Identification of a complete dibenzothiophene biodesulfurization operon and its regulator by functional metagenomics

Abstract

Dibenzothiophene biodesulfurization is an important process for removing sulfur from fossil fuels. Functional metagenomics identified a complete operon for biodesulfurization (dszEABC) with a different gene arrangement and containing a gene coding for the FMNdependent reductase that is missing in all other dsz operons. Regulation of the operon is fully characterized in vivo and in vitro and shown to be Ndependent, unlike other operons of cultured bacteria. The N-dependent promoter, the activator gene and the reductase-coding gene have been apparently recruited from a different pathway. An insulation phenomenon that could be relevant in nature to prevent spurious expression of "mobile" operons when laterally transferred to different replicons is also reported here and its mechanism elucidated: an insulator sequence in the promoter region binds to the ribosome binding site of the first gene dszE, thus preventing its translation, combined with translational coupling of the downstream dszABC genes.

Characterization of a dszEABC operon providing fast growth on dibenzothiophene and construction of broad‐host‐range biodesulfurization catalysts

“…The UPO112 fosmid also bears the dszR regulatory gene, previously identified in the UPO21 fosmid as a σ N ‐dependent transcriptional activator required for transcription from the dszE promoter (Martin‐Cabello et al ., 2020). Alignment of the dszE upstream regions in both fosmids also identified regions of very high similarity in the Shine–Dalgarno sequence, and also in the further upstream σ N ‐dependent promoter sequence and in the palindromic sequences identified in UPO21 as the DszR binding sites (Fig.…”

“…This rules out the possibility that DszR required the action of DszH to become active, a view also in contradiction with the fact that dszR can activate dszEABC expression in the absence of dszH if transcribed to sufficiently high levels (Fig. 8), or by the in vitro activity of purified DszR UPO21 (Martin‐Cabello et al ., 2020). It is possible that deletions remove an RNA binding site required for a protein to activate dszR translation.…”

“…More recently, a dszEABC operon, bearing all the genes required for DBT biodegradation, was identified by functional metagenomics and functionally characterized. In this case, the authors screened for the ability to oxygenate indole to produce the blue pigment indigo (Martin-Cabello et al, 2020). Now, we report on the isolation of a new fosmid able to sustain very fast growth on DBT as the only sulfur source to the surrogate E. coli strain hosting it.…”

The Alternating Growth of Bacteria within a Consortium During Desulfurization of Coal

“…This result could be due to the high concentration of sulfate from the previous step (before hour 36), resulting in repression of the desulfurization activity. The byproduct of desulfurization, including sulfate in a particular concentration, has an inhibitory effect on the activity of desulfurization enzymes and repression on the expression of dsz genes, although not on microbial growth (Martín-Cabello et al, 2020;Sousa et al, 2020).…”

“…The illustration in Figure 7 placed the bacteria members in the 4S pathway based on their activities and growth turnover in coal medium (Figures 1-6) and some references. The 4S is a well-known biodegradation pathway of dibenzothiophene (Jatoi et al, 2021;Li et al, 2019;Martín-Cabello et al, 2020;Mishra et al, 2016;Sousa et al, 2020). The most dominant role member in the coal consortium is M. osloensis COK1.…”