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

ArticleinApplied and Environmental Microbiology 73(9):3061-8 · June 2007with20 Reads
DOI: 10.1128/AEM.02296-06 · Source: PubMed
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.
    • "A single clone was isolated from the cytostat and designated M863 (Table 1). Using an approach as described previously [15], a library of clones was created that positioned random pieces of T. saccharolyticum DNA down-stream from a strong promoter integrated into the T. saccharolyticum chromosome, with the expectation that overexpression of some genes would lead to improved inhibitor tolerance. The library was selected on solid or liquid media containing extracts from pre-treated hardwood. "
    [Show abstract] [Hide abstract] ABSTRACT: Background The thermophilic, anaerobic bacterium Thermoanaerobacterium saccharolyticum digests hemicellulose and utilizes the major sugars present in biomass. It was previously engineered to produce ethanol at yields equivalent to yeast. While saccharolytic anaerobes have been long studied as potential biomass-fermenting organisms, development efforts for commercial ethanol production have not been reported. Results Here, we describe the highest ethanol titers achieved from T. saccharolyticum during a 4-year project to develop it for industrial production of ethanol from pre-treated hardwood at 51–55 °C. We describe organism and bioprocess development efforts undertaken to improve ethanol production. The final strain M2886 was generated by removing genes for exopolysaccharide synthesis, the regulator perR, and re-introduction of phosphotransacetylase and acetate kinase into the methyglyoxal synthase gene. It was also subject to multiple rounds of adaptation and selection, resulting in mutations later identified by resequencing. The highest ethanol titer achieved was 70 g/L in batch culture with a mixture of cellobiose and maltodextrin. In a “mock hydrolysate” Simultaneous Saccharification and Fermentation (SSF) with Sigmacell-20, glucose, xylose, and acetic acid, an ethanol titer of 61 g/L was achieved, at 92 % of theoretical yield. Fungal cellulases were rapidly inactivated under these conditions and had to be supplemented with cellulosomes from C. thermocellum. Ethanol titers of 31 g/L were reached in a 100 L SSF of pre-treated hardwood and 26 g/L in a fermentation of a hardwood hemicellulose extract. Conclusions This study demonstrates that thermophilic anaerobes are capable of producing ethanol at high yield and at titers greater than 60 g/L from purified substrates, but additional work is needed to produce the same ethanol titers from pre-treated hardwood.
    Full-text · Article · Dec 2016
    • "Butanol-tolerant strains have been mainly achieved by serial enrichment using increasing butanol concentrations and random chemical mutagenesis [2,[8][9][10]. The alternative directed mutagenesis of Clostridium spp. is still a difficult approach since solvent tolerance is a complex metabolic response involving a large number of genes [11][12][13][14][15], and often not enough genetic information about the target microorganisms is available. Furthermore , tools for directed mutagenesis specific for Clostridium spp. "
    [Show abstract] [Hide abstract] ABSTRACT: Butanol and 1,3-propanediol (1,3-PDO) are simultaneously produced by Clostridium pasteurianum from glycerol. In this study, random chemical mutagenesis of C. pasteurianum DSM 525 was conducted to improve its tolerance to butanol. Selected nutritional and operational parameters were evaluated to identify strategies that favour the production of each metabolite. From those experiments, it was possible to isolate cells able to produce 22% more butanol than the parent strain in serum bottles. The supplementation of the culture medium with 2 mg l−1 of iron increased the production of butanol by 163%, and the optimum inoculum age was found to be 12 h. Overall, the experiments conducted in bioreactor led to lower butanol titers than in serum bottles, which is attributed to the higher pressure present in the bottles. At pH 6.0, N2 sparging notoriously favoured the production of biomass and 1,3-PDO, while a lower pH (5.0) led to a higher butanol yield, although growth was negatively affected. The results herein gathered allowed the identification of specific conditions that favour the production of either butanol or 1,3-PDO. Furthermore, it was found that N2 sparging is a suitable strategy to maximize the titer, yield and productivity of 1,3-PDO using C. pasteurianum.
    Article · May 2016
    • "The 824D strain contains CAC1867, an XRE family regulator known to activate in the presence of xenobiotics [58]. Interestingly, an XRE response element, CAC1869 (in the same operon as CAC1867), was enriched in a similar 1-butanol challenge of C. acetobutylicum [9] . The amidohydrolase family of proteins like that of ENVG typically break nitrogen carbon bonds and can be found in proteins synthesizing glutamate, aspartic acid, and biotin. "
    [Show abstract] [Hide abstract] ABSTRACT: Advances in Raman spectroscopy are enabling more comprehensive measurement of microbial cell chemical composition. Advantages include results returned in near real-time and minimal sample preparation. In this research, Raman spectroscopy was used to analyze E. coli with engineered solvent tolerance, which is a multi-genic trait associated with complex and uncharacterized phenotypes that are of value to industrial microbiology. To generate solvent tolerant phenotypes, E. coli transformed with DNA libraries were serially enriched in the presence of 0.9% (v/v) and 1.1% (v/v) 1-butanol. DNA libraries were created using degenerate oligonucleotide primed PCR (DOP-PCR) from the genomic DNA of E. coli, Clostridium acetobutylicum ATCC 824, and the metagenome of a stream bank soil sample, which contained DNA from 72 different phyla. DOP-PCR enabled high efficiency library cloning (with no DNA shearing or end-polishing) and the inclusion un-culturable organisms. Nine strains with improved tolerance were analyzed by Raman spectroscopy and vastly different solvent-tolerant phenotypes were characterized. Common among these was improved membrane rigidity from increasing the fraction of unsaturated fatty acids at the expense of cyclopropane fatty acids. Raman spectroscopy offers the ability to monitor cell phenotype changes in near real-time and is adaptable to high-throughput screening, making it relevant to metabolic engineering.
    Full-text · Article · Jan 2016
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