title: 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.
authors.affiliation: Department of Biology, Illinois Institute of Technology, Chicago IL, United States of America.
authors.forename: Jia
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Biology, Illinois Institute of Technology, Chicago IL, United States of America.
authors.forename: Robert R
authors.initials: RR
authors.lastname: Butler
authors.affiliation: Department of Biology, Illinois Institute of Technology, Chicago IL, United States of America.
authors.forename: Fan
authors.initials: F
authors.lastname: Wu
authors.affiliation: Department of Biology, Illinois Institute of Technology, Chicago IL, United States of America.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Pombert
authors.affiliation: Department of Biology, Illinois Institute of Technology, Chicago IL, United States of America.
authors.forename: John J
authors.initials: JJ
authors.lastname: Kilbane
authors.affiliation: Department of Biology, Illinois Institute of Technology, Chicago IL, United States of America.
authors.forename: Benjamin C
authors.initials: BC
authors.lastname: Stark
mesh_terms: D000222:Adaptation, Physiological; D003001:Cloning, Molecular / Q000379:methods; D003470:Culture Media / Q000378:metabolism; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D019020:Directed Molecular Evolution; D005818:Genetic Engineering; D016680:Genome, Bacterial; D009876:Operon; D011401:Promoter Regions, Genetic; D012240:Rhodococcus / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013455:Sulfur / Q000378:metabolism*
chemical_list: D003470:Culture Media; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D013455:Sulfur