Targeted mutagenesis of the Clostridium acetobutylicum acetone–butanol–ethanol fermentation pathway

The production of the chemical solvents acetone and butanol by the bacterium Clostridium acetobutylicum was one of the first large-scale industrial processes to be developed, and in the first part of the last century ranked second in importance only to ethanol production. After a steep decline in its industrial use, there has been a recent resurgence of interest in the acetone–butanol–ethanol (ABE) fermentation process, with a particular emphasis on butanol production. In order to generate strains suitable for efficient use on an industrial scale, metabolic engineering is required to alter the AB ratio in favour of butanol, and eradicate the production of unwanted products of fermentation. Using ClosTron technology, a large-scale targeted mutagenesis in C. acetobutylicum ATCC 824 was carried out, generating a set of 10 mutants, defective in alcohol/aldehyde dehydrogenases 1 and 2 (adhE1, adhE2), butanol dehydrogenases A and B (bdhA, bdhB), phosphotransbutyrylase (ptb), acetate kinase (ack), acetoacetate decarboxylase (adc), CoA transferase (ctfA/ctfB), and a previously uncharacterised putative alcohol dehydrogenase (CAP0059). However, inactivation of the main hydrogenase (hydA) and thiolase (thl) could not be achieved. Constructing such a series of mutants is paramount for the acquisition of information on the mechanism of solvent production in this organism, and the subsequent development of industrial solvent producing strains. Unexpectedly, bdhA and bdhB mutants did not affect solvent production, whereas inactivation of the previously uncharacterised gene CAP0059 resulted in increased acetone, butanol, and ethanol formation. Other mutants showed predicted phenotypes, including a lack of acetone formation (adc, ctfA, and ctfB mutants), an inability to take up acids (ctfA and ctfB mutants), and a much reduced acetate formation (ack mutant). The adhE1 mutant in particular produced very little solvents, demonstrating that this gene was indeed the main contributor to ethanol and butanol formation under the standard batch culture conditions employed in this study. All phenotypic changes observed could be reversed by genetic complementation, with exception of those seen for the ptb mutant. This mutant produced around 100 mM ethanol, no acetone and very little (7 mM) butanol. The genome of the ptb mutant was therefore re-sequenced, together with its parent strain (ATCC 824 wild type), and shown to possess a frameshift mutation in the thl gene, which perfectly explained the observed phenotype. This finding reinforces the need for mutant complementation and Southern Blot analysis (to confirm single ClosTron insertions), which should be obligatory in all further ClosTron applications.     

New insights into the butyric acid metabolism of Clostridium acetobutylicum

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.     

Genetic engineering of Escherichia coli to enhance production of l-tryptophan

Reducing the accumulation of acetate in Escherichia coli cultures can decrease carbon efflux as by-products and reduce acetate toxicity, and therefore enable high cell density cultivation. The concentration of intracellular amino acids can be decreased by genetic modifications of the corresponding amino acid transport systems. This can increase the levels of amino acids in the fermentation broth by decreasing the feedback inhibition on the corresponding biosynthetic pathways. Here, the effects of genetic manipulation of phosphate acetyltransferase (pta), high affinity tryptophan transporter (mtr) and aromatic amino acid exporter (yddG) on l-tryptophan production were investigated. The pta mutants accumulated less acetate and showed higher capacity for producing l-tryptophan as compared with the parental strain. The strains lacking mtr, or overexpressed yddG, or with the both mtr knockout and yddG overexpression, accumulated lower concentrations of intracellular tryptophan but higher production of extracellular l-tryptophan. In the l-tryptohan fed-batch fermentation of an E. coli derived from TRTH0709/pMEL03 having deletion of pta-mtr and overexpression of yddG in a 30-L fermentor, the maximum concentration of l-tryptophan (48.68 g/L) was obtained, which represented a 15.96 % increase as compared with the parental strain. Acetate accumulated to a concentration of 0.95 g/L. The intracellular concentration of l-tryptophan was low, and the glucose conversion rate reached a high level of 21.87 %, which was increased by 15.53 % as compared with the parent strain.     

