Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus

Abstract

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.

Figure 1

Expression systems constructed in this work. (A) pSPGOX provides episomal expression of GOX under control of the ScPGK promoter; (B) pSPINGOX is an episomal vector and GOX expression is performed under control of the KmINU1 promoter; (C) pINGOXi is an integrative plasmid that confers the cells capacity of GOX expression under control of the KmINU1 promoter. (A) and (B) derive from pSPGK1 [48] and (C) corresponds to pNADFL11 [20] plus the expression cassette. Legend: p, promoter; t, terminator; SS, secretion signal sequence.

Figure 2

Coomassie blue stained SDS-PAGE for the analysis of the glycosylation pattern of GOX expressed in (A, B) K. marxianus and (C, D) K. lactis. (A) K. marxianus cell wall-associated GOX; (B) K. marxianus supernatant GOX; (C) K. lactis cell wall-associated GOX; (D) K. lactis supernatant GOX. Glyc: glycosylated enzyme. Deglyc: deglycosylated enzyme with PNGase F, as described in Methods. Construction codes are listed in Table 1. Standard: commercial GOX from A. niger (Product B6916, Sigma, USA), solubilized in water (Glyc) or deglycosylated (Deglyc) as described above. The arrows indicate the two bands resulting from the deglycosylation of GOX expressed in K. marxianus.

Figure 3

Effect of pH on heterologous GOX activity. (A) GOX activity was measured from pH 3 to 12; and (B) residual GOX activity was measured after 2 hours of enzyme incubation at the corresponding pH (here, activity measurements were always carried out at 37°C and pH 4.5). The following buffers were used: acetate (3.0, 4.0, 5.0); HEPES (6.0, 7.0, 8.0); Tris (9.0, 10.0, 11.0, 12.0). Each point is the mean of a duplicate assay. Km supernatant and Km cell wall: GOX produced by Km2 construction. Kl supernatant and Kl cell wall: GOX produced by Kl1 construction. Both clones were cultivated as described in the Methods section. Standard: commercial GOX from A. niger (Product B6916, Sigma, USA).

Figure 4

GOX stability at different temperatures. Time course of residual GOX activity was measured after incubation at (A) 37°C; (B) 55°C, and (C) 60°C. Activity measurements were always carried out at 37°C and pH 4.5. Each point is the mean of a duplicate assay. The error bars show standard deviations. Km supernatant and Km cell wall: GOX produced by Km2 construction. Kl supernatant and Kl cell wall: GOX produced by Kl1 construction. Both clones were cultivated as described in Methods section. Standard: commercial GOX from A. niger (Product B6916, Sigma, USA).