Expression of plasmid-encoded aad in Clostridium acetobutylicum M5 restores vigorous butanol production.

Mutant M5 of Clostridium acetobutylicum ATCC 824, which produces neither butanol nor acetone and is deficient in butyraldehyde dehydrogenase (BYDH), acetoacetate decarboxylase, and acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase activities, was transformed with plasmid pCAAD, which carries the gene aad (R. V. Nair, G. N. Bennett, and E. T. Papoutsakis, J. Bacteriol, 176:871-885, 1994). In batch fermentation studies, aad expression restored butanol formation (84 mM) in mutant M5 without any acetone formation or any significant increase in ethanol production. The corresponding protein (AAD) appeared as a ca. 96-kDa band in a denaturing protein gel. Expression of AAD in M5 resulted in restoration of BYDH activity and small increases in the activities of acetaldehyde dehydrogenase, butanol dehydrogenase, and ethanol dehydrogenase. These findings suggest that BYDH activity in C. acetobutylicum ATCC 824 resides largely in AAD, and that AAD's primary role is in the formation of butanol rather than of ethanol.     

Comparison between in vivo and in vitro enzyme activities in continuous and batch fermentations of Clostridium acetobutylicum

Summary In vitro activities of key enzymes and related parameters (ATP and ADP concentrations, intracellular pH (pH _i ), cell volume and the transmembrane pH) in various continuous and batch fermentations of Clostridium acetobutylicum were studied in order to investigate the regulation (genetic vs. enzyme level) of the solventogenesis process. In vitro activities varied significantly among an acidogenic (glucose limited) and three solventogenic (an iron limited, a CO gassed and a biomass recycle) continuous fermentations. However, in vitro enzyme activities did not correlate with in vivo specific production rates in continuous cultures indicating that solvent formation is regulated primarily at the enzyme level. Carbon monoxide (CO) gassing of an acidogenic continuous culture resulted in butyrate uptake without acetone formation due to inactivation of the acetoacetate decarboxylase by CO. In continuous, and to some extent in batch cultures, butyrate can be taken up via the reversal of the butyrate kinase and phosphotransbutyrylase pathway. Solvent formation in batch fermentations is both a result of enzyme induction and regulation. Acetone formation and the induction of acetoacetate decarboxylase occur simultaneously whereas both alcohol dehydrogenases are induced several hours before initiation of alcohol production. Finally, the levels of intracellular and related cell parameters (pH _i , pH, ATP and ADP concentrations) are discussed and related to the possible mechanisms of solventogenesis.     

兼性厌氧芽胞杆菌TSH1丁醇代谢途径中关键酶的检测

传统的丁醇生产菌均严格厌氧,本实验室分离了一株兼性厌氧的芽胞杆菌TSH1 (Bacillus sp.TSH1),丁醇梭菌具有相似的丁醇代谢通路及产物.通过研究乙醇和丁醇生成途径中关键酶的活性,分析乙醇脱氢酶、丁醇脱氢酶及丁醛脱氢酶的活性变化与产物生成的关系.结果表明,在发酵初期,3种酶的活性均迅速升高并在21h前达到最大值,丁醇、乙醇浓度也逐渐增加,乙醇脱氢酶在12h酶活达到最大值0.054 U/mg,丁醛脱氢酶在21h酶活达到最大值0.035 U/mg,丁醇脱氢酶则在15h酶活达到最大值0.055 U/mg.24 h后,3种酶活均开始下降,并维持在较低水平,而这段时间内产物浓度仍持续增长直至发酵结束.研究结果深化了对微生物丁醇代谢机理的认识,并为进一步研究芽胞杆菌丁醇代谢途径提供参考.     

Isolation and Characterization of Mutants of Clostridium acetobutylicum ATCC 824 Deficient in Acetoacetyl-Coenzyme A:Acetate/Butyrate:Coenzyme A-Transferase (EC 2.8.3.9) and in Other Solvent Pathway Enzymes

