of butanol. Both cell growth and butanol production of C. beijerinckii M11 were seriously inhibited when 0.9 g/L of ferulic acid was added into the P2 medium. Furthermore, C. beijerinckii M11 could produce 6.13 g/L of butanol using non-detoxified hemicellulosic hydrolysate from diluted sulfuric acid-treated corn fiber (SAHHC) as the carbon source. These results demonstrate that C. beijerinckii M11 has a high ferulic acid tolerance and is able to use non-detoxified SAHHC for butanol production., (© 2015 International Union of Biochemistry and Molecular Biology, Inc.)