(160aw) Expanding Promoter Options to Engineer an Environmental-Isolate of Bacillus Megaterium

With the recent shift toward the use of bioinformatics in metabolic engineering, developing sets of tools such as promoters, sigma factors, and ribosome binding sites is essential to controlling gene expression and enzyme activity in a predictable manner. In this study, we aim to identify and characterize a set of transcriptional promoters for a strain of Bacillus megaterium (SR7) previously isolated from a deep-subsurface carbon dioxide reservoir. The SR7 strain shows potential for culturing under high pressures of carbon dioxide, which permits in situ extraction of bioproducts to limit toxicity issues. As such, a broad set of constitutive promoters are desired to be active under aerobic and anaerobic conditions. Next-generation RNA sequencing was performed for SR7 grown under aerobic and anaerobic conditions (1 atm CO₂), and resulting sequences were aligned to a previously assembled and annotated genome. This analysis identified several promoters that are highly transcribed under all conditions as well as those differentially transcribed based on environment. Selected promoters were added to a common B. megaterium plasmid that lacked a promoter using golden gate assembly and were subsequently transformed into SR7 using a protoplast method. The effectiveness of these promoters was tested by monitoring the expression of fluorescent reporter proteins. In aerobic conditions, the green fluorescent protein (GFP) was used, whereas in anaerobic conditions either oxygen-insensitive flavin-based fluorescent protein (FbFp) or light, oxygen, or voltage-sensing domain (iLOV) were used. Using these indicator proteins, promoter activity is quantified throughout culture time to determine expression amount and dynamics. Promoter performance was compared to the widely used B. megaterium xylose-based promoter. The eventual goal of this work is to apply these promoters to metabolic pathways to control the expression of enzymes for bioproduction under different growth environments.