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Butanol inhibits bacterial activity by destroying the cell membrane of Clostridium acetobutylicum strains and altering functionality. Butanol toxicity also results in destruction of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS), thereby preventing glucose transport and phosphorylation and inhibiting transmembrane transport and assimilation of sugars, amino acids, and other nutrients. In this study, based on the addition of exogenous butanol, the tangible macro indicators of changes in the carbon ion beam irradiation-mutant Y217 morphology were observed using scanning electron microscopy (SEM). The mutant has lower microbial adhesion to hydrocarbon (MATH) value than C. acetobutylicum ATCC 824 strain. FDA fluorescence intensity and conductivity studies demonstrated the intrinsically low membrane permeability of the mutant membrane, with membrane potential remaining relatively stable. Monounsaturated FAs (MUFAs) accounted for 35.17% of the mutant membrane, and the saturated fatty acids (SFA)/unsaturated fatty acids (UFA) ratio in the mutant cell membrane was 1.65. In addition, we conducted DNA-level analysis of the mutant strain Y217. Expectedly, through screening, we found gene mutant sites encoding membrane-related functions in the mutant, including ATP-binding cassette (ABC) transporter-related genes, predicted membrane proteins, and the PTS transport system. It is noteworthy that an unreported predicted membrane protein (CAC 3309) may be related to changes in mutant cell membrane properties. KEY POINTS: • Mutant Y217 exhibited better membrane integrity and permeability. • Mutant Y217 was more resistant to butanol toxicity. • Some membrane-related genes of mutant Y217 were mutated.

Clostridium autoethanogenum and Clostridium ljungdahlii are physiologically and genetically very similar strict anaerobic acetogens capable of growth on carbon monoxide as sole carbon source. While exact nutritional requirements have not been reported, we observed that for growth, the addition of vitamins to media already containing yeast extract was required, an indication that these are fastidious microorganisms. Elimination of complex components and individual vitamins from the medium revealed that the only organic compounds required for growth were pantothenate, biotin and thiamine. Analysis of the genome sequences revealed that three genes were missing from pantothenate and thiamine biosynthetic pathways, and five genes were absent from the pathway for biotin biosynthesis. Prototrophy in C. autoethanogenum and C. ljungdahlii for pantothenate was obtained by the introduction of plasmids carrying the heterologous gene clusters panBCD from Clostridium acetobutylicum, and for thiamine by the introduction of the thiC-purF operon from Clostridium ragsdalei. Integration of panBCD into the chromosome through allele-coupled exchange also conveyed prototrophy. C. autoethanogenum was converted to biotin prototrophy with gene sets bioBDF and bioHCA from Desulfotomaculum nigrificans strain CO-1-SRB, on plasmid and integrated in the chromosome. The genes could be used as auxotrophic selection markers in recombinant DNA technology. Additionally, transformation with a subset of the genes for pantothenate biosynthesis extended selection options with the pantothenate precursors pantolactone and/or beta-alanine. Similarly, growth was obtained with the biotin precursor pimelate combined with genes bioYDA from C. acetobutylicum. The work raises questions whether alternative steps exist in biotin and thiamine biosynthesis pathways in these acetogens. Electronic supplementary material The online version of this article (10.1007/s00253-019-09763-6) contains supplementary material, which is available to authorized users.

Butyl butyrate (BB) has been widely used as a flavor and fragrance compound in the beverage, food, perfume, and cosmetic industries. Currently, BB is produced through two-step processes; butanol and butyrate are first produced and are used as precursors for the esterification reactions to yield BB in the next step. Recently, an alternative process to the current process has been developed by using microorganisms for the one-pot BB production. In the one-pot BB process, alcohol acyl transferases (AATs) and lipases play roles in the esterification of butanol together with their co-substrates butyryl-CoA and butyrate, respectively. In this paper, we review the characteristics of two enzymes including AAT and lipase in the esterification reaction. Also, we review the one-pot processes for BB production by employing the wild-type and engineered Clostridium species and the engineered Escherichia coli strains, with the combination of AATs and lipases.

Butanol inhibits bacterial activity by destroying the cell membrane of Clostridium acetobutylicum strains and altering functionality. Butanol toxicity also results in destruction of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS), thereby preventing glucose transport and phosphorylation and inhibiting transmembrane transport and assimilation of sugars, amino acids, and other nutrients. In this study, based on the addition of exogenous butanol, the tangible macro indicators of changes in the carbon ion beam irradiation-mutant Y217 morphology were observed using scanning electron microscopy (SEM). The mutant has lower microbial adhesion to hydrocarbon (MATH) value than C. acetobutylicum ATCC 824 strain. FDA fluorescence intensity and conductivity studies demonstrated the intrinsically low membrane permeability of the mutant membrane, with membrane potential remaining relatively stable. Monounsaturated FAs (MUFAs) accounted for 35.17% of the mutant membrane, and the saturated fatty acids (SFA)/unsaturated fatty acids (UFA) ratio in the mutant cell membrane was 1.65. In addition, we conducted DNA-level analysis of the mutant strain Y217. Expectedly, through screening, we found gene mutant sites encoding membrane-related functions in the mutant, including ATP-binding cassette (ABC) transporter-related genes, predicted membrane proteins, and the PTS transport system. It is noteworthy that an unreported predicted membrane protein (CAC 3309) may be related to changes in mutant cell membrane properties. KEY POINTS: • Mutant Y217 exhibited better membrane integrity and permeability. • Mutant Y217 was more resistant to butanol toxicity. • Some membrane-related genes of mutant Y217 were mutated.

