Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus

Saul N Rocha,María I González-Siso,José Abrahão-Neto,María E Cerdán,Andreas K Gombert

doi:10.1186/1475-2859-9-4

Saul N Rocha, María I González-Siso + Show 3 more

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https://doi.org/10.1186/1475-2859-9-4

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Abstract

BackgroundIn 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.ResultsThe 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.ConclusionsTaken 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.

Highlights

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
The use of S. cerevisiae elements for driving glucose oxidase (GOX) expression led to higher heterologous GOX levels in K. lactis than in K. marxianus
INU1 promoter and secretion signal were employed to drive heterologous gene expression, when compared to the use of a S. cerevisiae promoter with a K. lactis secretion signal. This agrees with the results reported by Bergkamp et al, who obtained 153 mg/L of heterologous alpha-galactosidase in K. marxianus, using the INU1 promoter and secretion signal, compared to 2 mg/L when the S. cerevisiae elements mentioned above

Summary

Introduction

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. There is no publicly available genome sequence, no commercial cloning system, and no strain adopted as a reference for basic research purposes for this species [4] It has been constantly pointed out as an attractive candidate microorganism for biotechnological applications, due to some of its physiological properties, such as thermotolerance, the capacity of catabolising a broader range of substrates than e.g. S_cerevisiae, a. Besides S. cerevisiae, the most common hosts for the expression of heterologous proteins are Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, and K. lactis [3,7,8,9,10,11] For many of these species, research is quite advanced, with most of the current works focusing on aspects such as the humanization of the secretory pathway, referred to as glycoengineering, essential for the production of therapeutic proteins, and the search for super-secreting phenotypes [12,13,14]

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