George N. Bennett

Genome Sequence and Comparative Analysis of the Solvent-Producing Bacterium Clostridium acetobutylicum

The genome sequence of the solvent-producing bacterium Clostridium acetobutylicum ATCC 824 has been determined by the shotgun approach. The genome consists of a 3.94-Mb chromosome and a 192-kb megaplasmid that contains the majority of genes responsible for solvent production. Comparison of C. acetobutylicum to Bacillus subtilis reveals significant local conservation of gene order, which has not been seen in comparisons of other genomes with similar, or, in some cases closer, phylogenetic proximity. This conservation allows the prediction of many previously undetected operons in both bacteria. However, the C. acetobutylicum genome also contains a significant number of predicted operons that are shared with distantly related bacteria and archaea but not with B. subtilis. Phylogenetic analysis is compatible with the dissemination of such operons by horizontal transfer. The enzymes of the solventogenesis pathway and of the cellulosome of C. acetobutylicum comprise a new set of metabolic capacities not previously represented in the collection of complete genomes. These enzymes show a complex pattern of evolutionary affinities, emphasizing the role of lateral gene exchange in the evolution of the unique metabolic profile of the bacterium. Many of the sporulation genes identified in B. subtilis are missing in C. acetobutylicum, which suggests major differences in the sporulation process. Thus, comparative analysis reveals both significant conservation of the genome organization and pronounced differences in many systems that reflect unique adaptive strategies of the two gram-positive bacteria.

Genetic manipulation of acid formation pathways by gene inactivation in Clostridium acetobutylicum ATCC 824

Integrational plasmid technology has been used to disrupt metabolic pathways leading to acetate and butyrate formation in Clostridium acetobutylicum ATCC 824. Non-replicative plasmid constructs, containing either clostridial phosphotransacetylase (pta) or butyrate kinase (buk) gene fragments, were integrated into homologous regions on the chromosome. Integration was assumed to occur by a Campbell-like mechanism, inactivating either pta or buk. Inactivation of the pta gene reduced phosphotransacetylase and acetate kinase activity and significantly decreased acetate production. Inactivation of the buk gene reduced butyrate kinase activity, significantly decreased butyrate production and increased butanol production.

Efficient succinic acid production from glucose through overexpression of pyruvate carboxylase in an Escherichia coli alcohol dehydrogenase and lactate dehydrogenase mutant

An adhE, ldhA double mutant Escherichia colistrain, SBS110MG, has been constructed to produce succinic acid in the presence of heterologous pyruvate carboxylase (PYC). The strategic design aims at diverting maximum quantities of NADH for succinate synthesis by inactivation of NADH competing pathways to increase succinate yield and productivity. Additionally an operational PFL enzyme allows formation of acetyl‐CoA for biosynthesis and formate as a potential source of reducing equivalents. Furthermore, PYC diverts pyruvate toward OAA to favor succinate generation. SBS110MG harboring plasmid pHL413, which encodes the heterologous pyruvate carboxylase from Lactococcus lactis, produced 15.6 g/L (132 mM) of succinate from 18.7 g/L (104 mM) of glucose after 24 h of culture in an atmosphere of CO2 yielding 1.3 mol of succinate per mole of glucose. This molar yield exceeded the maximum theoretical yield of succinate that can be achieved from glucose (1 mol/mol) under anaerobic conditions in terms of NADH balance. The current work further explores the importance of the presence of formate as a source of reducing equivalents in SBS110MG(pHL413). Inactivation of the native formate dehydrogenase pathway (FDH) in this strain significantly reduced succinate yield, suggesting that reducing power was lost in the form of formate. Additionally we investigated the effect of ptsGinactivation in SBS110MG(pHL413) to evaluate the possibility of a further increase in succinate yield. Elimination of the ptsG system increased the succinate yield to 1.4 mol/mol at the expense of a reduction in glucose consumption of 33%. In the presence of PYC and an efficient conversion of glucose to products, the ptsG mutation is not indispensable since PEP converted to pyruvate as a result of glucose phosphorylation by the glucose specific PTS permease EIICBglu can be rediverted toward OAA favoring succinate production.

