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Applied and Environmental Microbiology, February 2002, p. 691-698, Vol. 68, No. 2
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.2.691-698.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Joseph J. Arensdorf,* A. Katrina Loomis, Philip M. DiGrazia, Daniel J. Monticello, and Philip T. Pienkos

Enchira Biotechnology Corporation, The Woodlands, Texas 77381

Received 7 June 2001/ Accepted 14 November 2001

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.

* Corresponding author. Mailing address: Enchira Biotechnology Corporation, 4200 Research Forest Dr., The Woodlands, TX 77381. Phone: (281) 419-7000. Fax: (281) 364-6112. E-mail: jarensdorf{at}enchira.com.

Applied and Environmental Microbiology, February 2002, p. 691-698, Vol. 68, No. 2
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.2.691-698.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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