Butyl butyrate is widely used as a fragrance additive for foods and beverages. The first step in the currently used process is the production of precursors, including butanol and butyrate, from petroleum using chemical catalysts, followed by the conversion of precursors to butyl butyrate by immobilized lipase. In this work, we engineered Clostridium acetobutylicum for the selective, one-step production of butyl butyrate from glucose. C. acetobutylicum ATCC 824, possessing a strong carbon flux that yields butanol and butyryl-CoA, was selected as a host and was engineered by introducing alcohol acyltransferases (AATs) from Fragaria x ananassa (strawberry) or Malus sp. (apple). Batch culture of the engineered C. acetobutylicum strain CaSAAT expressing the strawberry SAAT gene produced 50.07 mg/L of butyl butyrate with a selectivity of 84.8% of total esters produced. Also, the engineered C. acetobutylicum strain CaAAAT expressing the apple AAAT gene produced 40.60 mg/L of butyl butyrate with a selectivity of 87.4%. This study demonstrated the feasibility of the one-step fermentation of butyl butyrate from glucose in the engineered C. acetobutylicum, as a proof of concept.

Butanol fermentation comprises two successive and distinct stages, namely acidogenesis and solventogenesis. The current lack of clarity regarding the underlying metabolic regulation of fermentation impedes improvements in biobutanol production. Here, a proteomics study was performed in the acidogenesis phase, the lowest pH point (transition point), and the solventogenesis phase in the butanol-producing symbiotic system TSH06. Forty-two Clostridium acetobutylicum proteins demonstrated differential expression levels at different stages. The protein level of butanol dehydrogenase increased in the solventogenesis phase, which was in accordance with the trend of butanol concentration. Stress proteins were upregulated either at the transition point or in the solventogenesis phase. The cell division-related protein Maf was upregulated at the transition point. We disrupted the maf gene in C. acetobutylicum TSH1, and Bacillus cereus TSH2 was added to form a new symbiotic system. TSH06△maf produced 13.9 ± 1.0 g/L butanol, which was higher than that of TSH06 (12.3 ± 0.9 g/L). Butanol was furtherly improved in fermentation at variable temperature with neutral red addition for both TSH06 and TSH06△maf. The butanol titer of the maf deletion strain was higher than that of the wild type, although the exact mechanism remains to be determined.

Despite the long-term interest in solventogenic clostridia-based ABE (acetone-butanol-ethanol) fermentation, clostridial butanol tolerance and its underlying mechanism remain poorly understood, which is a major obstacle hindering further improvements of this important fermentative process. In this study, a two-component system (TCS), BtrK/BtrR, was identified and demonstrated to positively regulate butanol tolerance and ABE solvent formation in Clostridium acetobutylicum, a representative species of solventogenic clostridia. The transcriptomic analysis results showed that BtrK/BtrR has a pleiotropic regulatory function, affecting a large number of crucial genes and metabolic pathways. Of the differentially expressed genes, btrTM, encoding a putative ABC-type transporter (named BtrTM), was shown to be under the direct control of BtrR, the response regulator of the BtrK/BtrR TCS. Furthermore, BtrTM was shown to contribute to more butanol tolerance (46.5% increase) by overexpression, revealing a novel regulatory mechanism consisting of the BtrK/BtrR TCS and the BtrTM transporter in C. acetobutylicum. Based on these findings, we achieved faster growth and solvent production of C. acetobutylicum by overexpressing BtrK/BtrR or its direct target BtrTM, although no significant improvement in the final butanol titer and yield. These results further confirm the importance of BtrK/BtrR and BtrTM in this organism. Also, of significance, a specific number of btrR-btrT-btrM-btrK-like gene clusters were identified in other Clostridium species, including the pathogens Clostridium perfringens and Clostridium botulinum, indicating a broad role for this regulatory module in the class Clostridia.

Clostridia are Gram-positive, spore-forming, obligate anaerobic bacteria that can produce solvents such as acetone, ethanol, and butanol, which can be used as biofuels or building block chemicals. Many successful attempts have been made to improve solvent yield and titer from sugars through metabolic engineering of solventogenic and acidogenic clostridia. More recently, cellulolytic and acetogenic clostridia have also attracted high interests for their ability to utilize low-cost renewable substrates such as cellulose and syngas. Process engineering such as in situ butanol recovery and consolidated bioprocessing (CBP) has been developed for improved solvent titer and productivity. This review focuses on metabolic and process engineering strategies for solvent production from sugars, lignocellulosic biomass, and syngas by various clostridia, including conventional solventogenic Clostridium acetobutylicum, engineered acidogens such as C. tyrobutyricum and C. cellulovorans, and carboxydotrophic acetogens such as C. carboxidivorans and C. ljungdahlii.

