Development of next generation sustainable dsRNA biocontrols

Global food demand is expected to rise by 30–62% in the coming decades, yet 20–40% of crop yields are lost annually to pests and plant diseases, resulting in losses of ~$540 billion. dsRNA biocontrols are emerging as sustainable and highly specific alternatives to broad-spectrum pesticides. However, realising their potential requires economic, large-scale manufacturing of high-quality dsRNA, which remains a challenge. This thesis aimed to optimise and develop novel microbial production strategies for dsRNA biocontrols enabled by synthetic biology approaches. Novel plasmid DNA constructs were designed using alternative multiple synthetic transcriptional terminators for dsRNA production in Escherichia coli Using triple transcriptional terminators, a 7.8-fold increase in dsRNA yield was achieved compared to constructs without terminators. Application of E. coli-expressed transcriptionally terminated dsRNA biocontrols led to increased insect mortality. Furthermore, the effect of convergent and divergent T7 RNA polymerase production of dsRNA via in vitro transcription and in vivo production in E. coli was investigated. In vivo, convergent promoters resulted in a 2.1-fold increase for larger dsRNAs (>400 bp). In contrast, a 2.2-fold increase for smaller dsRNAs (<250 bp) was obtained using divergent promoters. In vitro, a 6.46-fold increase was obtained using divergent promoters. Finally, building on the optimised microbial dsRNA production method, a novel approach for generating mRNA medicines in microbial systems was developed. The results demonstrated successful production of 3’ poly(A) tailed dsRNA, separation and isolation of poly(A) mRNA via denaturing IP-RP-HPLC, enzymatic 5’ capping and functional validation through GFP expression in HEK293 cells. This thesis has developed methods for increased dsRNA yield in microbial synthesis, providing mechanistic insight into transcriptional termination and RNA polymerase promoter design. These findings are relevant for the potential application of dsRNA-based biocontrols in agriculture. Furthermore, we present a new method that facilitates cost-effective large-scale manufacturing of mRNA medicines using microbial systems.