Dynamics of Genomic-Library Enrichment and Identification of Solvent Tolerance Genes for Clostridium acetobutylicum

Abstract

A Clostridium acetobutylicum ATCC 824 genomic library was constructed using randomly sheared DNA. Library inserts conferring increased tolerance to 1-butanol were isolated using two protocols. Protocol I utilized a single round of butanol challenges in batch culture, while protocol II, which gave clearly superior outcomes, was based on the serial transfer of stationary-phase cultures into progressively higher butanol concentrations. DNA microarray analysis made a high-resolution assessment of the dynamic process of library enrichment possible for the first time. Protocol I yielded a library insert containing the entire coding region of the gene CAC0003 (which codes for a protein of unknown function) but also several DNA fragments containing promoter regions. Protocol II enabled the successful identification of DNA fragments containing several intact genes conferring preferential growth under conditions of butanol stress. Since expression using the employed library is possible only from natural promoters, among the enriched genes, we identified 16 genes that constitute the first cistron of a transcriptional unit. These genes include four transcriptional regulators (CAC0977, CAC1463, CAC1869, and CAC2495). After subcloning plasmids carrying the CAC0003 and CAC1869 genes, strains 824(pCAC0003) and 824(pCAC1869) exhibited 13% and an 81% increases, respectively, in butanol tolerance relative to the plasmid control strain. 824(pCAC1869) consistently grew to higher cell densities in challenged and unchallenged cultures and exhibited prolonged metabolism. Our serial enrichment approach provided a more detailed understanding of the dynamic process of library enrichment under conditions of selective growth. Further characterization of the genes identified in this study will likely enhance our understanding of the complex phenotype of solvent tolerance.

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

“…Finally, we carried out Q-RT-PCR analyses of similar (but on purpose not identical in order to examine the reproducibility of the expression responses to various levels of stress; Tomas et al, 2004) biological experiments with acetate and butyrate stress for genes CAC0393, CAC0456, CAC0877, CAC3189, and CAC3288, which were selected based on the data presented below. For these experiments, we used the medium which includes 30 mM of acetate (i.e., CGM supplemented with 80 g/L glucose and 30 mM acetate), which is the standard medium for C. acetobutylicum cultures (Borden and Papoutsakis, 2007), as it provides better pH buffering, and reproducibility in metabolite profiles; the experiments of Figure 1 and of all the microarray data were carried out without this initial 30 mM acetate. We chose to use this standard medium to examine if key microarray data would be still reproducible at least in a qualitative and trend sense.…”

Engineering alternative butanol production platforms in heterologous bacteria

“…Additionally, stresses caused by butanol toxicity 60 have been attributed to the loss of pSOL1, a mega-plasmid encoding several essential 61 solvent-forming genes (Borden and Papoutsakis, 2007). Furthermore, the metabolic shift 62 from acidogenesis to solventogenesis in Clostridium presents additional complications 63 al., 2008; Knoshaug and Zhang, 2008).…”

Antibacterial photosensitization through activation of coproporphyrinogen oxidase

“…Phenotypic high-throughput screens using small-molecule libraries often result in the identification of numerous molecules with unknown targets. Several approaches have been successful in identifying intracellular targets of small molecules (26)(27)(28)(29)(30)(31)(32). Here, we report a genetic selection strategy based on the creation of a suicide strain that enables the identification of spontaneous resistant mutants to an activating compound that is typically nontoxic.…”