In this study, the integrated in situ extraction-gas stripping process was coupled with continuous ABE fermentation using immobilized Clostridium acetobutylicum. At the same time, oleyl alcohol was cocurrently flowed into the packed bed reactor with the fresh medium and then recycled back to the packed bed reactor after removing butanol in the stripper. A high glucose consumption of 52 g/L and a high butanol productivity of 11 g/L/h were achieved, resulting in a high butanol yield of 0.21 g-butanol/g-glucose. This can be attributed to both the high bacterial activity for solvent production as well as a threefold increase in the bacterial density inside the packed bed reactor. Also reported is that 64 % of the butanol produced can be recovered by the integrated in situ extraction-gas stripping process. A high butanol productivity and a high glucose consumption were simultaneously achieved.

Cyanobacteria are oxygen-evolving photosynthetic bacteria. Established genetic manipulation methods and recently developed gene-regulation tools have enabled the photosynthetic conversion of carbon dioxide to biofuels and valuable chemicals in cyanobacteria, especially in unicellular cyanobacteria. However, the oxygen sensitivity of enzyme(s) introduced into cyanobacteria hampers productivity in some cases. Anabaena sp. PCC 7120 is a filamentous cyanobacterium consisting of a few hundred of vegetative cells, which perform oxygenic photosynthesis. Upon nitrogen deprivation, heterocysts, which are specialized cells for nitrogen fixation, are differentiated from vegetative cells at semiregular intervals. The micro-oxic environment within heterocysts protects oxygen-labile nitrogenase from oxygen. This study aimed to repurpose the heterocyst as a host for the production of chemicals with oxygen-sensitive enzymes under photosynthetic conditions. Herein, Anabaena strains expressing enzymes of 1-butanol synthetic pathway from the anaerobe Clostridium acetobutylicum within heterocysts were created. A strain that expressed a highly oxygen-sensitive Bcd/EtfAB complex produced 1-butanol even under photosynthetic conditions. Furthermore, the 1-butanol production per heterocyst cell of a butanol-producing Anabaena strain was fivefold higher than that per cell of unicellular cyanobacterium with the same set of 1-butanol synthetic pathway genes. Thus, our study showed the usefulness of Anabaena heterocysts as a chassis for anaerobic production driven by oxygen-evolving photosynthesis.

Vitamin B1 (VB1) is an essential coenzyme for carbohydrate metabolism and involved in energy generation in most organisms. In this study, we found that insufficient biosynthesis of VB1 in Clostridium acetobutylicum ATCC 824 is a major limiting factor for efficient acetone-butanol-ethanol (ABE) fermentation. In order to improve the fermentation performance of C. acetobutylicum ATCC 824, the VB1 biosynthesis pathway was strengthened by overexpressing the thiC, thiG, and thiE genes. The engineered strain 824(thiCGE) showed enhanced VB1 and energy synthesis, resulting in better growth, faster sugar consumption, higher solvents production, and lower acids formation than the wild-type strain in both VB1 free and normal P2 medium (1 mg/L). Compared with the wild-type strain, 824(thiCGE) produced 13.0 ± 0.1% or 12.7 ± 1.2% more butanol in VB1 free P2 medium when glucose or xylose was used as the substrate, respectively. When mixed sugar (glucose:xylose = 2:1) was used as the substrate in VB1 free P2 medium, the xylose consumption rate and butanol titer of 824(thiCGE) were 45.8 ± 1.9% and 20.4 ± 0.3% higher than those of the wild-type strain. All these results demonstrated that this metabolic engineering strategy could provide a new and effective way to improve the cellular performance of solventogenic clostridia. In addition, it may have some potential application value in ABE fermentation using simple medium and/or lignocellulosic biomass.

Clostridium tyrobutyricum does not have the enzymes needed for using maltose or starch. Two extracellular α-glucosidases encoded by agluI and agluII from Clostridium acetobutylicum ATCC 824 catalyzing the hydrolysis of α-1,4-glycosidic bonds in maltose and starch from the non-reducing end were cloned and expressed in C. tyrobutyricum (Δack, adhE2), and their effects on n-butanol production from maltose and soluble starch in batch fermentations were studied. Compared to the parental strain grown on glucose, mutants expressing agluI showed robust activity in breaking down maltose and produced more butanol (17.2 vs. 9.5 g/L) with a higher butanol yield (0.20 vs. 0.10 g/g) and productivity (0.29 vs. 0.16 g/L h). The mutant was also able to use soluble starch as substrate, although at a slower rate compared to maltose. Compared to C. acetobutylicum ATCC 824, the mutant produced more butanol from maltose (17.2 vs. 11.2 g/L) and soluble starch (16.2 vs. 8.8 g/L) in batch fermentations. The mutant was stable in batch fermentation without adding antibiotics, achieving a high butanol productivity of 0.40 g/L h. This mutant strain thus can be used in industrial production of n-butanol from maltose and soluble starch.