Mutants of Clostridium acetobutylicum ATCC 824 exhibiting resistance to 2-bromobutyrate or rifampin were isolated after nitrosoguanidine treatment. Mutants were screened for solvent production by using an automated alcohol test system. Isolates were analyzed for levels of butanol, ethanol, acetone, butyrate, acetate, and acetoin in stationary-phase batch cultures. The specific activities of NADH- and NADPH-dependent butanol dehydrogenase and butyraldehyde dehydrogenase as well as those of acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase (butyrate-acetoacetate coenzyme A-transferase [EC 2.8.3.9]) (CoA-transferase), butyrate kinase, and phosphotransbutyrylase were measured at the onset of stationary phase. Rifampin-resistant strain D10 and 2-bromobutyrate mutant R were found to be deficient in only CoA-transferase, while several other mutants exhibited reduced butyraldehyde dehydrogenase and butanol dehydrogenase activities as well. The colony morphology of 2-bromobutyrate mutant R was similar to that of the parent on RCM medium; however, it had about 1/10 the level of CoA-transferase and increased levels of butanol dehydrogenase and butyraldehyde dehydrogenase. A nonsporulating, spontaneously derived degenerated strain exhibited reduced levels of butyraldehyde dehydrogenase, butanol, dehydrogenase, and CoA-transferase compared with those of the original strain. When C. acetobutylicum ATCC 824 was grown on medium containing low levels of 2-bromobutyrate, an altered colony morphology was observed. Not all strains resistant to 2-bromobutyrate (12 mM) were non-solvent-producing strains.     

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     

In situ extractive fermentation of acetone and butanol

The productivity of the acetone-butanol fermentation was increased by continuously removing acetone and butanol from the fermentation broth during fed-batch culture. Whole broth containing viable cells of Clostridium acetobutylicum was cycled to a Karr reciprocating plate extraction column in which acetone and butanol were extracted into oleyl alcohol flowing counter-currently through the column. By continuously removing these toxic metabolites from the broth, end product inhibition was reduced, and a concentrated feed solution containing 300 g/L glucose was fermented at an overall butanol productivity of 1.0 g/L h, 70% higher than the productivity of normal batch fermentation. The continuous extraction process provides flexible operation and lends itself to process scale-up.     

Enzymatic investigations on butanol dehydrogenase and butyraldehyde dehydrogenase in extracts of Clostridium acetobutylicum

Summary Reliable assay systems were developed for the detection and quantitation of butanol dehydrogenase and butyraldehyde dehydrogenase in extracts of Clostridium acetobutylicum. Butanol dehydrogenase was NADPH-dependent. The enzyme could be sparated by ultracentrifugation from a NADH-specific enzyme which probably represents the ethanol dehydrogenase but which also reacted with butyraldehyde to form butanol. Butyraldehyde dehydrogenase proved to be NADH-specific. All enzymes were induced shortly before butanol formation began. Specific activities decreased at the end of the fermentation process. An explanation for contradictory data in the literature is proposed.     

A new ternary complex between horse liver alcohol dehydrogenase, NAD+, and acetone

Acetone was found to form a dead-end ternary complex with horse liver alcohol dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD⁺) when the reactants were incubated for a long time at relatively high concentrations. The complex formation was demonstrated by measuring the increase in absorbance at 320 nm, the quenching of protein fluorescence, and the loss of enzyme activity. Since acetone is a substrate of liver alcohol dehydrogenase, and the presence of acetaldehyde or pyrazole prevents acetone from forming the dead-end complex with liver alcohol dehydrogenase and NAD⁺, the acetone molecule in the complex may be bound to the substrate binding site of liver alcohol dehydrogenase. The dissociation of the complex was demonstrated by prolonged dialysis or by addition of reduced nicotinamide adenine dinucleotide (NADH) and iso-butyramide. A modified nicotinamide adenine dinucleotide was obtained as a main product from the dead-end complex after dissociation of the complex or denaturation of the apoenzyme. The modified nicotinamide adenine dinucleotide was found to exhibit an absorption spectrum similar to that of NADH; however, it was not oxidizable by liver alcohol dehydrogenase in the presence of acetaldehyde and exhibited no fluorescence.     

Clostridium acetobutylicum mutants isolated for resistance to the pyruvate halogen analogs

Pyruvate halogen analogs, 3-fluoropyruvate and 3-bromopyruvate, are toxic towardClostridium acetobutylicum. After mutagenesis with nitrosoguanidine, mutants resistant to these compounds were selected. In a normal batch culture regulated at pH 4.8, mutants quickly initiated the acid-solvent transition, accumulating more acetoin and lactate than the wild type strain. The maximum rate of specific glucose uptake was higher in the mutants than in the wild type: 1.76 h^–1 and 0.9 h^–1 respectively. When the pH was uncontrolled, mutants converted glucose into solvents, essentially butanol, while the wild-type strain ceased its fermentation at the acidogenic stage. Enzymatic investigations revealed that acetate kinase, butyrate kinase, and acetoacetate decarboxylase activities decreased sooner in the mutants than in the parent strain.     