Characterization and Development of Two Reporter Gene Systems for Clostridium acetobutylicum

The use of lacZ from Thermoanaerobacterium thermosulfurigenes (encoding β-galactosidase) and lucB from Photinus pyralis (encoding luciferase) as reporter genes in Clostridium acetobutylicum was analyzed with promoters of genes required for solventogenesis and acidogenesis. Both systems proved to be well suited and allowed the detection of differences in promoter strength at least up to 100-fold. The luciferase assay could be performed much faster and comes close to online measurement. Resequencing of lacZ revealed a sequence error in the original database entry, which resulted in β-galactosidase with an additional 31 amino acids. Cutting off part of the gene encoding this C terminus resulted in decreased enzyme activity. The lacZ reporter data showed that bdhA (encoding butanol dehydrogenase A) is expressed during the early growth phase, followed by sol (encoding butyraldehyde/butanol dehydrogenase E and coenzyme A transferase) and bdhB (encoding butanol dehydrogenase B) expression. adc (encoding acetoacetate decarboxylase) was also induced early. There is about a 100-fold difference in expression between adc and bdhB (higher) and bdhA and the sol operon (lower). The lucB reporter activity could be increased 10-fold by the addition of ATP to the assay. Washing of the cells proved to be important in order to prevent a red shift of bioluminescence in an acidic environment (for reliable data). lucB reporter measurements confirmed the expression pattern of the sol and ptb-buk (encoding phosphotransbutyrylase and butyrate kinase) operons as determined by the lacZ reporter and showed that the expression level from the ptb promoter is 59-fold higher than that from the sol operon promoter.     

Genetics and Physiology of Acetate Metabolism by the Pta-Ack Pathway of Streptococcus mutans

In the dental caries pathogen Streptococcus mutans, phosphotransacetylase (Pta) catalyzes the conversion of acetyl coenzyme A (acetyl-CoA) to acetyl phosphate (AcP), which can be converted to acetate by acetate kinase (Ack), with the concomitant generation of ATP. A ΔackA mutant displayed enhanced accumulation of AcP under aerobic conditions, whereas little or no AcP was observed in the Δpta or Δpta ΔackA mutant. The Δpta and Δpta ΔackA mutants also had diminished ATP pools compared to the size of the ATP pool for the parental or ΔackA strain. Surprisingly, when exposed to oxidative stress, the Δpta ΔackA strain appeared to regain the capacity to produce AcP, with a concurrent increase in the size of the ATP pool compared to that for the parental strain. The ΔackA and Δpta ΔackA mutants exhibited enhanced (p)ppGpp accumulation, whereas the strain lacking Pta produced less (p)ppGpp than the wild-type strain. The ΔackA and Δpta ΔackA mutants displayed global changes in gene expression, as assessed by microarrays. All strains lacking Pta, which had defects in AcP production under aerobic conditions, were impaired in their abilities to form biofilms when glucose was the growth carbohydrate. Collectively, these data demonstrate the complex regulation of the Pta-Ack pathway and critical roles for these enzymes in processes that appear to be essential for the persistence and pathogenesis of S. mutans.     

