Expression and secretion of heterologous proteins in Kluyveromyces yeasts.

Author(s)

Saul Nitsche Rocha

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Escola Politécnica (EP/BC)
• Defense date: 2009-11-27

Examining board members

Andreas Karoly Gombert; José Abrahão Neto; Felipe Santiago Chambergo Alcalde; Beatriz Vahan Kilikian; Flávia Maria Lopes Passos

• Advisor: Andreas Karoly Gombert

Abstract

In spite of the advantageous physiological properties of the yeast Kluyveromyces marxianus to produce heterologous proteins, this species has not been widely explored for the synthesis of these biomolecules. On the other hand, more than 40 heterologous expression systems, including commercial ones, were developed for Kluyveromyces lactis. Moreover, there is no available literature concerning heterologous protein glycosylation in K. marxianus. Taking these facts into account, this work aimed at developing systems for the heterologous production of Aspergillus niger glucose oxidase (GOX) and of a thermophilic esterase (EST) from Thermus thermophilus in K. marxianus. The strain K. lactis CBS 2359 was utilized as a reference throughout the whole work. First, a physiological study was carried out in order to select one K. marxianus strain, out of three which had been chosen based on literature information, that exhibited the best physiological traits to be a heterologous expression host. The chosen strain was CBS 6556, based on a combination of the following properties: specific growth rate, metabolites formation, biomass yield on substrate, and secretion of the homologous enzyme inulinase. Subsequently, two episomal systems were constructed. In one of them, the heterologous gene was expressed under control of the S. cerevisiae PGK promoter, whereas in the other system, heterologous gene expression occurred under control of the K. marxianus INU1 promoter. An integrative expression system was also constructed, in which the KmINU1 promoter drove foreign gene expression. Both heterologous enzymes were characterized biochemically and also with respect to their glycosylation. The results attained with GOX led to an expression level of 1722 U/g DW (unit per gram of dry cell weight) in K. marxianus transformed with the episomal INU1-based system. The biochemical studies showed that the enzyme was very similar to the A. niger GOX. Furthermore, analysis of the glycosylation pattern showed a lower tendency of K. marxianus to hypermannosylate proteins, when compared to K. lactis. Higher levels of esterase (294 U/gDW) were obtained in K. lactis than in K. marxianus. However, the enzyme produced in the latter host presented a higher temperature for maximal activity ((50 °C), when compared to the former organism (45 °C). Both values are lower than the temperature for maximal activity of the homologous enzyme (65 °C), which can be explained by the glycans added by both yeast species to the peptide, resulting in a glycosylated protein, in contrast to the homologous esterase. Moreover, the yeast products presented three glycosylation patterns. In conclusion, the work presented in this thesis reached its aims, which were to develop these expression systems and to characterize biochemically the heterologous enzymes expressed, which included an analysis of the glycosylation pattern. The results presented here will certainly be of interest and aid the scientific community working on the expression of heterologous proteins in microorganisms. (AU)