Synthetic co-culture of autotrophic Clostridium carboxidivorans and chain elongating Clostridium kluyveri monitored by flow cytometry

Syngas fermentation with acetogens is known to produce mainly acetate and ethanol efficiently. Co-cultures with chain elongating bacteria making use of these products are a promising approach to produce longer-chain alcohols. Synthetic co-cultures with identical initial cell concentrations of Clostridium carboxidivorans and Clostridium kluyveri were studied in batch-operated stirred-tank bioreactors with continuous CO/CO₂-gassing and monitoring of the cell counts of both clostridia by flow cytometry after fluorescence in situ hybridization (FISH-FC). At 800 mbar CO, chain elongation activity was observed at pH 6.0, although growth of C. kluyveri was restricted. Organic acids produced by C. kluyveri were reduced by C. carboxidivorans to the corresponding alcohols butanol and hexanol. This resulted in a threefold increase in final butanol concentration and enabled hexanol production compared with a mono-culture of C. carboxidivorans. At 100 mbar CO, growth of C. kluyveri was improved; however, the capacity of C. carboxidivorans to form alcohols was reduced. Because of the accumulation of organic acids, a constant decay of C. carboxidivorans was observed. The measurement of individual cell concentrations in co-culture established in this study may serve as an effective tool for knowledge-based identification of optimum process conditions for enhanced formation of longer-chain alcohols by clostridial co-cultures.     

Genomic Analysis of Carbon Monoxide Utilization and Butanol Production by Clostridium carboxidivorans Strain P7T

Increasing demand for the production of renewable fuels has recently generated a particular interest in microbial production of butanol. Anaerobic bacteria, such as Clostridium spp., can naturally convert carbohydrates into a variety of primary products, including alcohols like butanol. The genetics of microorganisms like Clostridium acetobutylicum have been well studied and their solvent-producing metabolic pathways characterized. In contrast, less is known about the genetics of Clostridium spp. capable of converting syngas or its individual components into solvents. In this study, the type of strain of a new solventogenic Clostridium species, C. carboxidivorans, was genetically characterized by genome sequencing. C. carboxidivorans strain P7^T possessed a complete Wood-Ljungdahl pathway gene cluster, involving CO and CO₂ fixation and conversion to acetyl-CoA. Moreover, with the exception of an acetone production pathway, all the genetic determinants of canonical ABE metabolic pathways for acetate, butyrate, ethanol and butanol production were present in the P7^T chromosome. The functionality of these pathways was also confirmed by growth of P7^T on CO and production of CO₂ as well as volatile fatty acids (acetate and butyrate) and solvents (ethanol and butanol). P7^T was also found to harbour a 19 Kbp plasmid, which did not include essential or butanol production related genes. This study has generated in depth knowledge of the P7^T genome, which will be helpful in developing metabolic engineering strategies to improve C. carboxidivorans''s natural capacity to produce potential biofuels from syngas.     

Growth rate dependence of solventogenesis and solvents produced byClostridium beijerinckii

Summary Acidogenesis and solventogenesis byClostridium beijerinckii NRRL B-593 has been studied in batch growth, and in sucrose-limited chemostat and recycling fermentor growth. Cells grown in batch culture without pH control primarily produced either butyric and acetic acids, or these acids plus butanol, ethanol and isopropanol in ratios depending on the medium's content of reducing agent, calcium and iron. Cells in chemostat-culture at a mass doubling time (t_d) of 5.8 h produced primarily butyric and acetic acids at pH 6.8 and these acids plus butanol, ethanol and isopropanol at pH 4.8. Cells grown in a recycling fermentor (in which the t_d continuously increases) at pH 6.8 entered solventogenesis at a t_d of 43 h, producing primarily propanol, ethanol and butanol, along with butyric acid, but with greatly decreased production of acetic acid. Although clostridial form morphology, succeeded by sporulation, usually accompanied solventogenesis, the association was not invariant so that solventogenesis and sporogenesis can occur separately in this species.     

