Improving epoxide production of the ethene monooxygenase from Mycobacterium rhodesiae JS60

Abstract

Enzymes capable of producing epoxides are interesting for organic chemistry because epoxides are versatile intermediates. One such enzyme is the ethene monooxgyenase (MO) from Mycobacterium rhodesiae JS60. However, epoxide degradation occurs due to downstream metabolism by the organism and abiotic hydrolysis. This study focused on improving epoxide production from the M. rhodesiae JS60 ethene MO via three approaches: two-phase aqueous/organic systems, suppression of downstream metabolism, and mutagenesis of the enzyme itself. Whole-cell aqueous/organic systems have multiple benefits including the organic phase acting as a reservoir for toxic epoxides. Of the six organic phases tested, glyceryl trioctanoate was the best overall second phase increasing activity of the ethene MO dramatically (2-12 times higher) on three of the five substrates tested (styrene, cyclopentene, allyl alcohol). Coenzyme M (CoM) is responsible for moving epoxides into downstream metabolism. Although attempts to create a mutant incapable of synthesising CoM were unsuccessful, a reliable and efficient method for transforming M. rhodesiae JS60 cells was established and hygromycin resistance was found to be a good resistance marker in stain JS60 because the strain JS60 does not develop spontaneous resistance. Heterologous expression of the ethene MO in the host Mycobacterium smegmatis solves the issue of downstream metabolism but activity in such systems is low. Mutagenesis can increase activity but the bottleneck is screening the mutants generated. A two-step high throughput screen was developed with an initial high volume screen based on the indole assay. The second step is a quantitative screen of higher activity mutants based on 4-(nitrobenzyl)pyridine. A shuttle vector system was created for the cloning of EtnC mutant libraries, and enzyme activity was completely restored with the re-introduction of the native etnC gene. Due to time-constraints, generation and screening of ethene MO mutants was not performed but the basis for this work has been established.