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Rhodococcus strains were designed as model biocatalysts (BCs) for the production of acrylic acid and mixtures of acrylic monomers consisting of acrylamide, acrylic acid, and N-alkylacrylamide (N-isopropylacrylamide). To obtain BC strains, we used, among other approaches, adaptive laboratory evolution (ALE), based on the use of the metabolic pathway of amide utilization. Whole genome sequencing of the strains obtained after ALE, as well as subsequent targeted gene disruption, identified candidate genes for three new amidases that are promising for the development of BCs for the production of acrylic acid from acrylamide. New BCs had two types of amidase activities, acrylamide-hydrolyzing and acrylamide-transferring, and by varying the ratio of these activities in BCs, it is possible to influence the ratio of monomers in the resulting mixtures. Based on these strains, a prototype of a new technological concept for the biocatalytic synthesis of acrylic monomers was developed for the production of water-soluble acrylic heteropolymers containing valuable N-alkylacrylamide units. In addition to the possibility of obtaining mixtures of different compositions, the advantages of the concept are a single starting reagent (acrylamide), more unification of processes (all processes are based on the same type of biocatalyst), and potentially greater safety for personnel and the environment compared to existing chemical technologies., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Rhodococcus bacteria are a fast-growing platform for biocatalysis, biodegradation, and biosynthesis, but not a platform for molecular biology. That is, Rhodococcus are not convenient for genetic engineering. One major issue for the engineering of Rhodococcus is the absence of a publicly available, curated, and commented collection of sequences of genetic parts that are functional in biotechnologically relevant species of Rhodococcus ( R. erythropolis , R. rhodochrous , R. ruber , and R. jostii ). Here, we present a collection of genetic parts for Rhodococcus (vector replicons, promoter regions, regulators, markers, and reporters) supported by a thorough analysis of their functionality. We also highlight and discuss the gaps in Rhodococcus -related genetic parts and techniques, which should be filled in order to make these bacteria a full-fledged molecular biology platform independent of Escherichia coli . We conclude that all major types of required genetic parts for Rhodococcus are available now, except multicopy replicons. As for model Rhodococcus strains, there is a particular shortage of strains with high electrocompetence levels and strains designed for solving specific genetic engineering tasks. We suggest that these obstacles are surmountable in the near future due to an intensification of research work in the field of genetic techniques for non-conventional bacteria.

We describe here the 7.0-Mb draft genome sequence of Rhodococcus qingshengii strain TA37, which was obtained from samples of nitrile-contaminated soil collected in the Saratov Region (Russian Federation). This genomic resource will support the further development of biocatalysts for the inexpensive and green production of acrylic monomers.

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.

We report a 6.27-Mbp complete genome of Rhodococcus sp. strain M8, an originally discovered strain that is now under investigation for production of acrylic monomers. The genome consists of a 6.1-Mbp circular chromosome and a 173.2-kbp plasmid., (Copyright © 2021 Novikov et al.)

We describe here a 6.6-Mb draft genome sequence of Rhodococcus erythropolis strain HX7, which was obtained from soil samples collected from the northern Arkhangelsk region in the Russian Federation. This genomic resource will support further study of mechanisms of cold-resistant oil degradation in soil and potentially aid in soil bioremediation in cold oil-producing regions., (Copyright © 2021 Novikov et al.)

This study highlights the effect of heavy metal ions on the expression of cobalt-containing nitrile hydratase (NHase) in Rhodococcus strains, which over-produce this enzyme. Both metal-dependent derepression of transcription and maturation of NHase were considered. We demonstrated that nickel ions can derepress the NHase promoter in several Rhodococcus strains. The cblA gene of a cobalt-dependent transcriptional repressor was shown to be indispensable for nickel-mediated derepression. As for maturation, we showed that nickel ions could not replace cobalt ions during the synthesis of active NHase. We also revealed that the amount of β-subunit decreased during NHase expression without added cobalt. We showed this using three variants of NHase in vivo synthesis: by using nickel- or urea-induced synthesis in cblA+ strains, and by using metal-independent constitutive synthesis in cblA- strains. In all cases, we found that the amount of β-subunit was significantly lower than the amount of α-subunit. In contrast, equimolar amounts of both subunits were synthesized after growth in the presence of added cobalt. Nickel did not affect NHase synthesis in mixtures with cobalt. This suggests that the metal selectivity in cblA-dependent regulation of NHase transcription was too low to discriminate between cobalt and nickel, but the selectivity of the NHase maturation mechanism was high enough to do so. Moreover, we can assume that the β-subunit is more subject to proteolytic degradation without the addition of cobalt, than the α-subunit. This indicates that cobalt ions presumably play an unknown role in the stability of the β-subunit in vivo.