Development of a Gene Knockout System Using Mobile Group II Introns (Targetron) and Genetic Disruption of Acid Production Pathways in Clostridium beijerinckii

Clostridium beijerinckii is a well-known solvent-producing microorganism with great potential for biofuel and biochemical production. To better understand and improve the biochemical pathway to solvents, the development of genetic tools for engineering C. beijerinckii is highly desired. Based on mobile group II intron technology, a targetron gene knockout system was developed for C. beijerinckii in this study. This system was successfully employed to disrupt acid production pathways in C. beijerinckii, leading to pta (encoding phosphotransacetylase)- and buk (encoding butyrate kinase)-negative mutants. In addition to experimental characterization, the mutant phenotypes were analyzed in the context of our C. beijerinckii genome-scale model. Compared to those of the parental strain (C. beijerinckii 8052), acetate production in the pta mutant was substantially reduced and butyrate production was remarkably increased, while solvent production was dependent on the growth medium. The pta mutant also produced much higher levels of lactate, suggesting that disrupting pta influenced the energy generation and electron flow pathways. In contrast, acetate and butyrate production in the buk mutant was generally similar to that of the wild type, but solvent production was consistently 20 to 30% higher and glucose consumption was more rapid and complete. Our results suggest that the acid and solvent production of C. beijerinckii can be effectively altered by disrupting the acid production pathways. As the gene disruption method developed in this study does not leave any antibiotic marker in a disrupted allele, multiple and high-throughput gene disruption is feasible for elucidating genotype and phenotype relationships in C. beijerinckii.     

Development and Characterization of a Gene Expression Reporter System for Clostridium acetobutylicum ATCC 824

A gene expression reporter system (pHT3) for Clostridium acetobutylicum ATCC 824 was developed by using the lacZ gene from Thermoanaerobacterium thermosulfurogenes EM1 as the reporter gene. In order to test the reporter system, promoters of three key metabolic pathway genes, ptb (coding for phosphotransbutyrylase), thl (coding for thiolase), and adc (coding for acetoacetate decarboxylase), were cloned upstream of the reporter gene in pHT3 in order to construct vectors pHT4, pHT5, and pHTA, respectively. Detection of β-galactosidase activity in time course studies performed with strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA) demonstrated that the reporter gene produced a functional β-galactosidase in C. acetobutylicum. In addition, time course studies revealed differences in the β-galactosidase specific activity profiles of strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA), suggesting that the reporter system developed in this study is able to effectively distinguish between different promoters. The stability of the β-galactosidase produced by the reporter gene was also examined with strains ATCC 824(pHT4) and ATCC 824(pHT5) by using chloramphenicol treatment to inhibit protein synthesis. The data indicated that the β-galactosidase produced by the lacZ gene from T. thermosulfurogenes EM1 was stable in the exponential phase of growth. In pH-controlled fermentations of ATCC 824(pHT4), the kinetics of β-galactosidase formation from the ptb promoter and phosphotransbutyrylase formation from its own autologous promoter were found to be similar.     

Improving biobutanol production in engineered Saccharomyces cerevisiae by manipulation of acetyl-CoA metabolism

Recently, butanols (1-butanol, 2-butanol and iso-butanol) have generated attention as alternative gasoline additives. Butanols have several properties favorable in comparison to ethanol, and strong interest therefore exists in the reconstruction of the 1-butanol pathway in commonly used industrial microorganisms. In the present study, the biosynthetic pathway for 1-butanol production was reconstructed in the yeast Saccharomyces cerevisiae. In addition to introducing heterologous enzymes for butanol production, we engineered yeast to have increased flux toward cytosolic acetyl-CoA, the precursor metabolite for 1-butanol biosynthesis. This was done through introduction of a plasmid-containing genes for alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6), acetyl-CoA synthetase (ACS), and acetyl-CoA acetyltransferase (ERG10), as well as the use of strains containing deletions in the malate synthase (MLS1) or citrate synthase (CIT2) genes. Our results show a trend to increased butanol production in strains engineered for increased cytosolic acetyl-CoA levels, with the best-producing strains having maximal butanol titers of 16.3 mg/l. This represents a 6.5-fold improvement in butanol titers compared to previous values reported for yeast and demonstrates the importance of an improved cytosolic acetyl-CoA supply for heterologous butanol production by this organism.     

