Chemostat Approach for the Directed Evolution of Biodesulfurization Gain-of-Function Mutants

Abstract

Chemostat enrichment is a classical microbiological method that is well suited for use in directed-evolution strategies. We used a two-phase sulfur-limited chemostat to select for gain-of-function mutants with mutations in the biodesulfurization (Dsz) system of Rhodococcus erythropolis IGTS8, enriching for growth in the presence of organosulfur compounds that could not support growth of the wild-type strain. Mutations arose that allowed growth with octyl sulfide and 5-methylbenzothiophene as sole sulfur sources. An isolate from the evolved chemostat population was genetically characterized and found to contain mutations in two genes, dszA and dszC. A transversion (G to T) in dszC codon 261 resulted in a V261F mutation that was determined to be responsible for the 5-methylbenzothiophene gain-of-function phenotype. By using a modified RACHITT (random chimeragenesis on transient templates) method, mutant DszC proteins containing all possible amino acids at that position were generated, and this mutant set was assayed for the ability to metabolize 5-methylbenzothiophene, alkyl thiophenes, and dibenzothiophene. No mutant with further improvements in these catalytic activities was identified, but several clones lost all activity, confirming the importance of codon 261 for enzyme activity.

Use of a Novel Fluorinated Organosulfur Compound To Isolate Bacteria Capable of Carbon-Sulfur Bond Cleavage

“…These results are in agreement with recent work by Arensdorf et al (1), who used a chemostat to isolate mutants of strain IGTS8 that could utilize octyl sulfide, 5-methylbenzothiophene, and benzothiophene as sulfur sources. In their work, specific single-nucleotide changes in DszA were responsible for expanding the normally narrow substrate range of this enzyme.…”

“…The novelty of the desulfurization activity exhibited by strain JVH1 is best illustrated by comparison to known DBT-desulfurizing bacteria unable to utilize alkyl sulfides other than DMSO as sulfur sources (19). Interestingly, Arensdorf et al (1) reported that a Dsz-negative mutant of strain IGTS8 can transform DMSO, indicating that the DBT-desulfurization pathway is not required for this activity. In the results seen with this study, the detection of PFPSO in trace amounts (and the near stoichiometric recovery of PFPSO 2 from the four DBTdesulfurizing cultures) supports the hypothesis that these strains are unable to cleave COS bonds in alkyl chains.…”

Evolutionary Engineering of Saccharomyces cerevisiae for Anaerobic Growth on Xylose

“…Such strains were isolated in a long-term, multistep chemostat evolution experiment, which was initiated with the metabolically engineered S. cerevisiae strain TMB3001, which overexpresses the xylose utilization pathway of P. stipitis (7). The selection procedure was based on the well-known evolution of mutants with increased substrate affinity and utilization in chemostat cultures (1,6,24,25,34). However, the key to successful evolution was the fact that selection for aerobic xylose utilization and selection for anaerobic xylose utilization were decoupled (Fig.…”

Generation and characterisation of stable ethanol-tolerant mutants of Saccharomyces cerevisiae

“…This process can be more eVective when combined with a directed evolution procedure, such as chemostat incubation using an appropriate selection pressure to enrich for variants with desired characteristics [4]. Jimenez and Benitez [17] used a chemostat to select ethanol-tolerant yeast hybrids generated by crossing ethanol-tolerant wine yeast with laboratory yeast strains.…”