Complete Genome Sequence of Rhodococcus opacus Strain MoAcy1 (DSM 44186), an Aerobic Acetylenotroph Isolated from Soil

We report the genome of Rhodococcus opacus strain MoAcy1 ( DSM 44186 ), an aerobic soil isolate capable of using acetylene as its primary carbon and energy source (acetylenotrophy). The genome is composed of a single circular chromosome of ∼8 Mbp and two closed plasmids, with a G+C content of 67.3%.

Transcriptomic analysis of Rhodococcus opacus R7 grown on polyethylene by RNA-seq

Plastic waste management has become a global issue. Polyethylene (PE) is the most abundant synthetic plastic worldwide, and one of the most resistant to biodegradation. Indeed, few bacteria can degrade polyethylene. In this paper, the transcriptomic analysis unveiled for the first time Rhodococcus opacus R7 complex genetic system based on diverse oxidoreductases for polyethylene biodegradation. The RNA-seq allowed uncovering genes putatively involved in the first step of oxidation. In-depth investigations through preliminary bioinformatic analyses and enzymatic assays on the supernatant of R7 grown in the presence of PE confirmed the activation of genes encoding laccase-like enzymes. Moreover, the transcriptomic data allowed identifying candidate genes for the further steps of short aliphatic chain oxidation including alkB gene encoding an alkane monooxygenase, cyp450 gene encoding cytochrome P450 hydroxylase, and genes encoding membrane transporters. The PE biodegradative system was also validated by FTIR analysis on R7 cells grown on polyethylene.

Bacterial Transformation of Aromatic Monomers in Softwood Black Liquor

The valorization of lignin, a major component of plant-derived biomass, is essential to sustainable biorefining. We identified the major monoaromatic compounds present in black liquor, a lignin-rich stream generated in the kraft pulping process, and investigated their bacterial transformation. Among tested solvents, acetone extracted the greatest amount of monoaromatic compounds from softwood black liquor, with guaiacol, vanillin, and acetovanillone, in an approximately 4:3:2 ratio, constituting ~90% of the total extracted monoaromatic content. 4-Ethanol guaiacol, vanillate, and 4-propanol guaiacol were also present. Bacterial strains that grew on minimal media supplemented with the BL extracts at 1mM total aromatic compounds included Pseudomonas putida KT2442, Sphingobium sp. SYK-6, and Rhodococcus rhodochrous EP4. By contrast, the extracts inhibited the growth of Rhodococcus jostii RHA1 and Rhodococcus opacus PD630, strains extensively studied for lignin valorization. Of the strains that grew on the extracts, only R. rhodochrous GD01 and GD02, isolated for their ability to grow on acetovanillone, depleted the major extracted monoaromatics. Genomic analyses revealed that EP4, GD01, and GD02 share an average nucleotide identity (ANI) of 98% and that GD01 and GD02 harbor a predicted three-component carboxylase not present in EP4. A representative carboxylase gene was upregulated ~100-fold during growth of GD02 on a mixture of the BL monoaromatics, consistent with the involvement of the enzyme in acetovanillone catabolism. More generally, quantitative RT-PCR indicated that GD02 catabolizes the BL compounds in a convergent manner via the β-ketoadipate pathway. Overall, these studies help define the catabolic capabilities of potential biocatalytic strains, describe new isolates able to catabolize the major monoaromatic components of BL, including acetovanillone, and facilitate the design of biocatalysts to valorize under-utilized components of industrial lignin streams.

Characterization of Two Hydrogen Peroxide Resistant Peroxidases from Rhodococcus opacus 1CP

The dye-decolorizing peroxidases (DyP) are a family of heme-dependent enzymes present on a broad spectrum of microorganisms. While the natural function of these enzymes is not fully understood, their capacity to degrade highly contaminant pigments such as azo dyes or anthraquinones make them excellent candidates for applications in bioremediation and organic synthesis. In this work, two novel DyP peroxidases from the organism Rhodococcus opacus 1CP (DypA and DypB) were cloned and expressed in Escherichia coli. The enzymes were purified and biochemically characterized. The activities of the two DyPs via 2,2′-azino-bis [3-ethylbenzthiazoline-6-sulphonic acid] (ABTS) assay and against Reactive Blue 5 were assessed and optimized. Results showed varying trends for DypA and DypB. Remarkably, these enzymes presented a particularly high tolerance towards H2O2, retaining its activities at about 10 mM H2O2 for DypA and about 4.9 mM H2O2 for DypB.