The above paper (Yeast 8: [361][362][363][364][365][366][367][368][369][370][371][372] 1992) erroneously reported that the experiments carried out by Fellinger et al. (1991) used the strain leul-1. This reference should be Veale (1989). Fellinger et al. described results using the strain A 16, the development of which is described in this paper.

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.

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A strain of the methylotrophic yeast Hansenula polymorpha, A16, has been developed that expresses the guar alpha-galactosidase gene to 22.4 mg/g dry cell weight in chemostat cultures at a dilution rate of 0.1 h(-1). This corresponds to more than 13.1% of soluble cell protein, of which 56-62% is secreted into the medium. The alpha-galactosidase gene was flanked by the promoter and terminator sequences of the H.polymorpha mox gene, which can direct expression of the mox gene itself more than 30% of total cell protein under methanol growth. The expression cassette (pUR3510) based on the Saccharomyces cerevisiae plasmid, YEp13, was integrated into the genome. Such transformants were stable in chemostat cultures and exhibited 100% stability for both alpha-galactosidase+ and leu+ phenotypes. Chemostat cultures produced higher levels of alpha-galactosidase with higher specific productivities expressed as mg alpha-galactosidase g(-1) h(-1) compared to batch cultures.