Enhancement of Microbial Biodesulfurization via Genetic Engineering and Adaptive Evolution

Abstract

In previous work from our laboratories a synthetic gene encoding a peptide (“Sulpeptide 1” or “S1”) with a high proportion of methionine and cysteine residues had been designed to act as a sulfur sink and was inserted into the dsz (desulfurization) operon of Rhodococcus erythropolis IGTS8. In the work described here this construct (dszAS1BC) and the intact dsz operon (dszABC) cloned into vector pRESX under control of the (Rhodococcus) kstD promoter were transformed into the desulfurization-negative strain CW25 of Rhodococcus qingshengii. The resulting strains (CW25[pRESX-dszABC] and CW25[pRESX-dszAS1BC]) were subjected to adaptive selection by repeated passages at log phase (up to 100 times) in minimal medium with dibenzothiophene (DBT) as sole sulfur source. For both strains DBT metabolism peaked early in the selection process and then decreased, eventually averaging four times that of the initial transformed cells; the maximum specific activity achieved by CW25[pRESX-dszAS1BC] exceeded that of CW25[pRESX-dszABC]. Growth rates increased by 7-fold (CW25[pRESX-dszABC]) and 13-fold (CW25[pRESX-dszAS1BC]) and these increases were stable. The adaptations of CW25[pRESX-dszAS1BC] were correlated with a 3-5X increase in plasmid copy numbers from those of the initial transformed cells; whole genome sequencing indicated that during its selection processes no mutations occurred to any of the dsz, S1, or other genes and promoters involved in sulfur metabolism, stress response, or DNA methylation, and that the effect of the sulfur sink produced by S1 is likely very small compared to the cells’ overall cysteine and methionine requirements. Nevertheless, a combination of genetic engineering using sulfur sinks and increasing Dsz capability with adaptive selection may be a viable strategy to increase biodesulfurization ability.

Metabolic and process engineering for biodesulfurization in Gram-negative bacteria

“…The development of desulfurization-competent hosts, such as those adapted to express a sulfur-rich polypeptide (Pan et al, 2013;Wang et al, 2017), represents a new tool for studying the expression, protein folding, disulfide bond formation, and protein secretion of high-sulfur-content proteins. Polypeptides that contain a high concentration of cysteine/methionine include some of the most potent venoms and some therapeutic compounds such as bacteriocins, defensins, and various synthetic peptides with antimicrobial and anti-tumoral properties (Kilbane and Stark, 2016).…”

Combining co‐culturing of Paenibacillus strains and Vitreoscilla hemoglobin expression as a strategy to improve biodesulfurization

“…2008; Wang et al . 2017). Another approach involves using mixed cultures or co‐cultures of micro‐organisms, which could provide synergistic relationships resulting in better growth and more effective desulfurization (Kayser 1993; Wang et al .…”

Molybdenum Dioxide in Carbon Nanoreactors as a Catalytic Nanosponge for the Efficient Desulfurization of Liquid Fuels

“…[3,4] Therefore, a key current research challenge concerns the development of an efficient and economically viable process to remove sulfurcontaining contaminants from fuels in order to satisfy the ultralow sulfur diesel (ULSD) regulations of less than 10 ppm imposed by international policies. [9][10][11] Specifically, the use of ODS to remove the refractory sulfur compounds from fuels exploits key changes in the polarity of the oxidized products relative to the parent organosulfur contaminant during extraction and has already been established for the production of ULSD. [8] As a result, the development of other desulfurization processes has been more recently explored (Figure 1).…”