The Complete Genome Sequence and Structure of the Oleaginous Rhodococcus opacus Strain PD630 Through Nanopore Technology

Rhodococcus opacus PD630 is a model bacterial strain for the production of neutral lipids (mostly triacylglycerols,TAGs) from low-cost substrates and lignin biomass feedstocks. Presently, only a contig-level assembly of PD630 genome is available, after the exclusion from NCBI RefSeq of a ’complete’ version that was marked as ’anomalous assembly’. In this work, we achieved the R. opacus PD630 high-quality genome assembly by combining Oxford Nanopore sequencing with Illumina accurate short reads. Our work demonstrates that R. opacus PD630 has a 9.17 Mbp-long genome composed by one chromosome of 8.4 Mbp and three plasmids, i.e. one linear plasmid of 538 Kbp (pRoPD630 1) and two circular plasmids of 167.9 and 85 Kbp (pRoPD630 2 and pRoPD630 3). The comparison between this genome and the previous ”complete” version was performed to identify genetic and structural differences and to detect syntenic regions. The functional analysis of several R. opacus species plasmids showed that genes involved in lipid metabolism and xenobiotics’ degradation are generally clustered within specific plasmids. In the case of PD630, these genes are carried by the linear plasmid pRoPD630 1 whose structure has been assessed in this study. Our work defines the whole genome architecture and sequence of PD630, providing the groundwork to correctly interpret ’omics’ data and to design genetic engineering approaches for this model strain.