Construction and analysis of in vivo activity of E. coll promoter hybrids and promoter mutants that alter the −35 to −10 spacing

Abstract A series of promoter hybrids has been constructed by exchanging the − 35 and − 10 regions of lac UV5, tet , and trp promoters. These three promoters and the six hybrid promoters constructed from them have been inserted into a pKO plasmid which places galactokinase expression under the control of the inserted promoter. Additionally, promoter mutants were prepared which had altered the spacing between the − 35 and − 10 regions of the promoter. Derivatives of the tet promoter with one or two extra base pairs in this spacer region and constructions of the lac :: tet hybrid promoter with two different spacings have been inserted into the galactokinase expression plasmid. Measurements of galactokinase levels in strains harboring these plasmids permited the comparison of in vivo activities of the promoters. The strongest of the hybrid promoters (order: − 35, − 10) were trp :: lac and trp :: tet suggesting a high efficiency for the − 35 region of the trp promoter. The weakest promoters were tet :: trp , lac :: trp and lac :: tet indicating a weak − 10 region for the trp promoter and the importance of − 35 to − 10 spacing. Analysis of activity of related promoters with differences in spacing indicated that a distance of 19 bp yields a very weak promoter, and that 18 bp is less active than the 17-bp spacing, which is the most frequently found spacing in promoters.

Metabolic Engineering of Clostridium acetobutylicum ATCC 824 for Isopropanol-Butanol-Ethanol Fermentation

ABSTRACT Clostridium acetobutylicum naturally produces acetone as well as butanol and ethanol. Since acetone cannot be used as a biofuel, its production needs to be minimized or suppressed by cell or bioreactor engineering. Thus, there have been attempts to disrupt or inactivate the acetone formation pathway. Here we present another approach, namely, converting acetone to isopropanol by metabolic engineering. Since isopropanol can be used as a fuel additive, the mixture of isopropanol, butanol, and ethanol (IBE) produced by engineered C. acetobutylicum can be directly used as a biofuel. IBE production is achieved by the expression of a primary/secondary alcohol dehydrogenase gene from Clostridium beijerinckii NRRL B-593 (i.e., adh B-593) in C. acetobutylicum ATCC 824. To increase the total alcohol titer, a synthetic acetone operon (act operon; adc-ctfA-ctfB) was constructed and expressed to increase the flux toward isopropanol formation. When this engineering strategy was applied to the PJC4BK strain lacking in the buk gene (encoding butyrate kinase), a significantly higher titer and yield of IBE could be achieved. The resulting PJC4BK(pIPA3-Cm2) strain produced 20.4 g/liter of total alcohol. Fermentation could be prolonged by in situ removal of solvents by gas stripping, and 35.6 g/liter of the IBE mixture could be produced in 45 h.

Biodegradation of xenobiotics by anaerobic bacteria

Xenobiotic biodegradation under anaerobic conditions such as in groundwater, sediment, landfill, sludge digesters and bioreactors has gained increasing attention over the last two decades. This review gives a broad overview of our current understanding of and recent advances in anaerobic biodegradation of five selected groups of xenobiotic compounds (petroleum hydrocarbons and fuel additives, nitroaromatic compounds and explosives, chlorinated aliphatic and aromatic compounds, pesticides, and surfactants). Significant advances have been made toward the isolation of bacterial cultures, elucidation of biochemical mechanisms, and laboratory and field scale applications for xenobiotic removal. For certain highly chlorinated hydrocarbons (e.g., tetrachlorethylene), anaerobic processes cannot be easily substituted with current aerobic processes. For petroleum hydrocarbons, although aerobic processes are generally used, anaerobic biodegradation is significant under certain circumstances (e.g., O2-depleted aquifers, oil spilled in marshes). For persistent compounds including polychlorinated biphenyls, dioxins, and DDT, anaerobic processes are slow for remedial application, but can be a significant long-term avenue for natural attenuation. In some cases, a sequential anaerobic-aerobic strategy is needed for total destruction of xenobiotic compounds. Several points for future research are also presented in this review.

Succinate production in Escherichia coli.