The overexpression of CoA transferase (ctfAB), which catalyzes the reaction: acetate/butyrate + acetoacetyl-CoA → acetyl/butyryl-CoA + acetoacetate, was studied for its effects on acid reassimilation and butanol biosynthesis in Clostridium tyrobutyricum (Δack, adhE2). The plasmid pMTL007 was used to co-express adhE2 and ctfAB from Clostridium acetobutylicum ATCC 824. In addition, the sol operon containing ctfAB, adc (acetoacetate decarboxylase), and ald (aldehyde dehydrogenase) was also cloned from Clostridium beijerinckii NCIMB 8052 and expressed in C. tyrobutyricum (Δack, adhE2). Mutants expressing these genes were evaluated for their ability to produce butanol from glucose in batch fermentations at pH 5.0 and 6.0. Compared to C. tyrobutyricum (Δack, adhE2) without expressing ctfAB, all mutants with ctfAB overexpression produced more butanol, with butanol yield increased to 0.22 - 0.26 g/g (vs. 0.10 - 0.13 g/g) and productivity to 0.35 g/l h (vs. 0.13 g/l h) because of the reduced acetate and butyrate production. The expression of ctfAB also resulted in acetone production from acetoacetate through a non-enzymatic decarboxylation.

Biomass plastics are expected to contribute to the establishment of a carbon-neutral society by replacing conventional plastics derived from petroleum. The biomass-derived aromatic amine 4-aminocinnamic acid (4ACA) produced by recombinant bacteria is applied to the synthesis of high-performance biopolymers such as polyamides and polyimides. Here, we developed a microbial catalyst that hydrogenates the α,β-unsaturated carboxylic acid of 4ACA to generate 4-aminohydrocinnamic acid (4AHCA). The ability of 10 microbial genes for enoate and xenobiotic reductases expressed in Escherichia coli to convert 4ACA to 4AHCA was assessed. A strain producing 2-enoate reductase from Clostridium acetobutylicum (ca2ENR) reduced 4ACA to 4AHCA with a yield of > 95% mol mol-1 and reaction rates of 3.4 ± 0.4 and 4.4 ± 0.6 mM h-1 OD600-1 at the optimum pH of 7.0 under aerobic and anaerobic conditions, respectively. This recombinant strain reduced caffeic, cinnamic, coumaric, and 4-nitrocinnamic acids to their corresponding propanoic acid derivatives. We polycondensed 4AHCA generated from biomass-derived 4ACA by dehydration under a catalyst to form high-molecular-weight poly(4AHCA) with a molecular weight of M n = 1.94 MDa. This polyamide had high thermal properties as indicated by a 10% reduction in weight at a temperature of T d10 = 394 °C and a glass transition temperature of T g = 240 °C. Poly(4AHCA) derived from biomass is stable at high temperatures and could be applicable to the production of high-performance engineering plastics.

Clostridium tyrobutyricum is a promising organism for butyrate and n-butanol production, but cannot grow on sucrose. Three genes (scrA, scrB, and scrK) involved in the sucrose catabolic pathway, along with an aldehyde/alcohol dehydrogenase gene, were cloned from Clostridium acetobutylicum and introduced into C. tyrobutyricum (Δack) with acetate kinase knockout. In batch fermentation, the engineered strain Ct(Δack)-pscrBAK produced 14.8–18.8 g/L butanol, with a high butanol/total solvent ratio of ∼0.94 (w/w), from sucrose and sugarcane juice. Moreover, stable high butanol production with a high butanol yield of 0.25 g/g and productivity of 0.28 g/L∙h was obtained in batch fermentation without using antibiotics for selection pressure, suggesting that Ct(Δack)-pscrBAK is genetically stable. Furthermore, sucrose utilization by Ct(Δack)-pscrBAK was not inhibited by glucose, which would usually cause carbon catabolite repression on solventogenic clostridia. Ct(Δack)-pscrBAK is thus advantageous for use in biobutanol production from sugarcane juice and other sucrose-rich feedstocks.

Clostridium acetobutylicum is a model organism for the biotechnologically important acetone-butanol-ethanol (ABE) fermentation. With the objective to rationally develop strains with improved butanol production, detailed insights into the physiological and genetic mechanisms of solvent production are required. Therefore, pH-controlled phosphate-limited chemostat cultivation and DNA microarray technology were employed for an in-depth analysis of knockout mutants with defects in the central fermentative metabolism. The set of studied mutants included strains with inactivated phosphotransacetylase (pta), phosphotransbutyrylase (ptb), and acetoacetate decarboxylase (adc) encoding genes, as well as an adc/pta double knockout mutant. A comprehensive physiological characterization of the mutants was performed by continuous cultivation, allowing for a well-defined separation of acidogenic and solventogenic growth, combined with the advantage of the high reproducibility of steady-state conditions. The ptb-negative strain C. acetobutylicum ptb::int(87) exhibited the most striking metabolite profile: Sizable amounts of butanol (29 ± 1.3 mM) were already produced during acidogenic growth. The product patterns of the mutants as well as accompanying transcriptomic data are presented and discussed.

