BACKGROUND: Rhodococci are industrially important soil-dwelling Gram-positive bacteria that are well known for both nitrile hydrolysis and oxidative metabolism of aromatics. Rhodococcus rhodochrous ATCC BAA-870 is capable of metabolising a wide range of aliphatic and aromatic nitriles and amides. The genome of the organism was sequenced and analysed in order to better understand this whole cell biocatalyst. RESULTS: The genome of R. rhodochrous ATCC BAA-870 is the first Rhodococcus genome fully sequenced using Nanopore sequencing. The circular genome contains 5.9 megabase pairs (Mbp) and includes a 0.53 Mbp linear plasmid, that together encode 7548 predicted protein sequences according to BASys annotation, and 5535 predicted protein sequences according to RAST annotation. The genome contains numerous oxidoreductases, 15 identified antibiotic and secondary metabolite gene clusters, several terpene and nonribosomal peptide synthetase clusters, as well as 6 putative clusters of unknown type. The 0.53 Mbp plasmid encodes 677 predicted genes and contains the nitrile converting gene cluster, including a nitrilase, a low molecular weight nitrile hydratase, and an enantioselective amidase. Although there are fewer biotechnologically relevant enzymes compared to those found in rhodococci with larger genomes, such as the well-known Rhodococcus jostii RHA1, the abundance of transporters in combination with the myriad of enzymes found in strain BAA-870 might make it more suitable for use in industrially relevant processes than other rhodococci. CONCLUSIONS: The sequence and comprehensive description of the R. rhodochrous ATCC BAA-870 genome will facilitate the additional exploitation of rhodococci for biotechnological applications, as well as enable further characterisation of this model organism. The genome encodes a wide range of enzymes, many with unknown substrate specificities supporting potential applications in biotechnology, including nitrilases, nitrile hydratase, monooxygenases, cytochrome P450s, reductases, proteases, lipases, and transaminases.

Recent discoveries have shown that nanopatterns with feature sizes ≤100 nm could direct stem cell fate or kill bacteria. These effects could be used to develop orthopedic implants with improved osseointegration and decreased chance of implant-associated infections. The quest for osteogenic and bactericidal nanopatterns is ongoing but no controlled nanopatterns with dual osteogenic and bactericidal functionalities have been found yet. In this study, electron beam induced deposition (EBID) was used for accurate and reproducible decoration of silicon surfaces with four different types of nanopatterns. The features used in the first two nanopatterns (OST1 and OST2) were derived from osteogenic nanopatterns known to induce osteogenic differentiation of stem cells in the absence of osteogenic supplements. Two modifications of these nanopatterns were also included (OST2-SQ, OST2-H90) to study the effects of controlled disorder and lower nanopillar heights. An E. coli K-12 strain was used for probing the response of bacteria to the nanopatterns. Three nanopatterns (OST2, OST2-SQ, and OST2-H90) exhibited clear bactericidal behavior as evidenced by severely damaged cells and disrupted formation of extracellular polymeric substance. These findings indicate that controlled nanopatterns with features derived from osteogenic ones can have bactericidal activity and that EBID represents an enabling nanotechnology to achieve (multi)functional nanopatterns for bone implants.

Development of synthetic bactericidal surfaces is a drug-free route to the prevention of implant-associated infections. Surface nanotopographies with specific dimensions have been shown to kill various types of bacterial strains through a mechanical mechanism, while regulating stem cell differentiation and tissue regeneration. The effective ranges of dimensions required to simultaneously achieve both aims are in the <200 nm range. Here, a nanoscale additive manufacturing (=3D printing) technique called electron beam induced deposition (EBID) is used to fabricate nanopillars with reproducible and precisely controlled dimensions and arrangements that are within those effective ranges (i.e. a height of 190 nm, a diameter of 80 nm, and an interspacing of 170 nm). When compared to the flat surface, the nanopatterned surfaces show a significant bactericidal activity against both Escherichia coli and Staphylococcus aureus (with respective killing efficiencies of 97 ± 1% and 36 ± 5%). Direct penetration of nanopatterns into the bacterial cell wall leads to the disruption of the cell wall and cell death. The more rigid cell wall of S. aureus is consistent with the decreased killing efficiency. These findings support the development of nanopatterns with precisely controlled dimensions that are capable of killing both Gram-negative and Gram-positive bacteria.

Epimerization of cholic and chenodeoxycholic acid (CA and CDCA, respectively) is a notable conversion for the production of ursodeoxycholic acid (UDCA). Two enantiocomplementary hydroxysteroid dehydrogenases (7α- and 7β-HSDHs) can carry out this transformation fully selectively by specific oxidation of the 7α-OH group of the substrate and subsequent reduction of the keto intermediate to the final product (7β-OH). With a view to developing robust and active biocatalysts, novel NADH-active 7β-HSDH species are necessary to enable a solely NAD⁺-dependent redox-neutral cascade for UDCA production. A wild-type NADH-dependent 7β-HSDH from Lactobacillus spicheri (Ls7β-HSDH) was identified, recombinantly expressed, purified, and biochemically characterized. Using this novel NAD⁺-dependent 7β-HSDH enzyme in combination with 7α-HSDH from Stenotrophomonas maltophilia permitted the biotransformations of CA and CDCA in the presence of catalytic amounts of NAD⁺, resulting in high yields (>90 %) of UCA and UDCA.

Rhodococcus strains are ubiquitous in nature and known to metabolise a wide variety of compounds. At the same time, asymmetric reduction of C=C bonds is important in the production of high-valued chiral building blocks. In order to evaluate if Rhodococci can be used for this task, we have probed several Rhodococcus rhodochrous and R. erythropolis strains for ene-reductase activity. A series of substrates including activated ketones, an aldehyde, an imide and nitro-compound were screened using whole cells of seven Rhodococcus strains. This revealed that whole cells of all Rhodococcus strains showed apparent (S)-selectivity towards ketoisophorone, while most other organisms show (R)-selectivity for this compound. Three putative ene-reductases from R. rhodochrous ATCC 17895 were heterologously expressed in Escherichia coli. One protein was purified and its biocatalytic and biochemical properties were characterised, showing typical (enantioselective) properties for class 3 ene-reductases of the old yellow enzyme family.