Molecular regulation of methanol oxidase activity in continuous cultures of Hansenula polymorpha

Abstract

The regulation of methanol oxidase (MOX) in Hansenula polymorpha has been studied in continuous cultures using a mixture of glucose and methanol (4:1 w/w) as carbon source. The study focused on the identification of stages in the biosynthesis affecting the formation of active MOX in glucose-methanol-grown cells. The levels of MOX mRNA, MOX protein in monomeric and octameric from, the ratio FAD/MOX, and the actual MOX activity have been quantified as functions of the dilution rate D. Hybridization studies with MOX mRNA probes showed an induction of MOX mRNA formation up to D = 0.29 h(-1). The induction of MOX protein synthesis (up to 37% of the cellular protein) is determined at low D values on the transcriptional level. The MOX activity at high D values is tuned by FAD incorporation and (post-) translation. Despite the high levels of MOX mRNA, decreasing levels of MOX activity and MOX protein were found at D values ranging from 0.14 t 0.29 h(-1). The maximal ratio FAD/MOX(6) was determined at D = 0.1 h(-1), which correlated with the maximal specific activity of MOX. In glucose-methanol media both protein level and MOX activity are repressed by increasing levels of residual glucose at high D values.