Clostridium acetobutylicum Mutants That Produce Butyraldehyde and Altered Quantities of Solvents

Spontaneous mutants of Clostridium acetobutylicum NRRL B643 that were resistant to allyl alcohol (AA) were selected and characterized. These mutants contained 10- to 100-fold reduced activities of butanol and ethanol alcohol dehydrogenase. The AA mutants formed two groups and produced no ethanol. Type 1 AA mutants produced significant amounts of a new solvent, butyraldehyde, and contained normal levels of the coenzyme A-dependent butyraldehyde dehydrogenase (BAD). Type 2 AA mutants produced no significant butyraldehyde and lower levels of all solvents, and they contained 45- to 100-fold lower activity levels of BAD. Following ethyl methanesulfonate mutagenesis, low-acid-producing (Acid⁻) mutants were selected and characterized as superinduced solvent producers, yielding more than 99% of theoretical glucose carbon as solvents and only small amounts of acetate and butyrate. Following ethyl methanesulfonate mutagenesis, 13 sporulation-negative (Spo⁻) mutants were characterized; and 3 were found to produce only butyrate and acetate, a minor amount of acetone, and no alcohols. These Spo⁻ mutants contained reduced butanol dehydrogenase activity and no BAD enzyme activity. The data support the view that the type 2 AA, the Acid⁻, and the Spo⁻ mutants somehow alter normal regulated expression of the solvent pathway in C. acetobutylicum.     

Iron effect on acetone-butanol fermentation

WhenClostridium acetobutylicum was grown in batch culture under iron limitation (0.2 mg·l^–1) at a pH of 4.8, glucose was fermented, to butanol as the major fermentation end product, and small quantities of acetic acid were produced. The final conversion yield of glucose into butanol could be increased from 20% to 30% by iron limitation. The acetonebutanol ratio was changed from 3.7 (control) to 11.8. Hydrogenase specific activity was decreased by 40% and acetoacetate decarboxylase specific activity by 25% under iron limitation. Thus, iron limitation affects carbon and electron flow in addition to hydrogenase.     

Switching Clostridium acetobutylicum to an ethanol producer by disruption of the butyrate/butanol fermentative pathway

Solventogenic clostridia are well-known since almost a century due to their unique capability to biosynthesize the solvents acetone and butanol. Based on recently developed genetic engineering tools, a targeted 3-hydroxybutyryl-CoA dehydrogenase (Hbd)-negative mutant of Clostridium acetobutylicum was generated. Interestingly, the entire butyrate/butanol (C₄) metabolic pathway of C. acetobutylicum could be inactivated without a severe growth limitation and indicated the general feasibility to manipulate the central fermentative metabolism for product pattern alteration. Cell extracts of the mutant C. acetobutylicum hbd::int(69) revealed clearly reduced thiolase, Hbd and crotonase but increased NADH-dependent alcohol dehydrogenase enzyme activities as compared to the wildtype strain. Neither butyrate nor butanol were detected in cultures of C. acetobutylicum hbd::int(69), and the formation of molecular hydrogen was significantly reduced. Instead up to 16 and 20 g/l ethanol were produced in glucose and xylose batch cultures, respectively. Further sugar addition in glucose fed-batch fermentations increased the ethanol production to a final titer of 33 g/l, resulting in an ethanol to glucose yield of 0.38 g/g.     

Uptake and activation of acetate and butyrate in Clostridium acetobutylicum

Summary The pathway for uptake of acids during the solvent formation phase of an acetone-butanol fermentation by Clostridium acetobutylicum ATCC 824 was studied. ¹³C NMR investigations on actively metabolizing cells showed that butyrate can be taken up from the medium and quantitatively converted to butanol without accumulation of intermediates. The activities of acetate phosphotransacetylase, acetate kinase and phosphate butyryltransferase rapidly decreased to very low levels when the organism began to form solvents. This indicates that the uptake of acids does not occur via a reversal of these acid forming enzymes. No short-chain acyl-CoA synthetase activity or butyryl phosphate reducing activity could be detected. Based on our results and a critical analysis of literature data on acetone-butanol fermentations, it is suggested that an acetoacetyl-CoA: acetate (butyrate) CoA-transferase is solely responsible for uptake and activation of acetate and butyrate in C. acetobutylicum. The transferase exhibits a broad carboxylic acid specificity. The key enzyme in the uptake is acetoacetate decarboxylase, which is induced late in the fermentation and pulls the transferase reaction towards formation of acetoacetate. The major implication is that it is not feasible to obtain a batch-wise butanol fermentation without acetone formation and retention of a good yield of butanol.     