Disruption of the acetoacetate decarboxylase gene in solvent-producing Clostridium acetobutylicum increases the butanol ratio

A possible way to improve the economic efficacy of acetone–butanol–ethanol fermentation is to increase the butanol ratio by eliminating the production of other by-products, such as acetone. The acetoacetate decarboxylase gene (adc) in the hyperbutanol-producing industrial strain Clostridium acetobutylicum EA 2018 was disrupted using TargeTron technology. The butanol ratio increased from 70% to 80.05%, with acetone production reduced to approximately 0.21 g/L in the adc-disrupted mutant (2018adc). pH control was a critical factor in the improvement of cell growth and solvent production in strain 2018adc. The regulation of electron flow by the addition of methyl viologen altered the carbon flux from acetic acid production to butanol production in strain 2018adc, which resulted in an increased butanol ratio of 82% and a corresponding improvement in the overall yield of butanol from 57% to 70.8%. This study presents a general method of blocking acetone production by Clostridium and demonstrates the industrial potential of strain 2018adc.     

The glucose-starvation stimulon of Escherichia coli: induced and repressed synthesis of enzymes of central metabolic pathways and role of acetyl phosphate in gene expression and starvation survival

Proteins of the glucose-starvation stimulon were identified by using two-dimensional gel electrophoresis and the gene–protein database of Escherichia coli. Members of this stimulon Included enzymes of the Embden–Meyerhof–Parnas (EMP) pathway, phosphotransacetylase (Pta) and acetate kinase (AckA) of the acetyl phosphate/acetate production pathway, and formate transacetytase. The synthesis of these enzymes was found to be Induced concomitantly with the decreased synthesis of enzymes of the Krebs cycle. Thus, the modulation in the synthesis of specific proteins during aerobic glucose starvation is, In part, similar to the response of cells shifted to anaerobiosis. These modulations suggest that the glucose-starved cell increases the relative flow of carbon through the Pta–AckA pathway. Indeed, the ability to synthesize acetyl phosphate, an intermediate of the pathway, appears to be indispensable for glucose-starved cells as pta and pta–ackA double mutants were found to be impaired in their ability to survive glucose starvation. The survival characteristics of ackA mutants and the wild-type parent were indistinguishable. Moreover, the pta mutant failed to induce several proteins of the glucose-starvation stimulon.     

Phage Type 187 as a Separate Subunit MboI Restriction Site Within the Staphylococcus aureus Species

The aim of this study was to use MboI restriction of pta gene fragment to compare the strains of Staphylococcus aureus phage type 187 and other phage types of S. aureus isolated from humans and dogs, as well as canine S. intermedius group strains. The study included 395 human and canine staphylococcal strains representing S. aureus, S. intermedius, and S. pseudintermedius species. The strains were identified with classic phenotypic methods and by the presence of species-specific thermostable nuclease (nuc SA) gene. All the strains were subjected to the analysis of MboI restriction site of pta gene fragment with PCR–RFLP method. Nearly, all human and animal strains of S. aureus possessed 156- and 164-bp restriction fragments. One of the human strains lacked the 320-bp amplification product. In the case of all S. aureus phage type 187, the amplification product of pta gene was insusceptible to cutting with MboI restrictase. None of S. intermedius strains possessed restriction sites present in the product of amplification of pta gene, while all the strains of S. pseudintermedius had 213- and 107-bp restriction fragments. In conclusion, our findings regarding S. aureus phage type 187 reveal that within the population of strains of S. aureus species, these bacteria represent a group with distinct properties.     

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     

Solvent Production and Morphological Changes in Clostridium acetobutylicum

The morphological and cytological changes which occurred in Clostridium acetobutylicum P262 during the production of acetone, butanol, and ethanol in an industrial fermentation medium were identified and correlated with the growth and physiological changes. The swollen, cigar-shaped clostridial forms were involved in the conversion of acids to neutral solvents, and there was a correlation between the number of clostridial forms and the production of solvents. Sporulation mutants which were unable to form clostridial stages (cls mutants) did not produce solvents. Oligosporogenous mutants which showed reduced clostridial stage formation produced intermediate levels of solvents. Sporulation mutants blocked after the clostridial stage, which were unable to form mature spores (spo mutants), produced normal levels of solvents.     

