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Currently, most of the RNAs used in lab research are prepared by in vitro transcription or chemical synthesis, which can be costly. In vivo expression in bacterial cells is another approach to RNA preparation that allows large scale production at a lower cost. However, there are some obstacles in bacterial expression, including RNA degradation in host cell, as well as RNA extraction and purification. tRNA and 5S RNA have been reported as scaffolds to circumvent the degradation problem. These scaffolds can not only make the RNA product survive in the cell but also increase the stability after extraction. The packaging RNA (pRNA) of bacteriophage phi29 is a small non-coding RNA with a compact structure. The three-way junction (3WJ) region from pRNA is a thermodynamically stable RNA motif good for constructing therapeutic RNA nanoparticles. The 3WJ can not only integrate multiple RNA modules, but also stabilize them. Here I report a series of approaches made to express recombinant RNAs based on pRNA or 3WJ in bacteria, including 1) Investigating the mechanism of RNA folding in vitro and in vivo using 3WJ. 3WJ-based RNAs were expressed in E. coli using pET system. The results show that the folding of RNA is affected by both overall and regional energy landscape. 2) Expression of an RNA nanoparticle harboring multiple functional modules, a model of therapeutic RNA, in E. coli using a combination of tRNA scaffold and pRNA-3WJ. The expression was successful and all of the RNA modules were functional. 3) Expression of pRNA-based recombinant RNAs in B. subtilis. This is a novel system of expressing recombinant RNAs in Gram-positive bacteria.

Some bacteria produce a potent antifungal agent (pyrrolnitrin) from tryptophan using four dioxygen dependent steps to outcompete other microbes. Each step of this process is catalyzed by an oxygenase encoded by the prnABCD cassette. The first enzymatic step in pyrrolnitrin biosynthesis is the regioselective chlorination of tryptophan to form 7-chlorotryptophan. This halogenation is catalyzed by PrnA, a Flavin dependent oxygenase, which has been isolated and characterized from P. fluorescens. The pyrrolnitrin biosynthesis pathway (prnABCD) has been also observed in the Burkholderia genus. This thesis comprises my studies on the expression, purification, and characterization of PrnA from Burkholderia ambifaria. Beyond the comparative preliminary data on B. ambifaria PrnA, we report an NADPH-dependent activity assay for PrnA coupling the oxidative chlorination of tryptophan (and related substrates) with NADPH consumption. The steady-state kinetic parameters associated with PrnA of kcat, KM, and catalytic efficiency of enzymes are also reported for this system under defined experimental conditions.

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