Enzyme Pattern and Aerobic Growth of Saccharomyces cerevisiae Under Various Degrees of Glucose Limitation

The enzyme pattern of Saccharomyces cerevisiae was followed during batch growth and in continuous culture in a synthetic medium limited for glucose under aerobic conditions. Seven enzymes were measured: succinate-cytochrome c oxidoreductase, malate dehydrogenase, nicotinamide adenine dinucleotide-linked glutamate dehydrogenase, malate synthase, isocitrate lyase, aldolase, and nicotinamide adenine dinucleotide phosphate (NADP(+))-linked glutamate dehydrogenase. During fermentation of glucose and high growth rate (mu) during the first log phase in batch experiments, the first five enzymes (group I) were repressed, and aldolase and NADP(+)-linked glutamate dehydrogenase (group II) were derepressed. During growth on the accumulated ethyl alcohol and lower mu, the group I enzymes were preferentially formed and the other two were repressed. A sequence of derepression of the group I enzymes was found during the shift from glucose to ethyl alcohol metabolism, which can be correlated with a strong increase in the percentage of single (nonbudding) cells in the population. A correlation between the state of cells in the budding cycle and enzyme repression and derepression is suggested. In continuous culture, the enzyme pattern was shown to be related to the growth rate. The group I enzymes were repressed at high growth rates, while the group II enzymes were derepressed. Each enzyme exhibits a different dependence. The enzyme pattern is shown to depend on the rate of substrate consumption as well as on the type of metabolism and to be correlated with the budding cycle. The enzyme pattern is considered to be controlled by changes of intracellular catabolic or metabolic conditions inherent in the division cycle.     

Isolation of a new butanol-producing Clostridium strain: High level of hemicellulosic activity and structure of solventogenesis genes of a new Clostridium saccharobutylicum isolate

New isolates of solventogenic bacteria exhibited high hemicellulolytic activity. They produced butanol and acetone with high selectivity for butanol (about 80% of butanol from the total solvent yield). Their 16S rDNA sequence was 99% identical to that of Clostridium saccharobutylicum. The genes responsible for the last steps of solventogenesis and encoding crotonase, butyryl-CoA dehydrogenase, electron-transport protein subunits A and B, 3-hydroxybutyryl-CoA dehydrogenase, alcohol dehydrogenase, CoA-transferase (subunits A and B), acetoacetate decarboxylase, and aldehyde dehydrogenase were identified in the new C. saccharobutylicum strain Ox29 and cloned into Escherichia coli. The genes for crotonase, butyryl-CoA dehydrogenase, electron-transport protein subunits A and B, and 3-hydroxybutyryl-CoA dehydrogenase composed the bcs-operon. A monocistronic operon containing the alcohol dehydrogenase gene was located downstream of the bcs-operon. Genes for aldehyde dehydrogenase, CoA-transferase (subunits A and B), and acetoacetate decarboxylase composed the sol-operon. The gene sequences and the gene order within the sol- and bcs-operons of C. saccharobutylicum Ox29 were most similar to those of Clostridium beijerinckii. The activity of some of the bcs-operon genes, expressed in heterologous E. coli, was determined.     

