Metabolite stress and tolerance in the production of biofuels and chemicals: gene-expression-based systems analysis of butanol, butyrate, and acetate stresses in the anaerobe Clostridium acetobutylicum.

Metabolite accumulation has pleiotropic, toxic, or beneficial effects on cell physiology, but such effects are not well understood at the molecular level. Cells respond and adapt to metabolite stress by mechanisms largely unexplored, especially in the context of multiple and simultaneous stresses. Solventogenic and related clostridia have an inherent advantage for production of biofuels and chemicals directly from cellulosic material and other complex carbohydrates, but issues of product/metabolite tolerance and related culture productivities remain. Using DNA microarray-based gene expression analysis, the transcriptional-stress responses of Clostridium acetobutylicum to fermentation acids acetate and butyrate and the solvent product butanol were analyzed and compared in the context of cell physiology. Ontological analysis demonstrated that stress by all three metabolites resulted in upregulation of genes related to post-translational modifications and chaperone activity, and downregulation of the translation-machinery genes. Motility genes were downregulated by acetate-stress only. The general metabolite stress included upregulation of numerous stress genes (dnaK, groES, groEL, hsp90, hsp18, clpC, and htrA), the solventogenic operon aad-ctfA-ctfB, and other solventogenic genes. Acetate stress downregulated expression of the butyryl-CoA- and butyrate-formation genes, while butyrate stress downregulated expression of acetate-formation genes. Pyrimidine-biosynthesis genes were downregulated by most stresses, but purine-biosynthesis genes were upregulated by acetate and butyrate, possibly for thiamine and histidine biosynthesis. Methionine-biosynthesis genes were upregulated by acetate stress, indicating a possibly conserved stress response mechanism also observed in Escherichia coli. Nitrogen-fixation gene expression was upregulated by acetate stress. Butyrate stress upregulated many iron-metabolism genes, riboflavin-biosynthesis genes, and several genes related to cellular repair from oxidative stress, such as perR and superoxide dismutases. Butanol stress upregulated the glycerol metabolism genes glpA and glpF. Surprisingly, metabolite stress had no apparent effect on the expression of the sporulation-cascade genes. It is argued that the list of upregulated genes in response to the three metabolite stresses includes several genes whose overexpression would likely impart tolerance, thus making the information generated in this study, a valuable source for the development of tolerant recombinant strains.

A comparative genomic view of clostridial sporulation and physiology.

Clostridia are anaerobic, endospore-forming prokaryotes that include strains of importance to human and animal health and physiology, cellulose degradation, solvent production and bioremediation. Their differentiation and related developmental programmes are not well understood at the molecular level. Recent genome sequencing and transcriptional-profiling studies have offered a glimpse of their inner workings and indicate that a better understanding of the orchestration of the molecular events that underlie their unique physiology, capabilities and diversity will pay major dividends.

Transcriptional program of early sporulation and stationary-phase events in Clostridium acetobutylicum.

DNA microarray analysis of Clostridium acetobutylicum was used to examine the genomic-scale gene expression changes during the shift from exponential-phase growth and acidogenesis to stationary phase and solventogenesis. Self-organizing maps were used to identify novel expression patterns of functional gene classes, including aromatic and branched-chain amino acid synthesis, ribosomal proteins, cobalt and iron transporters, cobalamin biosynthesis, and lipid biosynthesis. The majority of pSOL1 megaplasmid genes (in addition to the solventogenic genes aad-ctfA-ctfB and adc) had increased expression at the onset of solventogenesis, suggesting that other megaplasmid genes may play a role in stationary-phase phenomena. Analysis of sporulation genes and comparison with published Bacillus subtilis results indicated conserved expression patterns of early sporulation genes, including spo0A, the sigF operon, and putative canonical genes of the sigma(H) and sigma(F) regulons. However, sigE expression could not be detected within 7.5 h of initial spo0A expression, consistent with the observed extended time between the appearance of clostridial forms and endospore formation. The results were compared with microarray comparisons of the wild-type strain and the nonsolventogenic, asporogenous M5 strain, which lacks the pSOL1 megaplasmid. While some results were similar, the expression of primary metabolism genes and heat shock proteins was higher in M5, suggesting a difference in metabolic regulation or a butyrate stress response in M5. The results of this microarray platform and analysis were further validated by comparing gene expression patterns to previously published Northern analyses, reporter assays, and two-dimensional protein electrophoresis data of metabolic genes (including all major solventogenesis genes), sporulation genes, heat shock proteins, and other solventogenesis-induced gene expression.