SpoIIE regulates sporulation but does not directly affect solventogenesis in Clostridium acetobutylicum ATCC 824

Using gene expression reporter vectors, we examined the activity of the spoIIE promoter in wild-type and spo0A-deleted strains of Clostridium acetobutylicum ATCC 824. In wild-type cells, the spoIIE promoter is active in a transient manner during late solventogenesis, but in strain SKO1, where the sporulation initiator spo0A is disrupted, no spoIIE promoter activity is detectable at any stage of growth. Strains 824(pMSpo) and 824(pASspo) were created to overexpress spoIIE and to decrease spoIIE expression via antisense RNA targeted against spoIIE, respectively. Some cultures of strains 824(pMSpo) degenerated during fermentations by losing the pSOL1 megaplasmid and hence did not produce the solvents ethanol, acetone, and butanol. The frequent degeneration event was shown to require an intact copy of spoIIE. Nondegenerate cultures of 824(pMSpo) exhibited normal growth and solvent production. Strain 824(pASspo) exhibited prolonged solventogenesis characterized by increased production of ethanol (225%), acetone (43%), and butanol (110%). Sporulation in strains harboring pASspo was significantly delayed, with sporulating cells exhibiting altered morphology. These results suggest that SpoIIE has no direct effect on the control of solventogenesis and that the changes in solvent production in spoIIE-downregulated cells are mediated by effects on the cell during sporulation.     

Deciphering mixotrophic Clostridium formicoaceticum metabolism and energy conservation: Genomic analysis and experimental studies

Clostridium formicoaceticum, a Gram-negative mixotrophic homoacetogen, produces acetic acid as the sole metabolic product from various carbon sources, including fructose, glycerol, formate, and CO₂. Its genome of 4.59-Mbp contains a highly conserved Wood-Ljungdahl pathway gene cluster with the same layout as that in other mixotrophic acetogens, including Clostridium aceticum, Clostridium carboxidivorans, and Clostridium ljungdahlii. For energy conservation, C. formicoaceticum does not have all the genes required for the synthesis of cytochrome or quinone used for generating proton gradient in H⁺-dependent acetogens such as Moorella thermoacetica; instead, it has the Rnf system and a Na⁺-translocating ATPase similar to the one in Acetobacterium woodii. Its growth in both heterotrophic and autotrophic media were dependent on the sodium concentration. C. formicoaceticum has genes encoding acetaldehyde dehydrogenases, alcohol dehydrogenases, and aldehyde oxidoreductases, which could convert acetyl-CoA and acetate to ethanol and butyrate to butanol under excessive reducing equivalent conditions.     

Characterization of enzymes involved in the ethanol production of <Emphasis Type="Italic">Moorella</Emphasis> sp. HUC22-1

Since the thermophilic bacterium Moorella sp. HUC22-1 produces 120 mM acetate and 5.2 mM ethanol from H₂–CO₂, several candidate genes, which were predicted to code for three alcohol dehydrogenases (AdhA, B, C) and one acetaldehyde dehydrogenase (Aldh), were cloned from HUC22-1. The cloned genes were subcloned into a His-tagged expression vector and expressed in Escherichia coli. Recombinant AdhA and B were both dependent on NADP(H) but independent of NAD(H), and their reduction activities from aldehyde to alcohol were higher than their oxidation activities. In contrast with AdhA and B, no activity of AdhC was observed in either reaction. On the other hand, Aldh was active toward both NADP(H) and NAD(H). The enzyme activity of Aldh was directed toward the thioester cleavage and the thioester condensation. When 50 μg of AdhA and 50 μg Aldh were added to the buffer solution (pH 8.0) containing NADPH, NADH and acetyl-CoA at 60°C, 1.6 mM ethanol was produced from 3 mM acetyl-CoA after 90 min. Expression analysis of the mRNAs revealed that the expression level of aldh was threefold higher in the H₂–CO₂ culture than that in the fructose culture, but levels of adhA, B and C were decreased.     

