Biodesulfurization enhancement via targeted re-insertion of the flavin reductase dszD in the genome of the model strain Rhodococcus qingshengii IGTS8

Abstract

Biodesulfurization (BDS) has emerged as an alternative to the excessively costly hydrodesulfurization of recalcitrant heterocyclic sulfur compounds, such as dibenzothiophene (DBT) and its derivatives. The model desulfurizing strain Rhodococcus qingshengii IGTS8 is responsible for the removal of sulfur through the 4S metabolic pathway, operating through a plasmid-borne dszABC operon, as well as the chromosomal gene for the flavin reductase, d szD. However, naturally occurring biocatalysts do not exhibit the required BDS activity to be useful for industrial applications and for this reason, genetic modifications are currently being explored. Here, we constructed a genetically modified R. qingshengii IGTS8 strain, which carries an additional copy of the flavin reductase gene dszD under the control of the rhodococcal promoter P _kap1 , inserted in the neutral chromosomal genetic locus crtI. We conducted a comparative study of the growth and biodesulfurization capabilities of P _kap1 -dszD, wild-type and crtIΔ strains, grown on different types and concentrations of carbon and sulfur sources. A significant enhancement of biodesulfurization activity, maximum calculated biomass, and dszD transcript levels in the presence of DBT as the sole sulfur source was achieved for the P _kap1 -dszD strain paving the way for further studies that could lead to a more viable commercial biodesulfurization process.

Keywords: Actinomycetes; Biodesulfurization; Dibenzothiophene; FMN reductase; Genetic engineering; Model biocatalyst; Whole-cell biocatalyst.

Re-insertion of the flavin reductase dszD in the chromosomal carotenoid biosynthesis locus crtI of the model biodesulfurization strain Rhodococcus qingshengii IGTS8, resulted in the generation of a phenotypic mutant strain that exhibits up to 9-fold enhanced dszD expression and presents significantly enhanced dibenzothiophene biodesulfurization activity during the exponential growth phase.

Fig. 1

Genomic and enzymatic background of the 4S desulfurization pathway in R. qingshengii IGTS8.

Fig. 2

Organization of wild-type (wt) and engineered dszD and crtI loci within the chromosome of R. qingshengii IGTS8.

Fig. 3

Effect of different carbon sources on strain growth (Biomass, g/L) and desulfurization activity (Units 2-HBP/mg Dry Cell Weight [DCW]) for the wt (A), crtIΔ (B), and P_kap1-dszD (C) strains grown in the presence of 110 mM glycerol, or 165 mM ethanol as the sole carbon source. The sole sulfur source was DMSO (2 mM). The biodesulfurization activity was measured in resting-cell assays after 20, 45, and 65 h of growth. See also Table 3.

Fig. 4

Growth (Biomass, g/L) and desulfurization activity (Units 2-HBP/mg_DCW) of strains wt, crtIΔ, and P_kap1-dszD, grown in the presence of 1 mM (A), 2 mM (B), or 4 mM (C) DMSO as the sole sulfur source. The sole carbon source was ethanol (165 mM). The biodesulfurization activity was monitored with resting-cell assays after 20, 45, and 65 h of growth. See also Table 3.

Fig. 5

Growth (Biomass, g/L) and desulfurization activity (Units 2-HBP/mg_DCW) of wt, crtIΔ, and P_kap1-dszD strains grown in the presence of 1 mM (A), or 2 mM (B) sulfate as the sole sulfur source. Ethanol was supplemented as the sole carbon source (165 mM). The biodesulfurization activity was monitored with resting-cell assays after 20, 45, and 65 h of growth. See also Table 3.

Fig. 6

Growth (Biomass, g/L) and desulfurization activity (Concentration of 2-HBP in μM) of strains wt, crtIΔ, and P_kap1-dszD, grown in the presence of 0.1 mM DBT as the sole sulfur source. The sole carbon source was ethanol (165 mM). The levels of 2-HBP in samples collected from the growing DBT cultures were quantified after 24, 48, 72, 96, 120, 144, and 168 h of growth.

Fig. 7

Transcript level comparison within dszA, dszB, dszC, and dszD genes, for the wt and genetically engineered strains crtIΔ and P_kap1-dszD, grown in the presence of (A) 2 mM DMSO or (B) 0.1 mM DBT as the sole sulfur source. The sole carbon source was ethanol (165 mM). Samples were collected from cultures in the mid-log growth phase. A logarithmic scale was applied. Fold changes are presented relative to the lowest expression of each gene (set as 1 AU; arbitrary unit). Asterisks indicate P-values (∗∗, P < 0.01; ∗∗∗, P < 0.001).