Transcriptional analysis of spo0A overexpression in Clostridium acetobutylicum and its effect on the cell's response to butanol stress.

Spo0A is the regulator of stationary-phase events and is required for transcription of solvent formation genes in Clostridium acetobutylicum. In order to elucidate the role of spo0A in differentiation, we performed transcriptional analysis of 824(pMSPOA) (a spo0A-overexpressing C. acetobutylicum strain with enhanced sporulation) against a plasmid control strain. DNA microarray data were contrasted to data from a spo0A knockout strain (SKO1) that neither sporulates nor produces solvents. Transcripts of fatty acid metabolism genes, motility and chemotaxis genes, heat shock protein genes, and genes encoding the Fts family of cell division proteins were differentially expressed in the two strains, suggesting that these genes play roles in sporulation and the solvent stress response. 824(pMSPOA) alone showed significant downregulation of many glycolytic genes in stationary phase, which is consistent with metabolic flux analysis data. Surprisingly, spo0A overexpression resulted in only nominal transcriptional changes of regulatory genes (abrB and sigF) whose expression was significantly altered in SKO1. Overexpression of spo0A imparted increased tolerance and prolonged metabolism in response to butanol stress. While most of the differentially expressed genes appear to be part of a general stress response (similar to patterns in two plasmid control strains and a groESL-overexpressing strain), several genes were expressed at higher levels at early time points after butanol challenge only in 824(pMSPOA). Most of these genes were related to butyryl coenzyme A and butyrate formation and/or assimilation, but they also included the cell division gene ftsX, the gyrase subunit-encoding genes gyrB and gyrA, DNA synthesis and repair genes, and fatty acid synthesis genes, all of which might play a role in the immediate butanol stress response, and thus in enhanced butanol tolerance.

DNA array-based transcriptional analysis of asporogenous, nonsolventogenic Clostridium acetobutylicum strains SKO1 and M5.

The large-scale transcriptional program of two Clostridium acetobutylicum strains (SKO1 and M5) relative to that of the parent strain (wild type [WT]) was examined by using DNA microarrays. Glass DNA arrays containing a selected set of 1,019 genes (including all 178 pSOL1 genes) covering more than 25% of the whole genome were designed, constructed, and validated for data reliability. Strain SKO1, with an inactivated spo0A gene, displays an asporogenous, filamentous, and largely deficient solventogenic phenotype. SKO1 displays downregulation of all solvent formation genes, sigF, and carbohydrate metabolism genes (similar to genes expressed as part of the stationary-phase response in Bacillus subtilis) but also several electron transport genes. A major cluster of genes upregulated in SKO1 includes abrB, the genes from the major chemotaxis and motility operons, and glycosylation genes. Strain M5 displays an asporogenous and nonsolventogenic phenotype due to loss of the megaplasmid pSOL1, which contains all genes necessary for solvent formation. Therefore, M5 displays downregulation of all pSOL1 genes expressed in the WT. Notable among other genes expressed more highly in WT than in M5 were sigF, several two-component histidine kinases, spo0A, cheA, cheC, many stress response genes, fts family genes, DNA topoisomerase genes, and central-carbon metabolism genes. Genes expressed more highly in M5 include electron transport genes (but different from those downregulated in SKO1) and several motility and chemotaxis genes. Most of these expression patterns were consistent with phenotypic characteristics. Several of these expression patterns are new or different from what is known in B. subtilis and can be used to test a number of functional-genomic hypotheses.