Effects of zinc on the production of alcohol by <Emphasis Type="Italic">Clostridium carboxidivorans</Emphasis> P7 using model syngas

Renewable energy, including biofuels such as ethanol and butanol from syngas bioconversed by Clostridium carboxidivorans P7, has been drawing extensive attention due to the fossil energy depletion and global eco-environmental issues. Effects of zinc on the growth and metabolites of C. carboxidivorans P7 were investigated with model syngas as the carbon source. The cell concentration was doubled, the ethanol content increased 3.02-fold and the butanol content increased 7.60-fold, the hexanol content increased 44.00-fold in the medium with 280 μM Zn²⁺, when comparing with those in the control medium [Zn²⁺, (7 μM)]. Studies of the genes expression involved in the carbon fixation as well as acid and alcohol production in the medium with 280 μM Zn²⁺ indicated that fdhII was up-regulated on the second day, acs A, fdhII, bdh35 and bdh50 were up-regulated on the third day and bdh35, acsB, fdhI, fdhIII, fdhIV, buk, bdh10, bdh35, bdh40 and bdh50 were up-regulated on the fourth day. The results indicated that the increased Zn²⁺ content increased the alcohol production through increase in the gene expression of the carbon fixation and alcohol dehydrogenase.     

Enzymes limiting butanol and acetone formation in continuous and batch cultures of Clostridium acetobutylicum

Summary The formation of butanol in continuous cultures of Clostridium acetobutylicum is regulated at the genetic level via expression of butyraldehyde dehydrogenase since increased in vitro activities of this key enzyme are associated with increased in vivo butanol formation rates in both acidogenic and solventogenic fermentations. Addition of glucose, butyric acid and carbon monoxide results in induction of butyraldehyde dehydrogenase. The production of acetone in continuous fermentation is also controlled at the genetic level through expression of coenzyme A (CoA)-transferase; this enzyme is induced by glucose. Carbon monoxide inactivates acetoacetate decarboxylase. In controlled-pH batch fermentation solventogenesis does not correlate with in vitro activities of butyraldehyde dehydrogenase. Instead, initiation of alcohol formation is accompanied by increased activities of both reduced nicotine adenine dinucleotide (NADH)- and reduced nicotine adenine dinucleotide phosphate (NADPH)-specific alcohol dehydrogenases. The production of acetone in batch fermentation is regulated at the genetic level through combined induction of both CoA-transferase and acetoacetate decarboxylase. These two enzymes are not detected in either batch or continuous culture at or above pH 6.0. This finding explains the inability of the cells to produce acetone at elevated culture pH.     

Metabolic engineering of Clostridium acetobutylicum M5 for highly selective butanol production

To improve butanol selectivity, Clostridium acetobutylicum M5(pIMP1E1AB) was constructed by adhE1-ctfAB complementation of C. acetobutylicum M5, a derivative strain of C. acetobutylicum ATCC 824, which does not produce solvents due to the lack of megaplasmid pSOL1. The gene products of adhE1-ctfAB catalyze the formation of acetoacetate and ethanol/butanol with acid re-assimilation in solventogenesis. Effects of the adhE1-ctfAB complementation of M5 were studied by batch fermentations under various pH and glucose concentrations, and by flux balance analysis using a genome-scale metabolic model for this organism. The metabolically engineered M5(pIMP1E1AB) strain was able to produce 154 mM butanol with 9.9 mM acetone at pH 5.5, resulting in a butanol selectivity (a molar ratio of butanol to total solvents) of 0.84, which is much higher than that (0.57 at pH 5.0 or 0.61 at pH 5.5) of the wild-type strain ATCC 824. Unlike for C. acetobutylicum ATCC 824, a higher level of acetate accumulation was observed during fermentation of the M5 strain complemented with adhE1 and/or ctfAB. A plausible reason for this phenomenon is that the cellular metabolism was shifted towards acetate production to compensate reduced ATP production during the largely growth-associated butanol formation by the M5(pIMP1E1AB) strain.     