Rhodococcus strains are important biocatalysts used for biotechnological production of acrylamide catalysed by a nitrile hydratase (NHase) containing cobalt. This metalloenzyme is present at high intracellular concentrations representing up to 50% of the soluble proteins in Rhodococcus rhodochrous M8 strain. Cobalt ions were formerly reported to be essential for the synthesis of the NHase subunits, encoded by nhmBA structural genes in R. rhodochrous M8. To understand the regulatory mechanisms enabling high expression of the NHase structural genes by cobalt, two reporter genes coding for an acylamidase from Rhodococcus erythropolis TA37 and a nitrilase from Alcaligenes denitrificans C-32 were fused to the nhmBA promoter. It was shown that cobalt-dependent regulation of transcription occurs independently of another regulatory genes, nhmCD, involved in substrate-dependent regulation of transcription. Cobalt ions led to an increase (up to five-fold) in transcription of reporter genes correlated with synthesis of corresponding enzymes in R. rhodochrous recombinant strains. This led to identification of a new transcriptional regulator from the ArsR family, named CblA. Using a cblA mutant strain, it was established that CblA acted as a repressor by preventing transcription of the NHase operon promoter in the absence of cobalt ions., (Copyright © 2018 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

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., (Copyright © 2018 Novikov et al.)

Gammaproteobacterium Thalassolituus oleivorans plays an important role in oil degradation in sea water through emulsifying crude oil and alkanes at low temperatures in polar sea environment. Here we report the complete genome sequence of K-188 strain (VKPM B-9394) isolated in the Barents Sea and compare it with other known Thalassolituus oleivorans strains. The Thalassolituus strains are differed in orthologs number of the genes of alkane degradation, transport proteins, genes of sugar utilization, endonucleases, signaling proteins, transcriptional regulators and presence of CRISPR/Cas locus. Also only the genome of K-188 contains the 3-hydroxyalkanoate synthetase., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The nucleotide sequence of cryptic plasmid (designated as pBL90) detected in the cells of Brevibacterium lactofermentum DSM 1412 was determined. The length of plasmid DNA is 67826 bp. Comparison of the nucleotide sequence of pBL90 with known plasmid sequences showed no long regions of significant homology. Computer analysis of the plasmid DNA revealed 29 open reading frames (ORFs). The amino acid sequences of 15 ORFs (approximately 25% of plasmid length) have a high (>70%) level of identity to proteins from different plasmids of Corynebacterium representatives, including replicative proteins. Unusual in pBL90 is the presence of replicative genes from two different families and types of replication.

The expression of a new acylamidase gene from R. erythropolis 37 was studied in Rhodococcus erythropolis strains. This acylamidase, as a result of its unique substrate specificity, can hydrolyse N-substituted amides (4'-nitroacetanilide, N-isopropylacrylamide, N'N-dimethylaminopropylacrylamide). A new expression system based on the use of the promoter region of nitrilhydratase genes from R. rhodochrous M8 was created to achieve constitutive synthesis of acylamidase in R. erythropolis cells. A fourfold improvement in the acylamidase activity of recombinant R. erythropolis cells as compared with the parent wild-type strain was obtained through the use of the new expression system.

Nucleotide sequence of cryptic plasmid pN30 from a Rhodococcus erythropolis 30 soil isolate was determined. Plasmid DNA consists of 5403 nucleotide pairs and contains about 62% GC pairs, which is typical of Rhodococcus DNA. No significant homology was determined between the pN30 DNA sequence and those of known plasmids. Computer-aided analysis of pN30 sequence revealed open reading frames that encode proteins strongly homologous to replicative proteins encoded by small cryptic plasmids of different actinomycetes.

Static photorefractive gratings are recorded by a running interference pattern in the ferroelectric crystal Ba(2)NaNb(5)O(15) with an externally applied ac field. Both two- and four-wave mixing based on this recording mechanism are investigated.