The renewed interests in clostridial acetone-butanol-ethanol (ABE) fermentation as a next-generation biofuel source led to significantly intensified research in the past few years. This mini-review focuses on the current status of metabolic engineering techniques available for the model organism of ABE fermentation, Clostridium acetobutylicum. A comprehensive survey of various application examples covers two general issues related to both basic and applied research questions: (i) how to improve biofuel production and (ii) what information can be deduced from respective genotype/phenotype manipulations. Recently developed strategies to engineer C. acetobutylicum are summarized including the current portfolio of altered gene expression methodologies, as well as systematic (rational) and explorative (combinatorial) metabolic engineering approaches.

Solvent screening for in situ liquid extraction of products from acetone-butanol-ethanol (ABE) fermentation was carried out, taking into account biological parameters (biocompatibility, bioavailability, and product yield) and extraction performance (partition coefficient and selectivity) determined in real fermentation broth. On the basis of different solvent characteristics obtained from literature, 16 compounds from different chemical families were selected and experimentally evaluated for their extraction capabilities in a real ABE fermentation broth system. From these compounds, nine potential solvents were also tested for their biocompatibility towards Clostridium acetobutylicum. Moreover, bioavailability and differences in substrate consumption and total n-butanol production with respect to solvent-free fermentations were quantified for each biocompatible solvent. Product yield was enhanced in the presence of organic solvents having higher affinity for butanol and butyric acid. Applying this methodology, it was found that the Guerbet alcohol 2-butyl-1-octanol presented the best extracting characteristics (the highest partition coefficient (6.76) and the third highest selectivity (644)), the highest butanol yield (27.4 %), and maintained biocompatibility with C. acetobutylicum.

This study elucidated the importance of two critical enzymes in the regulation of butanol production in Clostridium acetobutylicum ATCC 824. Overexpression of both the 6-phosphofructokinase (pfkA) and pyruvate kinase (pykA) genes increased intracellular concentrations of ATP and NADH and also resistance to butanol toxicity. Marked increases of butanol and ethanol production, but not acetone, were also observed in batch fermentation. The butanol and ethanol concentrations were 29.4 and 85.5 % higher, respectively, in the fermentation by double-overexpressed C. acetobutylicum ATCC 824/pfkA+pykA than the wild-type strain. Furthermore, when fed-batch fermentation using glucose was carried out, the butanol and total solvent (acetone, butanol, and ethanol) concentrations reached as high as 19.12 and 28.02 g/L, respectively. The reason for improved butanol formation was attributed to the enhanced NADH and ATP concentrations and increased tolerance to butanol in the double-overexpressed strain.

In response to changing extracellular pH levels, phosphate-limited continuous cultures of Clostridium acetobutylicum reversibly switches its metabolism from the dominant formation of acids to the prevalent production of solvents. Previous experimental and theoretical studies have revealed that this pH-induced metabolic switch involves a rearrangement of the intracellular transcriptomic, proteomic and metabolomic composition of the clostridial cells. However, the influence of the population dynamics on the observations reported has so far been neglected. Here, we present a method for linking the pH shift, clostridial growth and the acetone-butanol-ethanol fermentation metabolic network systematically into a model which combines the dynamics of the external pH and optical density with a metabolic model. Furthermore, the recently found antagonistic expression pattern of the aldehyde/alcohol dehydrogenases AdhE1/2 and pH-dependent enzyme activities have been included into this combined model. Our model predictions reveal that the pH-induced metabolic shift under these experimental conditions is governed by a phenotypic switch of predominantly acidogenic subpopulation towards a predominantly solventogenic subpopulation. This model-driven explanation of the pH-induced shift from acidogenesis to solventogenesis by population dynamics casts an entirely new light on the clostridial response to changing pH levels. Moreover, the results presented here underline that pH-dependent growth and pH-dependent specific enzymatic activity play a crucial role in this adaptation. In particular, the behaviour of AdhE1 and AdhE2 seems to be the key factor for the product formation of the two phenotypes, their pH-dependent growth, and thus, the pH-induced metabolic switch in C. acetobutylicum.

In this study, a novel system for synthesis of 2-butanone from levulinic acid (γ-keto-acid) via an enzymatic reaction was developed. Acetoacetate decarboxylase (AADC; E.C. 4.1.1.4) from Clostridium acetobutylicum was selected as a biocatalyst for decarboxylation of levulinic acid. The purified recombinant AADC from Escherichia coli successfully converted levulinic acid to 2-butanone with a conversion yield of 8.4-90.3 % depending on the amount of AADC under optimum conditions (30 °C and pH 5.0) despite that acetoacetate, a β-keto-acid, is a natural substrate of AADC. In order to improve the catalytic efficiency, an AADC-mediator system was tested using methyl viologen, methylene blue, azure B, zinc ion, and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as mediators. Among them, methyl viologen showed the best performance, increasing the conversion yield up to 6.7-fold in comparison to that without methyl viologen. The results in this study are significant in the development of a renewable method for the synthesis of 2-butanone from biomass-derived chemical, levulinic acid, through enzymatic decarboxylation.