Intracellular metabolite profiling and the evaluation of metabolite extraction solvents for Clostridium carboxidivorans fermenting carbon monoxide

Clostridium carboxidivorans ferments CO, CO₂, and H₂ via the Wood-Ljungdahl pathway. CO, CO_2, and H₂ are unique substrates, unlike other carbon sources like glucose, so it is necessary to analyze intracellular metabolite profiles for gas fermentation by C. carboxidivorans for metabolic engineering. Moreover, it is necessary to optimize the metabolite extraction solvent specifically for C. carboxidivorans fermenting syngas. In comparison with glucose media, the gas media allowed significant abundance changes of 38 and 34 metabolites in the exponential and stationary phases, respectively. Especially, C. carboxidivorans cultivated in the gas media showed changes of fatty acid metabolism and higher levels of intracellular fatty acid synthesis possibly due to cofactor imbalance and slow metabolism. Meanwhile, the evaluation of extraction solvents revealed the mixture of water-isopropanol-methanol (2:2:5, v/v/v) to be the best extraction solvent, which showed a higher extraction capability and reproducibility than pure methanol, the conventional extraction solvent. This is the first metabolomic study to demonstrate the unique intracellular metabolite profiles of the gas fermentation compared to glucose fermentation, and to evaluate water-isopropanol-methanol as the optimal metabolite extraction solvent for C. carboxidivorans on gas fermentation.     

New insights and novel developments in clostridial acetone/butanol/isopropanol fermentation

Clostridial acetone/butanol fermentation used to rank second only to ethanol fermentation by yeast in its scale of production and thus is one of the largest biotechnological processes known. Its decline since about 1950 has been caused by increasing substrate costs and the availability of much cheaper feedstocks for chemical solvent synthesis by the petrochemical industry. The so-called oil crisis in 1973 led to renewed interest in novel fermentation and product recovery technologies as well as in the metabolism and genetics of the bacterial species involved. As a consequence, almost all of the enzymes leading to solvent formation are known, their genes have been sequenced (in fact, Clostridium acetobutylicum has been recently included in the microbial genome sequencing project), the regulatory mechanisms controlling solventogenesis have begun to emerge and recombinant DNA techniques have been developed for these clostridia to construct specific production strains. In parallel, cheap agricultural-waste-based feedstocks have been exploited for their potential as novel substrates, continuous culture methods have been successfully established and new on-line product recovery technologies are now available, such as gas stripping, liquid/liquid extraction, and membrane-based methods. In combination with these achievements, a reintroduction of acetone/butanol fermentation on an industrial scale seems to be economically feasible, a view that is supported by a new pilot plant in Austria recently coming into operation. Received: 18 December 1997 / Received revision: 27 January 1998 / Accepted: 27 January 1998     

Controlling the oxidoreduction potential of the culture of <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> leads to an earlier initiation of solventogenesis,thus increasing solvent productivity

Fermentative production of solvents (acetone, butanol, and ethanol) by Clostridium acetobutylicum is generally a biphasic process consisting of acidogenesis and solventogenesis. We report that the biphasic metabolism of C. acetobutylicum could be changed by oxidoreduction potential (ORP) regulation. When using air to control the ORP of the fermentation broth at −290 mV, an earlier initiation of solventogenesis was achieved. Solvent production reached 25.6 g l⁻¹ (2.8 g acetone l⁻¹, 16.8 g butanol l⁻¹, 6.0 g ethanol l⁻¹), a 35% increase compared with the ORP uncontrolled process. Metabolic flux analysis revealed that there was a general increase of the central carbon flux in the first 24 h of fermentation when ORP was controlled at −290 mV, compared with the control. Specifically, the solvent ratio (acetone:butanol:ethanol) was changed from 25:64:11 to 11:66:23 at ORP level of −290 mV, which might have resulted from the rigidity at acetyl-CoA node and the flexibility at acetoacetyl-CoA and butyryl-CoA nodes in response to ORP regulation.     

