Enhancement of Microbial Biodesulfurization via Genetic Engineering and Adaptive Evolution

Area of Science:

• Microbial Biotechnology
• Genetic Engineering
• Bioremediation

Background:

• Previous design of a synthetic gene encoding a sulfur sink peptide (Sulpeptide 1 or S1) rich in methionine and cysteine.
• Insertion of the S1 gene into the desulfurization (dsz) operon of Rhodococcus erythropolis IGTS8.

Purpose of the Study:

• To evaluate the efficacy of combining genetic engineering with a sulfur sink and adaptive selection for enhanced biodesulfurization.
• To assess the impact of the dszAS1BC construct and intact dsz operon on dibenzothiophene (DBT) metabolism in Rhodococcus qingshengii.

Main Methods:

• Transformation of desulfurization-negative Rhodococcus qingshengii strain CW25 with dszAS1BC and dszABC constructs.
• Adaptive selection through repeated passages in minimal medium with DBT as the sole sulfur source.
• Analysis of DBT metabolism, growth rates, and genetic stability via whole genome sequencing.

Main Results:

• Both engineered strains showed increased DBT metabolism, averaging four times that of initial cells, with CW25[pRESX-dszAS1BC] exhibiting higher activity.
• Stable 7-fold and 13-fold increases in growth rates were observed for CW25[pRESX-dszABC] and CW25[pRESX-dszAS1BC], respectively.
• Adaptations in CW25[pRESX-dszAS1BC] correlated with increased plasmid copy numbers; no mutations were detected in key genes.

Conclusions:

• The combination of genetic engineering with sulfur sinks and adaptive selection is a viable strategy for enhancing biodesulfurization capabilities.
• The observed improvements in DBT metabolism and growth rates were achieved without detrimental mutations, indicating a robust adaptation mechanism.