Efficient regeneration of NADPH is one of the limiting factors that constrain the productivity of biotransformation processes. In order to increase the availability of NADPH for enhanced biotransformation by engineered Escherichia coli, modulation of the pentose phosphate pathway and amplification of the transhydrogenases system have been conventionally attempted as primary solutions. Recently, other approaches for stimulating NADPH regeneration during glycolysis, such as replacement of native glyceradehdye-3-phosphate dehydrogenase (GAPDH) with NADP-dependent GAPDH from Clostridium acetobutylicum and introduction of NADH kinase catalyzing direct phosphorylation of NADH to NADPH from Saccharomyces cerevisiae, were attempted and resulted in remarkable impacts on NADPH-dependent bioprocesses. This review summarizes several metabolic engineering approaches used for improving the NADPH regenerating capacity in engineered E. coli for whole-cell-based bioprocesses and discusses the key features and progress of those attempts.

Clostridial acetone-butanol-ethanol (ABE) fermentation features a remarkable shift in the cellular metabolic activity from acid formation, acidogenesis, to the production of industrial-relevant solvents, solventogensis. In recent decades, mathematical models have been employed to elucidate the complex interlinked regulation and conditions that determine these two distinct metabolic states and govern the transition between them. In this review, we discuss these models with a focus on the mechanisms controlling intra- and extracellular changes between acidogenesis and solventogenesis. In particular, we critically evaluate underlying model assumptions and predictions in the light of current experimental knowledge. Towards this end, we briefly introduce key ideas and assumptions applied in the discussed modelling approaches, but waive a comprehensive mathematical presentation. We distinguish between structural and dynamical models, which will be discussed in their chronological order to illustrate how new biological information facilitates the ‘evolution’ of mathematical models. Mathematical models and their analysis have significantly contributed to our knowledge of ABE fermentation and the underlying regulatory network which spans all levels of biological organization. However, the ties between the different levels of cellular regulation are not well understood. Furthermore, contradictory experimental and theoretical results challenge our current notion of ABE metabolic network structure. Thus, clostridial ABE fermentation still poses theoretical as well as experimental challenges which are best approached in close collaboration between modellers and experimentalists.

Butanol is an ideal renewable biofuel which possesses superior fuel properties. Previously, butanol-producing symbiotic system TSH06 was isolated in our lab, with microoxygen tolerance ability. To boost butanol yield for large-scale industrial production, TSH06 was used as parental strain and subjected to atmospheric and room temperature plasma (ARTP) and four rounds of genome shuffling (GS). ARTP mutant and GS strain were co-cultured with facultative anaerobic Bacillus cereus TSH2 to form a symbiotic system with microoxygen tolerance, which was then subjected to fermentation. Relative messenger RNA (mRNA) level of key enzyme gene was measured by real-time PCR. The highest butanol titer of TS4-30 reached 15.63 g/L, which was 34% higher than TSH06 (12.19 g/L). Compared with parental strain, mRNA of acid-forming gene in TS4-30 decreased in acidogenesis phase, while solvent-forming gene increased in solventogenesis phase. This gene expression pattern was consistent with high butanol yield and low acid level in TS4-30. In summary, symbiotic system TS4-30 was obtained with butanol titer improvement and microoxygen tolerance.

Clostridium acetobutylicum is an important industrial microorganism for n-butanol bioproduction, and its transcription factor AbrB0310 regulates various important cellular processes. However, the roles of two abrB homologues, abrB1941 and abrB3647, have not been determined because they appear inactive during transcription. Here, we performed a detailed investigation into the function of abrB1941 and abrB3647 in C. acetobutylicum. Interestingly, we observed that AbrB3647 exerts an important influence on biphasic fermentation that opposes the influence of AbrB0310, while AbrB1941 might not be essential. When abrB3647 was disrupted using the Targetron system, a greatly improved cellular growth occurred. The following analysis shows that all three AbrBs participated in metabolically regulating acidogenesis, solventogenesis, and a two-phase transition in C. acetobutylicum, but the AbrB0310 and AbrB3647 functions were the most important. Moreover, the target genes subject to AbrB0310 and AbrB3647 regulation closely overlap. Based on these results, we will better understand the roles of the three AbrBs in regulating solventogenic clostridia cell physiology.