Succinate has been recognized as an important platform chemical that can be produced from biomass. While a number of organisms are capable of succinate production naturally, this review focuses on the engineering of Escherichia coli for the production of four‐carbon dicarboxylic acid. Important features of a succinate production system are to achieve an optimal balance of reducing equivalents generated by consumption of the feedstock, while maximizing the amount of carbon channeled into the product. Aerobic and anaerobic production strains have been developed and applied to production from glucose and other abundant carbon sources. Metabolic engineering methods and strain evolution have been used and supplemented by the recent application of systems biology and in silico modeling tools to construct optimal production strains. The metabolic capacity of the production strain, the requirement for efficient recovery of succinate, and the reliability of the performance under scaleup are important in the overall process. The costs of the overall biorefinery‐compatible process will determine the economic commercialization of succinate and its impact in larger chemical markets.

Effect of Sorghum vulgare phosphoenolpyruvate carboxylase and Lactococcus lactis pyruvate carboxylase coexpression on succinate production in mutant strains of Escherichia coli

Sorghum vulgare phosphoenolpyruvate carboxylase (PEPC) and Lactococcus lactis pyruvate carboxylase (PYC) were overexpressed in Escherichia coli concurrently to improve the production of succinate, a valuable industrial specialty chemical. This coexpression system was also applied to E. coli mutant strains strategically designed by inactivating the competing pathways of succinate formation. The highest level of succinate production was observed in E. coli strains coexpressing both PEPC and PYC when compared with E. coli strains individually overexpressing either PEPC or PYC. Lactate production was also significantly reduced with PEPC and PYC coexpression. Lactate and acetate pathways were inactivated to eliminate the competing pathways of succinate formation. Results showed that inactivation of both the lactate and acetate pathways with the coexpression of PEPC and PYC was most effective in improving succinate production. Inactivating the lactate or acetate pathway alone only caused a majority of the carbon flux to shift to other metabolites rather than succinate. Coexpression of PEPC and PYC was also applied to an E. coli mutant strain deficient in lactate dehydrogenase and pyruvate:formate lyase that accumulated a substantial amount of the intermediate metabolite pyruvate during growth. Results showed that PEPC and PYC coexpression was effective in depleting pyruvate accumulation and increasing the production of metabolites.

Effect of Overexpression of a Soluble Pyridine Nucleotide Transhydrogenase (UdhA) on the Production of Poly(3-hydroxybutyrate) in Escherichia coli

A soluble pyridine nucleotide transhydrogenase (UdhA) has been used to increase the productivity and yield of PHB in vivo. By inducing a high level of UdhA, which can transfer reducing equivalents between NAD and NADP, we have increased NADPH availability, resulting in high yield and productivity of PHB in Escherichia coli. Coexpression of the phboperon from Alcaligenes eutrophusH16 and the native udhAfrom E. coli from high copy plasmids resulted in an increase in PHB yield from 49 to 66% g of PHB per gram of total cell dry weight and an increase in final concentration from 3.52 to 6.42 g/L; the PHB concentration of the udhA carrying strain is almost twice that of the control strain expressing only the phb operon. The results of this study demonstrate the effectiveness of cofactor manipulation and its application as a tool in metabolic engineering.

Increasing the Acetyl-CoA Pool in the Presence of Overexpressed Phosphoenolpyruvate Carboxylase or Pyruvate Carboxylase Enhances Succinate Production in Escherichia coli

An in vivo strategy to apply the activation effect of acetyl‐CoA on phosphoenolpyruvate carboxylase (PEPC) and pyruvate carboxylase (PYC) to increase succinate production in Escherichia coli was studied. This approach relies on the increased intracellular acetyl‐CoA and CoA levels by overexpressing E. coli pantothenate kinase (PANK). The results showed that coexpression of PANK and PEPC, and PANK and PYC, did improve succinate production compared to the individual expression of PEPC and PYC, respectively. The intracellular acetyl‐CoA and CoA levels were also measured, and each showed a significant increase when the PANK was overexpressed. Another effect observed was a decrease in lactate production. The least amount of lactate was produced when PANK and PEPC, and PANK and PYC, were coexpressed. This result showed increased competitiveness of the succinate pathway at the phosphoenolpyruvate and pyruvate nodes for the carbon flux, as a result reducing the carbon flux toward the lactate pathway. The study also demonstrates a feasible method for metabolic engineering to modulate enzyme activity in vivo through specific activators and inhibitors.