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Two new bacterial strains, Pseudomonas marginales MA32 and Pseudomonas putida MA113, containing nitrile hydratases resistant to cyanide were isolated from soil samples by an enrichment procedure. In contrast to known nitrile hydratases, which rapidly lose activity at low to moderate cyanide concentrations, the enzymes described in this paper tolerate up to 50 mM cyanide. They show a broad substrate spectrum including not only small substrates like acrylonitrile but also nitriles with longer side chains and even nitriles with quarternary alpha‐carbon atoms. Both characteristics are essential for the transformation of ketone cyanohydrins, which are much more instable and therefore releasing much higher amounts of prussic acid than cyanohydrins formed from aldehydes. P. marginales MA32 was used as a whole cell biocatalyst for the hydration of acetone cyanohydrin to α‐Hydroxyisobutyramide, which is a precursor of methacrylamide, an important pre‐polymer. After optimization of the process conditions a maximum amide concentration of more than 1.6 M could be reached within 5 hours with 5 g/L biocatalyst referred to cell dry weight.

Metal-dependent cblA-mediated mechanism of transcription regulation of NHase could not discriminate Ni and Co, but mechanism of NHase enzyme maturation could do this.

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Rhodococcus sp. strain M8 is a nitrile-degrading bacterium isolated from acrylonitrile-contaminated sites. This strain produces the enzymes for sequential nitrile degradation, cobalt-type nitrile hydratase, and amidase in large amounts. Its draft genome sequence, announced here, has an estimated size of 6.3 Mbp.

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.

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