Enhancement of butanol tolerance and butanol yield in Clostridium acetobutylicum mutant NT642 obtained by nitrogen ion beam implantation

As a promising alternative biofuel, biobutanol can be produced through acetone/butanol/ethanol (ABE) fermentation. Currently, ABE fermentation is still a small-scale industry due to its low production and high input cost. Moreover, butanol toxicity to the Clostridium fermentation host limits the accumulation of butanol in the fermentation broth. The wild-type Clostridium acetobutylicum D64 can only produce about 13 g butanol/L and tolerates less than 2% (v/v) butanol. To improve the tolerance of C. acetobutylicum D64 for enhancing the production of butanol, nitrogen ion beam implantation was employed and finally five mutants with enhanced butanol tolerance were obtained. Among these, the most butanol tolerant mutant C. acetobutylicum NT642 can tolerate above 3% (v/v) butanol while the wide-type strain can only withstand 2% (v/v). In batch fermentation, the production of butanol and ABE yield of C. acetobutylicum NT642 was 15.4 g/L and 22.3 g/L, respectively, which were both higher than those of its parental strain and the other mutants using corn or cassava as substrate. Enhancing butanol tolerance is a great precondition for obtaining a hyper-yield producer. Nitrogen ion beam implantation could be a promising biotechnology to improve butanol tolerance and production of the host strain C. acetobutylicum.     

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.     

Genetics of alcohol dehydrogenase in Saccharomyces cerevisiae: I. Isolation and genetic analysis of adh mutants

On the basis of allyalcohol resistance, Saccharomyces cerevisiae mutanta were isolated that were deficient in alcohol dehydrogenase (ADH). The mutants were divided into three classes by their different ADH isozyme pattern obtained after starch-gel electrophoresis: adc mutants that did not produce the constitutive ADH, adr mutants from which the glucose repressible enzyme (ADHII) was absent, and adm mutants deficient in ADH activity associated with the mitochondria.Genetic analysis showed that two genes control synthesis of the glucose repressible enzyme ADHII, one gene the constitutive ADHI and a fourth nuclear gene the mitochondrial ADH. None of these four genes showed any linkage.The various mutant types did not show drastic effects on yeast growth on media containing glucose or ethanol as sole carbon sources.     

New options to engineer biofuel microbes: Development and application of a high-throughput screening system

The number of recent efforts on rational metabolic engineering approaches to increase butanol production in Clostridium acetobutylicum are quite limited, demonstrating the physiological complexity of solventogenic clostridia. Since multiple largely unknown parameters determine a particular phenotype, an inverse strategy to select a phenotype of interest can be useful. However, the major constraint for explorative or combinatorial metabolic engineering approaches is the availability of a feasible screening method to select the desired phenotype from a large population in a high-throughput manner. Therefore, a semi-quantitative assay was developed to monitor alcohol production in microtiter cultures of C. acetobutylicum. The applicability of the screening system was evaluated by two examples. First, C. acetobutylicum ATCC 824 was chemically mutagenized and subjected to high butanol concentrations as a pre-selection step. Screening of the butanol-tolerant population resulted in the identification of mutants with >20% increased butanol production as compared to the wildtype. The second application example was based on a pre-engineered C. acetobutylicum strain with low acetone biosynthetic activity, but concomitantly reduced butanol titer. After chemical mutagenesis, a total of 4390 clones was analyzed and mutants with significantly increased butanol concentrations and similarly low acetone levels as the parental strain were selected. Thus, the suitability of the semi-quantitative screening system was validated, opening up new perspectives for combinatorial strategies to improve solventogenic clostridia and other biofuel microbes.