The transformation of trinitrotoluene (TNT) by several mutant strains of Clostridium acetobutylicum has been examined to analyze the maximal rate of initial transformation, determine the effects of metabolic mutations of the host on transformation rate, and to assess the cell metabolic changes brought about during TNT transformation. Little difference in the maximal rate of TNT degradation in early acid phase cultures was found between the parental ATCC 824 strain and strains altered in the acid forming pathways (phosphotransacetylase, or butyrate kinase) or in a high-solvent-producing strain (mutant B). This result is in agreement with the previous findings of a similar degradation rate in a degenerate strain (M5) that had lost the ability to produce solvent. A series of antisense constructs were made that reduced the expression of hydA, encoding the Fe-hydrogenase, or hydE and hydF, genes encoding hydrogenase maturating proteins. While the antisense hydA strain had only ∼30 % of the activity of wild type, the antisense hydE strain exhibited a TNT degradation rate around 70 % that of the parent. Overexpression of hydA modestly increased the TNT degradation rate in acid phase cells, suggesting the amount of reductant flowing into hydrogenase rather than the hydrogenase level itself was a limiting factor in many situations. The redox potential, hydrogen evolution, and organic acid metabolites produced during rapid TNT transformation in early log phase cultures were measured. The redox potential of the acid-producing culture decreased from -370 to -200 mV immediately after addition of TNT and the hydrogen evolution rate decreased, lowering the hydrogen to carbon dioxide ratio from 1.4 to around 1.1 for 15 min. During the time of TNT transformation, the treated acidogenic cells produced less acetate and more butyrate. The results show that during TNT transformation, the cells shift metabolism away from hydrogen formation to reduction of TNT and the resulting effects on cell redox cofactors generate a higher proportion of butyrate.

In this study, we employed TiO₂ enrichment and high accuracy liquid chromatography-mass spectrometry-mass spectrometry to identify the phosphoproteome of Clostridium acetobutyicum ATCC824 in acidogenesis and solventogenesis. As many as 82 phosphopeptides in 61 proteins, with 107 phosphorylated sites on serine, threonine, or tyrosine, were identified with high confidence. We detected 52 phosphopeptides from 44 proteins in acidogenesis and 70 phosphopeptides from 51 proteins in solventogenesis, respectively. Bioinformatic analysis revealed most of the phosphoproteins located in cytoplasm and participated in carbon metabolism. Based on comparison between the two stages, we found 27 stage-specific phosphorylated proteins (10 in acidogenesis and 17 in solventogenesis), some of which were solvent production-related enzymes and metabolic regulators, showed significantly different phosphorylated status. Further analysis indicated that protein phosphorylation could be involved in the shift of stages or in solvent production pathway directly. Comparison against several other organisms revealed the evolutionary diversity among them on phosphorylation level in spite of their high homology on protein sequence level.

Biosynthesis of acetone and n-butanol is naturally restricted to the group of solventogenic clostridia with Clostridium acetobutylicum being the model organism for acetone-butanol-ethanol (ABE) fermentation. According to limited genetic tools, only a few rational metabolic engineering approaches were conducted in the past to improve the production of butanol, an advanced biofuel. In this study, a phosphotransbutyrylase-(Ptb) negative mutant, C. acetobutylicum ptb::int(87), was generated using the ClosTron methodology for targeted gene knock-out and resulted in a distinct butyrate-negative phenotype. The major end products of fermentation experiments without pH control were acetate (3.2 g/l), lactate (4.0 g/l), and butanol (3.4 g/l). The product pattern of the ptb mutant was altered to high ethanol (12.1 g/l) and butanol (8.0 g/l) titers in pH ≥ 5.0-regulated fermentations. Glucose fed-batch cultivation elevated the ethanol concentration to 32.4 g/l, yielding a more than fourfold increased alcohol to acetone ratio as compared to the wildtype. Although butyrate was never detected in cultures of C. acetobutylicum ptb::int(87), the mutant was still capable to take up butyrate when externally added during the late exponential growth phase. These findings suggest that alternative pathways of butyrate re-assimilation exist in C. acetobutylicum, supposably mediated by acetoacetyl-CoA:acyl-CoA transferase and acetoacetate decarboxylase, as well as reverse reactions of butyrate kinase and Ptb with respect to previous studies.

Solventogenic clostridia are characterised by their biphasic fermentative metabolism, and the main final product n-butanol is of particular industrial interest because it can be used as a superior biofuel. During exponential growth, Clostridium acetobutylicum synthesises acetic and butyric acids which are accompanied by the formation of molecular hydrogen and carbon dioxide. During the stationary phase, the solvents acetone, butanol and ethanol are produced. However, the molecular mechanisms of this metabolic switch are largely unknown so far. In this study, in silico, in vitro and in vivo analyses were performed to elucidate the function of the CAC2713-encoded redox-sensing transcriptional repressor Rex and its role in the solventogenic shift of C. acetobutylicum ATCC 824. Electrophoretic mobility shift assays showed that Rex controls the expression of butanol biosynthetic genes as a response to the cellular NADH/NAD(+) ratio. Interestingly, the Rex-negative mutant C. acetobutylicum rex::int(95) produced high amounts of ethanol and butanol, while hydrogen and acetone production were significantly reduced. Both ethanol and butanol (but not acetone) formation started clearly earlier than in the wild type. In addition, the rex mutant showed a de-repression of the bifunctional aldehyde/alcohol dehydrogenase 2 encoded by the adhE2 gene (CAP0035) as demonstrated by increased adhE2 expression as well as high NADH-dependent alcohol dehydrogenase activities. The results presented here clearly indicated that Rex is involved in the redox-dependent solventogenic shift of C. acetobutylicum.

