Abstract
Hansenula polymorpha is a methylotrophic yeast widely employed in biotechnology as a ''protein factory''. Most promoters used for heterologous protein expression, like MOX (methanol oxidase) and DAS (di-hydroxy acetone synthase), are involved in the peroxisomal methanol metabolism (C1 metabolism) and are under strong glucose repression. Interestingly, the MOX promoter is subjected to glucose regulation also in Saccharomyces cerevisiae, a non-methylotrophic yeast in which this phenomenon is well studied. In this species, the transcription factor Tup1p plays an essential role in glucose repression of several genes. This effect is counteracted by the activator Snf1p when glucose is exhausted from medium. Therefore, to test whether this regulatory circuit has been conserved in H. polymorpha, HpTUP1 and HpSNF1 were partially cloned and disrupted. Deletion of HpTUP1 did not affect glucose repression of the major C1 metabolism genes (MOX, DAS). Thus, though conserved, HpTUP1 does not seem to take part in a general glucose repression in H. polymorpha. In contrast, the deletion of HpSNF1 led to significant decreases in the activation of these genes in the absence of glucose. Therefore, the effect of HpSNF1 in transcriptional activation may be through an HpTUP1- independent circuit.
Highlights
Mutants lacking HpSNF1 presented lower MOX and DAS induction on methanol, when compared to wild-type cells. These results indicate that the participation of Snf1p in the regulation of the major H. polymorpha C1 metabolism genes occurs in a Tup1p-independent manner, implying a minor involvement of these conserved proteins in the mechanism of glucose repression of methanol metabolism
We did not observe differences in either glucose or methanol utilization among the three strains. These results indicate that, contrarily to S. cerevisiae, tup1 or snf1 disruptions failed to lead to any direct effect in the carbon utilization pathways of H. polymorpha
We investigated possible contributions of the conserved H. polymorpha TUP1 and SNF1 genes in the glucose repression mechanism
Summary
The expression of the major enzymes accounting for peroxisomal methanol metabolism (C1 metabolism), namely methanol oxidase (Moxp), catalase (Catp) and di-hydroxi acetone synthase (Dasp), is controlled at the transcriptional level The genes encoding these three enzymes are fully induced when cells are grown on methanol, and repressed to different extents in the presence of glucose (Genu et al, 2003; Janowicz et al, 1985; Roggenkamp et al, 1984). The MOX promoter has been widely employed for heterologous protein expression, and H. polymorpha has been considered a “versatile protein factory” (Gellissen, 2000; van Dijk et al, 2000) It is a very suitable source of carbon for general industrial fermentation, glucose cannot be regularly employed for protein expression in H. polymorpha. It would be a significant technological improvement if the MOX promoter could function in glucose-containing media
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