Production of biofuels from C1-gases with Clostridium and related bacteria—Recent advances

Clostridium spp. are suitable for the bioconversion of C₁-gases (e.g., CO₂, CO and syngas) into different bioproducts. These products can be used as biofuels and are reviewed here, focusing on ethanol, butanol and hexanol, mainly. The production of higher alcohols (e.g., butanol and hexanol) has hardly been reviewed. Parameters affecting the optimization of the bioconversion process and bioreactor performance are addressed as well as the pathways involved in these bioconversions. New aspects, such as mixotrophy and sugar versus gas fermentation, are also reviewed. In addition, Clostridia can also produce higher alcohols from the integration of the Wood-Ljungdahl pathway and the reverse ß-oxidation pathway, which has also not yet been comprehensively reviewed. In the latter process, the acetogen uses the reducing power of CO/syngas to reduce C₄ or C₆ fatty acids, previously produced by a chain elongating microorganism (commonly Clostridium kluyveri), into the corresponding bioalcohol.     

Purification and characterization of an alcohol dehydrogenase from Lithospermum erythrorhizon cell cultures

An NAD-dependent alcohol dehydrogenase has been purified to apparent homogeneity from cell suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), using protamine sulphate and ammonium sulphate precipitation and chromatography on DEAE-Sephacel, Superdex 200, hydroxyapatite and HiTrap blue. The enzyme is a homodimer with a M_r of ca. 77,000. Each subunit with a M_r of 40,000 contains two zinc atoms. Its isoelectric point was found at pH 5.0. The best alcohol substrate of the enzyme is ethanol. The pH optimum for ethanol oxidation is at pH 8.7 and for acetaldehyde reduction at pH 4.6. The Michaelis constants for ethanol and NAD are 2.49 and 0.05 (pH 8.7), and for acetaldehyde and NADH 2.2 and 0.078 mM (pH 4.6), respectively. Partial amino acid sequences of the purified enzyme showed high homology to alcohol dehydrogenases from other plants.Abbreviations ADH alcohol dehydrogenase - DTT dithiothreitol - PMSF dephenylmethylsulfonyl fluoride - PVPP polyvinylpolypyrrolidone - IAA indole-3-acetic acid - TFA trifluoroacetic acid     

Problems with the microbial production of butanol

With the incessant fluctuations in oil prices and increasing stress from environmental pollution, renewed attention is being paid to the microbial production of biofuels from renewable sources. As a gasoline substitute, butanol has advantages over traditional fuel ethanol in terms of energy density and hygroscopicity. A variety of cheap substrates have been successfully applied in the production of biobutanol, highlighting the commercial potential of biobutanol development. In this review, in order to better understand the process of acetone–butanol–ethanol production, traditional clostridia fermentation is discussed. Sporulation is probably induced by solvent formation, and the molecular mechanism leading to the initiation of sporulation and solventogenesis is also investigated. Different strategies are employed in the metabolic engineering of clostridia that aim to enhancing solvent production, improve selectivity for butanol production, and increase the tolerance of clostridia to solvents. However, it will be hard to make breakthroughs in the metabolic engineering of clostridia for butanol production without gaining a deeper understanding of the genetic background of clostridia and developing more efficient genetic tools for clostridia. Therefore, increasing attention has been paid to the metabolic engineering of E. coli for butanol production. The importation and expression of a non-clostridial butanol-producing pathway in E. coli is probably the most promising strategy for butanol biosynthesis. Due to the lower butanol titers in the fermentation broth, simultaneous fermentation and product removal techniques have been developed to reduce the cost of butanol recovery. Gas stripping is the best technique for butanol recovery found so far.     

