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

Abstract

Rhodococcus 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 Rhodococcus rhodochrous 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 tuf (elongation factor Tu) and P sod (superoxide dismutase) from Corynebacterium glutamicum ATCC13032, P cpi (isocitrate lyase) from Rhodococcus erythropolis PR4, and P nh (nitrile hydratase) from R. rhodochrous 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 cpi < P sod ≪ P tuf < P nh ), covering 10 3 -fold range, and the most active P nh and P tuf 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 nh 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 Rhodococcus was also proposed. Concerning regulation, P tuf could not be regulated (2-fold change), while others were tunable (6-fold for P sod , 79-fold for P nh , and 44-fold for P cpi ). 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 tuf and P cpi did not change their activity profile under different growth conditions, whereas the P sod and P nh profiles changed depending on the growth media. The results allow flexible construction of Rhodococcus strains using the studied promoters, and demonstrate a valuable approach for complex characterization of promoters intended for biotechnological strain construction.

An Integrative Toolbox for Synthetic Biology in Rhodococcus

“…Two growth phases were examined, as promoter activity can vary significantly with growth phase. 47 Of the tested promoters, P M6 , P tet , and P T1 were the strongest, although P M6 yielded ∼2.5−3 times greater fluorescence than the other two. We had identified P M6 previously and developed P T1 as a derivative.…”

“…Integrative promoter constructs were transformed into RHA1, and promoter activity was determined in mid exponential and early stationary phase by using fluorescence normalized to OD 600 as a readout (Figure C and D). Two growth phases were examined, as promoter activity can vary significantly with growth phase . Of the tested promoters, P M6 , P tet , and P T1 were the strongest, although P M6 yielded ∼2.5–3 times greater fluorescence than the other two.…”

Further Studies on the 3-Ketosteroid 9α-Hydroxylase of Rhodococcus ruber Chol-4, a Rieske Oxygenase of the Steroid Degradation Pathway

“…Recently, a review on the advances in genetic toolkits and methods for engineering Rhodococcus has been published, and, in parallel, a set of Rhodococcus promoter-RBS combinations with fine-tuning of different activity levels have been built and compiled [35,45]. The Rhodococcus promoter regions described here can contribute to enrich the set of genetic parts for gene expression control in this strain.…”

“…The studies done with a series of R. ruber mutants (∆kshB and single, double and triple ∆kshA mutants) have probed that KshA2 isoform is needed for the degradation of steroid substrates with short side chain; KshA3 works on those molecules with longer side chains and KshA1 acts as a more versatile enzyme related to the cholic acid (ACHO) catabolism and collaborating with KshA2 or KshA3 activities [31]. Although these enzymes are important within the steroid catabolism, there are not many studies done on genetic regulation of these activities, just for KstD promoters [32], a partial study on Ksh2 in Rhodococcus erythropolis strain SQ1 [33] and general studies on Rhodococcus gene promoters [34,35]. The findings of how the steroid clusters are regulated could be useful in metabolic engineering of Rhodococcus strains.…”