A Set of Active Promoters with Different Activity Profiles for Superexpressing Strain.

bacteria are a promising platform for biodegradation, biocatalysis, and biosynthesis, but the use of rhodococci is hampered by the insufficient number of both platform strains for expression and promoters that are functional and thoroughly studied in these strains. To expand the list of such strains and promoters, we studied the expression capability of the M33 strain, and the functioning of a set of recombinant promoters in it. We showed that the strain supports superexpression of the target enzyme (nitrile hydratase) using alternative inexpensive feedings-acetate and urea-without growth factor supplementation, thus being a suitable expression platform. The promoter set included P (elongation factor Tu) and P (superoxide dismutase) from ATCC13032, P (isocitrate lyase) from PR4, and P (nitrile hydratase) from M8. Activity levels, regulation possibilities, and growth-phase-dependent activity profiles of these promoters were studied in derivatives of the M33 strain. The activities of the promoters were significantly different (P < P ≪ P < P), covering 10-fold range, and the most active P and P produced up to a 30-50% portion of target protein in soluble intracellular proteins. On the basis of the mRNA quantification and amount of target protein, the production level of P was positioned close to the theoretical upper limit of expression in a bacterial cell. A selection method for the laboratory evolution of such active promoters directly in was also proposed. Concerning regulation, P could not be regulated (2-fold change), while others were tunable (6-fold for P, 79-fold for P, and 44-fold for P). The promoters possessed four different activity profiles, including three with peak of activity at different growth phases and one with constant activity throughout the growth phases. P and P did not change their activity profile under different growth conditions, whereas the P and P profiles changed depending on the growth media. The results allow flexible construction of strains using the studied promoters, and demonstrate a valuable approach for complex characterization of promoters intended for biotechnological strain construction.