Engineering a homobutanol fermentation pathway in Escherichia coli EG03

A homobutanol fermentation pathway was engineered in a derivative of Escherichia coli B (glucose [glycolysis] => 2 pyruvate + 2 NADH; pyruvate [pyruvate dehydrogenase] => acetyl-CoA + NADH; 2 acetyl-CoA [butanol pathway enzymes] + 4 NADH => butanol; summary stoichiometry: glucose => butanol). Initially, the native fermentation pathways were eliminated from E. coli B by deleting the genes encoding for lactate dehydrogenase (ldhA), acetate kinase (ackA), fumarate reductase (frdABCD), pyruvate formate lyase (pflB), and alcohol dehydrogenase (adhE), and the pyruvate dehydrogenase complex (aceEF-lpd) was anaerobically expressed through promoter replacement. The resulting strain, E. coli EG03 (ΔfrdABCD ΔldhA ΔackA ΔpflB Δ adhE ΔpdhR ::pflBp6-aceEF-lpd ΔmgsA), could generate 4 NADH for every glucose oxidized to two acetyl-CoA through glycolysis and the pyruvate dehydrogenase complex. However, EG03 lost its ability for anaerobic growth due to the lack of NADH oxidation pathways. When the butanol pathway genes that encode for acetyl-CoA acetyltransferase (thiL), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), butyryl-CoA dehydrogenase (bcd, etfA, etfB), and butyraldehyde dehydrogenase (adheII) were cloned from Clostridium acetobutylicum ATCC 824, and expressed in E. coli EG03, a balanced NADH oxidation pathway was established for homobutanol fermentation (glucose => 4 NADH + 2 acetyl-CoA => butanol). This strain was able to convert glucose to butanol (1,254 mg l(-1)) under anaerobic condition.     

Acetone production in solventogenic <Emphasis Type="Italic">Clostridium</Emphasis> species: new insights from non-enzymatic decarboxylation of acetoacetate

Development of a butanologenic strain with high selectivity for butanol production is often proposed as a possible route for improving the economics of biobutanol production by solventogenic Clostridium species. The acetoacetate decarboxylase (aadc) gene encoding acetoacetate decarboxylase (AADC), which catalyzes the decarboxylation of acetoacetate into acetone and CO₂, was successfully disrupted by homologous recombination in solventogenic Clostridium beijerinckii NCIMB 8052 to generate an aadc ⁻ mutant. Our fermentation studies revealed that this mutant produces a maximum acetone concentration of 3 g/L (in P2 medium), a value comparable to that produced by wild-type C. beijerinckii 8052. Therefore, we postulated that AADC-catalyzed decarboxylation of acetoacetate is not the sole means for acetone generation. Our subsequent finding that non-enzymatic decarboxylation of acetoacetate in vitro, under conditions similar to in vivo acetone–butanol–ethanol (ABE) fermentation, produces 1.3 to 5.2 g/L acetone between pH 6.5 and 4 helps rationalize why various knock-out and knock-down strategies designed to disrupt aadc in solventogenic Clostridium species did not eliminate acetone production during ABE fermentation. Based on these results, we discuss alternatives to enhance selectivity for butanol production.     

Metabolic engineering of Clostridium acetobutylicum ATCC 824 for increased solvent production by enhancement of acetone formation enzyme activities using a synthetic acetone operon

The ability to genetically alter the product-formation capabilities of Clostridium acetobutylicum is necessary for continued progress toward industrial production of the solvents butanol and acetone by fermentation. Batch fermentations at pH 4.5, 5.5, or 6.5 were conducted using C. acetobutylicum ATCC 824 (pFNK6). Plasmid pFNK6 contains a synthetic operon (the "ace operon") in which the three homologous acetone-formation genas (adc, ctfA, and ctfB) are transcribed from the adc promoter. The corresponding enzymes (acetoacetate decarboxylase and CoA-transferase) were best expressed in pH 4.5 fermentations. However, the highest levels of solvents were attained at pH 5.5. Relative to the plasmid-free control strain at pH 5.5, ATCC 824 (pFNK6) produced 95%, 37%, and 90% higher final concentrations of acetone, butanol, and ethanol, respectively; a 50% higher yield (g/g) of solvents on glucose; and a 22-fold lower mass of residual carboxylic acids. At all pH values, the acetone-formation enzymes were expressed earlier with ATCC 824 (pFNK6) than in control fermentations, leading to earlier induction of acetone formation. Furthermore, strain ATCC 824 (pFNK6) produced butanol significantly earlier in the fermentation and produced significant levels of solvents at pH 6.5. Only trace levels of solvents were produced by strain ATCC 824 at pH 6.5. Compared with ATCC 824, a plasmid-control strain containing a vector without the ace operon also produced higher levels of solvents [although lower than those of strain ATCC 824 (pFNK6)] and lower levels of acids. Strains containing plasmid-borne derivatives of the ace operon, in which either the acetoacetate decarboxylase or CoA-transferase alone were expressed at elevated levels, produced acids and solvents at levels similar to those of the plasmid-control strain. (c) 1993 John Wiley & Sons, Inc.