Genome Sequence of the Solvent-Producing Bacterium Clostridium carboxidivorans Strain P7T

Clostridium carboxidivorans strain P7^T is a strictly anaerobic acetogenic bacterium that produces acetate, ethanol, butanol, and butyrate. The C. carboxidivorans genome contains all the genes for the carbonyl branch of the Wood-Ljungdahl pathway for CO₂ fixation, and it encodes enzymes for conversion of acetyl coenzyme A into butanol and butyrate.Clostridium carboxidivorans strain P7^T (equivalent to ATCC BAA-624^T and DSM 15243^T) is an obligate anaerobe that can grow autotrophically with H₂ and CO₂ or CO (fixing carbon via the Wood-Ljungdahl pathway), or it can grow chemoorganotrophically with simple sugars (1). Acetate, ethanol, butanol, and butyrate are end products of metabolism.For slow-growing strict anaerobes such as Clostridium carboxidivorans, genome sequencing provides a rapid theoretical characterization of its metabolism compared to traditional methods. We isolated and amplified genomic C. carboxidivorans DNA using the Wizard genomic DNA purification kit (Promega, Madison, WI) and the REPLI-g kit (Qiagen). A single shotgun pyrosequencing run using a Genome Sequencer FLX system (454 Life Sciences, Branford, CT) resulted in 429,680 high-quality reads (mean read length, 231.6 bp) that were assembled using Newbler software (454 Life Sciences) into 225 contigs >500 bp long. Paired-end sequencing produced 111,154 reads (mean read length, 256.3 bp). Assembly of the paired-end and shotgun reads produced 73 scaffolds containing 216 large contigs with a mean sequence depth of 16.33 reads. PCR amplification and Sanger sequencing were conducted, followed by scaffold assembly using Sequencher (Gene Codes, Ann Arbor, MI). The 4.4-Mb final assembly has 33 scaffolds containing 69 contigs with a Phred-equivalent quality score of 40 or above (accuracy, >99.99%) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"ADEK00000000","term_id":"295883218","term_text":"ADEK00000000"}}ADEK00000000).The sequence was annotated using Annotation Engine (J. Craig Venter Institute) and manually curated using Manatee (http://manatee.sourceforge.net/). The genome has 29.7% G+C content and contains 4,174 protein-coding sequences, 3 rRNA operons, 1 tmRNA (dual tRNA-like and mRNA-like nature), 6 noncoding RNAs (ncRNAs), and 48 tRNA genes. (6). Comparison of 16S rRNA genes showed that C. carboxidivorans is closely related to Clostridium scatologenes ATCC 25775^T (97% sequence identity) and Clostridium drakei type strain SL1^T (99% sequence identity). C. carboxidivorans shares 94% 16S rRNA sequence identity with Clostridium ljungdahlii (4.6 Mb), another solventogenic species.Pathway analyses indicated that C. carboxidivorans is similar to other anaerobic acetogens, such as Moorella thermoacetica (8), in having an incomplete reductive tricarboxylic acid (TCA) cycle where fumarate reductase is absent. Like other acetogenic clostridia, C. carboxidivorans uses the Wood-Ljungdahl pathway for fixing carbon dioxide to organic carbon via acetyl coenzyme A (acetyl-CoA) (5). Two of these genes encode carbon monoxide dehydrogenase (CODH) and acetyl-CoA synthase (ACS), which form a complex to catalyze the carbonyl branch of the pathway for carbon fixation and acetyl-CoA production. C. carboxidivorans has genes that encode phosphotransacetylase and acetate kinase for converting acetyl-CoA into acetate, yielding ATP (2).C. carboxidivorans is unique among other known acetogenic clostridia because it can fix carbon via the Wood-Ljungdahl pathway and convert acetyl-CoA into butanol, which is more energy dense than ethanol. Both C. carboxidivorans and Clostridium acetobutylicum encode NADPH-dependent butanol dehydrogenase (74% identity) to convert acetyl-CoA into butanol (3, 4), but C. acetobutylicum cannot fix CO₂ or CO into acetyl-CoA. Conversely, C. ljungdahlii can fix CO and CO₂, but it lacks butanol dehydrogenase and cannot convert acetyl-CoA into butanol. Therefore, P7 includes beneficial properties of both these industrially important strains. The genome sequence of C. carboxidivorans P7 could potentially accelerate research allowing its industrial application for biofuel production or to enable some of its pathways to be used directly in synthetic biology for biofuel production.     