The working temperature of a photobioreactor under sunlight can be elevated above the optimal growth temperature of a microorganism. To improve the biohydrogen productivity of photosynthetic bacteria at higher temperatures, a [FeFe]-hydrogenase gene from the thermophile Clostridium thermocellum was expressed in the mesophile Rhodopseudomonas palustris CGA009 (strain CGA-CThydA) using a log-phase expression promoter P( pckA ) to drive the expression of heterogeneous hydrogenase gene. In contrast, a mesophilic Clostridium acetobutylicum [FeFe]-hydrogenase gene was also constructed and expressed in R. palustris (strain CGA-CAhydA). Both transgenic strains were tested for cell growth, in vivo hydrogen production rate, and in vitro hydrogenase activity at elevated temperatures. Although both CGA-CThydA and CGA-CAhydA strains demonstrated enhanced growth over the vector control at temperatures above 38 °C, CGA-CThydA produced more hydrogen than the other strains. The in vitro hydrogenase activity assay, measured at 40 °C, confirmed that the activity of the CGA-CThydA hydrogenase was higher than the CGA-CAhydA hydrogenase. These results showed that the expression of a thermophilic [FeFe]-hydrogenase in R. palustris increased the growth rate and biohydrogen production at elevated temperatures. This transgenic strategy can be applied to a broad range of purple photosynthetic bacteria used to produce biohydrogen under sunlight.

Continuous production of acetone, n-butanol, and ethanol (ABE) was carried out using immobilized cells of Clostridium acetobutylicum DSM 792 using glucose and sugar mixture as a substrate. Among various lignocellulosic materials screened as a support matrix, coconut fibers and wood pulp fibers were found to be promising in batch experiments. With a motive of promoting wood-based bio-refinery concept, wood pulp was used as a cell holding material. Glucose and sugar mixture (glucose, mannose, galactose, arabinose, and xylose) comparable to lignocellulose hydrolysate was used as a substrate for continuous production of ABE. We report the best solvent productivity among wild-type strains using column reactor. The maximum total solvent concentration of 14.32 g L(-1) was obtained at a dilution rate of 0.22 h(-1) with glucose as a substrate compared to 12.64 g L(-1) at 0.5 h(-1) dilution rate with sugar mixture. The maximum solvent productivity (13.66 g L(-1) h(-1)) was obtained at a dilution rate of 1.9 h(-1) with glucose as a substrate whereas solvent productivity (12.14 g L(-1) h(-1)) was obtained at a dilution rate of 1.5 h(-1) with sugar mixture. The immobilized column reactor with wood pulp can become an efficient technology to be integrated with existing pulp mills to convert them into wood-based bio-refineries.

The complex changes in the life cycle of Clostridium acetobutylicum, a promising biofuel producer, are not well understood. During exponential growth, sugars are fermented to acetate and butyrate, and in the transition phase, the metabolism switches to the production of the solvents acetone and butanol accompanied by the initiation of endospore formation. Using phosphate-limited chemostat cultures at pH 5.7, C. acetobutylicum was kept at a steady state of acidogenic metabolism, whereas at pH 4.5, the cells showed stable solvent production without sporulation. Novel proteome reference maps of cytosolic proteins from both acidogenesis and solventogenesis with a high degree of reproducibility were generated. Yielding a 21% coverage, 15 protein spots were specifically assigned to the acidogenic phase, and 29 protein spots exhibited a significantly higher abundance in the solventogenic phase. Besides well-known metabolic proteins, unexpected proteins were also identified. Among these, the two proteins CAP0036 and CAP0037 of unknown function were found as major striking indicator proteins in acidogenic cells. Proteome data were confirmed by genome-wide DNA microarray analyses of the identical cultures. Thus, a first systematic study of acidogenic and solventogenic chemostat cultures is presented, and similarities as well as differences to previous studies of batch cultures are discussed. Electronic supplementary material The online version of this article (doi:10.1007/s00253-010-2741-x) contains supplementary material, which is available to authorized users.

A Lactobacillus brevis strain with the ability to synthesize butanol from glucose was constructed by metabolic engineering. The genes crt, bcd, etfB, etfA, and hbd, composing the bcs-operon, and the thl gene encode the enzymes of the lower part of the clostridial butanol pathway (crotonase, butyryl-CoA-dehydrogenase, two subunits of the electron transfer flavoprotein, 3-hydroxybutyryl-CoA dehydrogenase, and thiolase) of Clostridium acetobutylicum. They were cloned into the Gram-positive/Gram-negative shuttle plasmid vector pHYc. The two resulting plasmids pHYc-thl-bcs and pHYc-bcs (respectively, with and without the clostridial thl gene) were transferred to Escherichia coli and L. brevis. The recombinant L. brevis strains were able to synthesize up to 300 mg l(-1) or 4.1 mM of butanol on a glucose-containing medium. A L. brevis strain carrying the clostridial bcs-operon has the ability to synthesize butanol with participation of its own thiolase, aldehyde dehydrogenase, and alcohol dehydrogenase. The particular role of the enzymes involved in butanol production and the suitability of L. brevis as an n-butanol producer are discussed.