Improved Efficiency of the Desulfurization of Oil Sulfur Compounds in Escherichia coli Using a Combination of Desensitization Engineering and DszC Overexpression

Abstract

The 4S pathway of biodesulfurization, which can specifically desulfurize aromatic S-heterocyclic compounds without destroying their combustion value, is a low-cost and environmentally friendly technology that is complementary to hydrodesulfurization. The four Dsz enzymes convert the model compound dibenzothiophene (DBT) into the sulfur-free compound 2-hydroxybiphenyl (HBP). Of these four enzymes, DszC, the first enzyme in the 4S pathway, is the most severely affected by the feedback inhibition caused by HBP. This study is the first attempt to directly modify DszC to decrease its inhibition by HBP, with the results showing that the modified protein is insensitive to HBP. On the basis of the principle that the final HBP product could show a blue color with Gibbs reagent, a high-throughput screening method for its rapid detection was established. The screening method and the combinatorial mutagenesis generated the mutant AKWC (A101K/W327C) of DszC. After the IC 50 was calculated, the feedback inhibition of the AKWC mutant was observed to have been substantially reduced. Interestingly, the substrate inhibition of DszC had also been reduced as a result of directed evolution. Finally, the recombinant BL21(DE3)/ BADC*+C* (C* represents AKWC) strain exhibited a specific conversion rate of 214.84 μmol HBP /g DCW /h, which was 13.8-fold greater than that of the wild-type strain. Desensitization engineering and the overexpression of the desensitized DszC protein resulted in the elimination of the feedback inhibition bottleneck in the 4S pathway, which is practical and effective progress toward the production of sulfur-free fuel oil. The results of this study demonstrate the utility of desensitization of feedback inhibition regulation in metabolic pathways by protein engineering.

Isolation and Identification of Dibenzothiophene Desulfurizing Bacteria Occurring in Oil Contaminated Soils of Mechanical Workshops

“…This was confirmed by the development of blue coloration during Gibbs assay indicating the production of 2-HBP which is the end product of 4S pathway (Rahpeyma et al, 2017). The Gibb's assay was used to identify the desulfurization activity of bacteria through 4S pathway by several earlier workers (Sadare et al, 2017;Shahaby and El-din, 2017;Li et al, 2019). The other isolates of our study which were negative for Gibb's assay may be utilizing the other pathways viz., Kodama pathway and Van Afferden pathway for metabolizing DBT as they were also grown abundantly on basal salt medium supplemented with glucose and DBT as sole sulfur source.…”

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

“…One of the main limitations of DBT biodegradation efficiency via the 4S pathway is the concomitant production of 2‐HBP, which has been shown to inhibit growth and DBT biodegradation itself (Abin‐Fuentes et al ., 2013; Li et al ., 2019). To test how 2‐HBP accumulation could affect E .…”

“…Toxicity of 2-HBP in E. coli and Pseudomonas One of the main limitations of DBT biodegradation efficiency via the 4S pathway is the concomitant production of 2-HBP, which has been shown to inhibit growth and DBT biodegradation itself (Abin-Fuentes et al, 2013;Li et al, 2019). To test how 2-HBP accumulation could affect E. coli growth, a specialized strain bearing the UPO112 fosmid was grown in minimal medium with the different sulfur sources, and the effect on growth of increasing concentrations of 2-HBP was tested.…”

“…One of the technical problems is that DBT biodegradation is repressed by other preferred sulfur sources, which has prompted to construct engineered strains expressing dsz genes from ectopic promoters (Gallardo et al ., 1997; Takada et al ., 2005; Alves et al ., 2007; Khosravinia et al ., 2018). In addition, other problems preclude development of biocatalysts, including substrate bioavailability, final product (2‐HBP) toxicity, or enzyme product inhibition (Abin‐Fuentes et al ., 2013; Li et al ., 2019). Therefore there is a need to pursue the search for new enzymes and novel bacterial strain characteristics to develop an efficient biocatalyst.…”

Enhancing biodesulfurization by engineering a synthetic dibenzothiophene mineralization pathway

“…The FMNH 2 required for the oxidation steps is supplied by a NADH-FMN oxidoreductase (DszD; Figure 1A), which is usually chromosomally encoded (Gupta et al, 2005;Mohebali and Ball, 2016;Kilbane, 2017;Parveen et al, 2020;Hirschler et al, 2021;Martín-Cabello et al, 2022). One of the major bottlenecks of the biodesulfurization of DBT through the 4S route is the inhibitory effect of the final product, 2HBP, on the initial Dsz enzymes (Xu et al, 2006;Chen et al, 2008;Akhtar et al, 2009;Abin-Fuentes et al, 2013;Kilbane, 2017;Li et al, 2019a). To deal with this limitation, different approaches have been applied over the years.…”