Butanol formation byClostridium thermosaccharolyticum at neutral pH

Summary Clostridium thermosaccharolyticum can produce up to 40 mM butanol. The formation of ethanol and butanol from starch and glucose by strain DSM 571 and by the new isolate 021 was compared. The ratios for ethanol/acetate and butanol/butyrate were higher during growth at neutral pH than at acidic pH. Butanol formation was greatly stimulated by the addition of butyrate.     

Engineering Clostridium acetobutylicum for production of kerosene and diesel blendstock precursors

Processes for the biotechnological production of kerosene and diesel blendstocks are often economically unattractive due to low yields and product titers. Recently, Clostridium acetobutylicum fermentation products acetone, butanol, and ethanol (ABE) were shown to serve as precursors for catalytic upgrading to higher chain-length molecules that can be used as fuel substitutes. To produce suitable kerosene and diesel blendstocks, the butanol:acetone ratio of fermentation products needs to be increased to 2–2.5:1, while ethanol production is minimized. Here we show that the overexpression of selected proteins changes the ratio of ABE products relative to the wild type ATCC 824 strain. Overexpression of the native alcohol/aldehyde dehydrogenase (AAD) has been reported to primarily increase ethanol formation in C. acetobutylicum. We found that overexpression of the AAD^D485G variant increased ethanol titers by 294%. Catalytic upgrading of the 824(aad^D485G) ABE products resulted in a blend with nearly 50 wt%≤C₉ products, which are unsuitable for diesel. To selectively increase butanol production, C. beijerinckii aldehyde dehydrogenase and C. ljungdhalii butanol dehydrogenase were co-expressed (strain designate 824(Cb ald-Cl bdh)), which increased butanol titers by 27% to 16.9 g L⁻¹ while acetone and ethanol titers remained essentially unaffected. The solvent ratio from 824(Cb ald-Cl bdh) resulted in more than 80 wt% of catalysis products having a carbon chain length≥C₁₁ which amounts to 9.8 g L⁻¹ of products suitable as kerosene or diesel blendstock based on fermentation volume. To further increase solvent production, we investigated expression of both native and heterologous chaperones in C. acetobutylicum. Expression of a heat shock protein (HSP33) from Bacillus psychrosaccharolyticus increased the total solvent titer by 22%. Co-expression of HSP33 and aldehyde/butanol dehydrogenases further increased ABE formation as well as acetone and butanol yields. HSP33 was identified as the first heterologous chaperone that significantly increases solvent titers above wild type C. acetobutylicum levels, which can be combined with metabolic engineering to further increase solvent production.     

Tn916-induced mutants of Clostridium acetobutylicum defective in regulation of solvent formation

Sixteen Tn916-induced mutants of Clostridium acetobutylicum were selected that were defective in the production of acetone and butanol. Formation of ethanol, however, was only partially affected. The strains differed with respect to the degree of solvent formation ability and could be assigned to three different groups. Type I mutants (2 strains) were completely defective in acetone and butanol production and contained one or three copies of Tn916 in the chromosome. Analysis of the mutants for enzymes responsible for solvent production revealed the presence of a formerly unknown, specific acetaldehyde dehydrogenase. The data obtained also strongly indicate that the NADP⁺-dependent alcohol dehydrogenase is in vivo reponsible for ethanol formation, whereas the NAD⁺-dependent alcohol dehydrogenase is probably involved in butanol production. No activity of this enzyme together with all other enzymes in the acetone and butanol pathway could be found in type I strains. All tetracycline-resistant mutants obtained did no longer sporulate.Non-standard abbreviations AADC acetoacetate decarboxylase - AcaDH acetaldehyde dehydrogenase - BuaDH butyraldehyde dehydrogenase - CoA-TF acetoacetyl coenzyme A: acetate/butyrate: coenzyme A transferase - NAD-ADH, NAD⁺ dependent alcohol dehydrogenase - NADP-ADH, NADP⁺ dependent alcohol dehydrogenase