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.

FIG. 1.

Proposed Dsz pathway for BDS of DBT by R. erythropolis IGTS8. DBTO, DBT sulfoxide; DBTO₂, DBT sulfone; HPBS, 2-(2"-hydroxyphenyl)-benzene sulfinate; HBP, 2-hydroxybiphenyl; DBT-MO, DBT monooxygenase; DBTO2-MO, DBTO₂ monooxygenase; FMN, flavin mononucleotide.

FIG. 2.

A synthetic oligonucleotide (upper strand) was 5" phosphorylated to allow ligation repair from upstream sequence. The oligonucleotide was randomized at codon 261, and the MunI site was removed with two silent mutations.