Bio-crude transcriptomics: Gene discovery and metabolic network reconstruction for the biosynthesis of the terpenome of the hydrocarbon oil-producing green alga, Botryococcus braunii race B (Showa)*
AffiliationNatural Products Center, School of Natural Resources and the Environment, The University of Arizona, 250 E. Valencia Rd, Tucson, AZ, 85739, USA
Bio5 Institute, The University of Arizona, 1657 E. Helen St, Tucson, AZ, 85721, USA
Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, P. O. Box 951606, Los Angeles, CA, 90095, USA
Department of Ecology and Evolutionary Biology, The University of Arizona, 1041 E. Lowell St, Tucson, AZ, 85721, USA
Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, TX, 77843, USA
Fatty acid biosynthesis
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CitationMolnár et al. BMC Genomics 2012, 13:576 http://www.biomedcentral.com/1471-2164/13/576
Rights© 2012 Molnár et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0)
Collection InformationThis item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at email@example.com.
AbstractBACKGROUND:Microalgae hold promise for yielding a biofuel feedstock that is sustainable, carbon-neutral, distributed, and only minimally disruptive for the production of food and feed by traditional agriculture. Amongst oleaginous eukaryotic algae, the B race of Botryococcus braunii is unique in that it produces large amounts of liquid hydrocarbons of terpenoid origin. These are comparable to fossil crude oil, and are sequestered outside the cells in a communal extracellular polymeric matrix material. Biosynthetic engineering of terpenoid bio-crude production requires identification of genes and reconstruction of metabolic pathways responsible for production of both hydrocarbons and other metabolites of the alga that compete for photosynthetic carbon and energy.RESULTS:A de novo assembly of 1,334,609 next-generation pyrosequencing reads form the Showa strain of the B race of B. braunii yielded a transcriptomic database of 46,422 contigs with an average length of 756 bp. Contigs were annotated with pathway, ontology, and protein domain identifiers. Manual curation allowed the reconstruction of pathways that produce terpenoid liquid hydrocarbons from primary metabolites, and pathways that divert photosynthetic carbon into tetraterpenoid carotenoids, diterpenoids, and the prenyl chains of meroterpenoid quinones and chlorophyll. Inventories of machine-assembled contigs are also presented for reconstructed pathways for the biosynthesis of competing storage compounds including triacylglycerol and starch. Regeneration of S-adenosylmethionine, and the extracellular localization of the hydrocarbon oils by active transport and possibly autophagy are also investigated.CONCLUSIONS:The construction of an annotated transcriptomic database, publicly available in a web-based data depository and annotation tool, provides a foundation for metabolic pathway and network reconstruction, and facilitates further omics studies in the absence of a genome sequence for the Showa strain of B. braunii, race B. Further, the transcriptome database empowers future biosynthetic engineering approaches for strain improvement and the transfer of desirable traits to heterologous hosts.
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Characterization of an N-acyl-L-homoserine lactone-mediated regulatory system controlling phenazine biosynthesis in Pseudomonas aureofaciens 30-84: In vitro and in situ analysisPierson, Leland S., III; Wood, Derek William, 1965- (The University of Arizona., 1997)Pseudomonas aureofaciens 30-84 is a soilborne bacterium that colonizes the wheat rhizosphere. This strain produces three phenazine antibiotics which are responsible for both suppression of take-all disease of wheat caused by Gaeumannomyces graminis var. tritici and enhanced survival of 30-84 within the wheat rhizosphere in competition with other organisms. A gene (phzR) was identified just prior to the start of this work that is required for phenazine production by 30-84. PhzR was identified as a positive regulator of the phenazine biosynthetic operon. During the course of this dissertation it was discovered that PhzR belongs to the LuxR family of N-acyl- scL-homoserine lactone-responsive transcriptional regulators and that phenazine production in P. aureofaciens 30-84 is mediated by a diffusible signal molecule. The gene responsible for production of this signal (phzI) was identified. Both phzI and phzR are required for the production of phenazines in vitro. Together these two proteins (PhzR/PhzI) comprise a N-acyl- scL-homoserine lactone (AHL) response system that controls phenazine antibiotic production in P. aureofaciens 30-84. Classic AHL-mediated regulatory systems consist of two proteins, a LuxR homolog (PhzR) which transcriptionally activates target gene expression in response to AHL produced by the second protein, the LuxI homolog (PhzI). Using HPLC coupled with high resolution mass spectroscopy, the specific AHL produced via PhzI has been identified as N-hexanoyl- scL-homoserine lactone (HHL). It has been determined that PhzR activates phenazine production in conjunction with HHL produced by PhzI via transcriptional activation of the phenazine biosynthetic gene phzB. A variety of synthetic AHLs restore transcription of phzB and phenazine production in phzI mutants suggesting that phzI mutants can be used to detect the presence of exogenous AHLs. This ability was exploited to show that HHL is required for phenazine expression in situ and is an effective interpopulation signal molecule in the wheat rhizosphere. The work presented in this dissertation is the first to show that AHL-mediated regulation, previously only examined in vitro, can operate within the natural habitat of a bacterium.