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Background  In spite of its advantageous physiological properties for bioprocess applications, the use of the yeast  Kluyveromyces marxianus  as a host for heterologous protein production has been very limited, in constrast to its close relative  Kluyveromyces   lactis . In the present work, the model protein glucose oxidase (GOX) from  Aspergillus niger  was cloned into  K. marxianus  CBS 6556 and into  K. lactis  CBS 2359 using three different expression systems. We aimed at verifying how each expression system would affect protein expression, secretion/localization, post-translational modification, and biochemical properties.    Results  The highest GOX expression levels (1552 units of secreted protein per gram dry cell weight) were achieved using an episomal system, in which the  INU1  promoter and terminator were used to drive heterologous gene expression, together with the  INU1  prepro sequence, which was employed to drive secretion of the enzyme. In all cases, GOX was mainly secreted, remaining either in the periplasmic space or in the culture supernatant. Whereas the use of genetic elements from  Saccharomyces cerevisiae  to drive heterologous protein expression led to higher expression levels in  K. lactis  than in  K. marxianus , the use of  INU1  genetic elements clearly led to the opposite result. The biochemical characterization of GOX confirmed the correct expression of the protein and showed that  K. marxianus  has a tendency to hyperglycosylate the protein, in a similar way as already observed for other yeasts, although this tendency seems to be smaller than the one of e.g.  K. lactis  and  S. cerevisiae . Hyperglycosylation of GOX does not seem to affect its affinity for the substrate, nor its activity.    Conclusions  Taken together, our results indicate that  K. marxianus  is indeed a good host for the expression of heterologous proteins, not only for its physiological properties, but also because it correctly secretes and folds these proteins.

Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus