New Strategies in the Localization of Natural Product Biosynthetic Pathways and Achieving Heterologous Expression
Kim, Eun Jin
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Natural products have long furnished medical science playing a significant role in drug discovery and development. Their importance notwithstanding, it is estimated that less than 1% of microorganisms can be cultivated from environmental sources using standard laboratory techniques. It is therefore necessary to develop biochemical and genetic techniques to access these uncultivable genomes. Here as a point of departure toward this goal, two cDNA libraries of two microorganisms were constructed along with five fosmid libraries with DNA isolated from marine environmental samples. We describe the construction of cDNA libraries from marine microbial species and detail experiments to exploit these libraries for their natural product biosynthetic pathways and other metabolic enzymes they harbor. However, no useful biosynthetic pathways were detected within the cDNA libraries. Genetic selection by complementation was additionally explored as a method to identify and localize biosynthetic gene clusters within marine microbial DNA libraries. Genetic selection is a fast and economic method which utilizes selection of a part of a pathway to represent the presence of an entire pathway for the complementation of known mutant strains. We describe genetic selection to localize biotin biosynthetic pathways of Hon6 (Chromohalobacter sp.) as a proof of principle experiment for the identification and localization of biosynthetic pathways in general. Instead of developing purification methods or manipulating cultivation conditions, large fragments of non-culturable bacterial genomes can be cloned and expressed using recombinant DNA technology. A strong transcriptional promoter to control high-level gene expression is required in recombinant expression plasmids. We aimed to develop new tools to control gene expression through the use of riboswitches. Riboswitches are metabolite-sensing ribonucleic acid (RNA) elements that possess the remarkable ability to control gene expression. The thiamine pyrophosphate (TPP) riboswitch system was chosen as it will enable use of E. coli as a suitable host strain. This system is particularly attractive because it has one of the simplest structures among the riboswitches elucidated to date. The use of the TPP riboswitch will also enable modulation of pathway gene expression by varying the TPP coccentration as many gene products are toxic. The violacein gene cluster from Chromobacterium violaceum was selected and placed under the control of this riboswitch. We describe modulation of heterologous gene expression by the ThiC/Riboswitch and detail experiments to investigate the expression and manipulation of the gene cluster under various promoters.