Expression of xyloglucan endotransglycosylases of Gerbera hybrida and Betula pendula in Pichia pastoris

• Published in: Journal of biotechnology Vol. 130; no. 2; pp. 161 - 170
• Main Authors: Toikkanen, Jaana H., Niku-Paavola, Marja-Leena, Bailey, Michael, Immanen, Juha, Rintala, Eija, Elomaa, Paula, Helariutta, Yrjö, Teeri, Teemu H., Fagerström, Richard
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 15.06.2007; Amsterdam Elsevier; New York, NY
• Subjects: Amino Acid Sequence; Asteraceae - enzymology; Asteraceae - genetics; Betula - enzymology; Betula - genetics; Betula pendula; Biological and medical sciences; Biomass; Bioreactor cultivation; Bioreactors; Biotechnology; Biotechnology - methods; Cell Culture Techniques; Culture Media; enzyme activity; Filtration; Fundamental and applied biological sciences. Psychology; Gene Expression; gene transfer; genes; genetic transformation; Gerbera; Gerbera hybrida; Glycosyltransferases - analysis; Glycosyltransferases - biosynthesis; Glycosyltransferases - genetics; Glycosyltransferases - isolation & purification; Glycosyltransferases - metabolism; Heterologous expression; hexosyltransferases; Methanol - metabolism; Molecular Sequence Data; Pichia - enzymology; Pichia - genetics; Pichia - growth & development; Pichia pastoris; Sequence Homology, Amino Acid; Temperature; xyloglucan endotransglycosylase; Xyloglucan endotransglycosylase (XET); Xyloglucan endotransglycosylase (XET); Bioreactor cultivation; Pichia pastoris; Betula pendula; Heterologous expression; Gerbera hybrida; Compositae; Gene expression; Gerbera; Fungi; Bioreactor; Dicotyledones; Angiospermae; Ascomycetes; Spermatophyta; Betulaceae; Culture; Thallophyta
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2007.03.004

The plant enzyme xyloglucan endotransglycosylase (XET; EC 2.4.1.207, xyloglucan:xyloglucosyl transferase) participates in selective modification of plant cell walls during cell growth. XETs are potential catalysts in various applications. Here, sequences encoding two XETs from Gerbera hybrida and Betula pendula are reported. The encoded proteins, which are 51% identical at the amino acid level, were expressed in the yeast Pichia pastoris in secreted form with the aid of mating factor alpha signal sequence. XET production in shake flask cultivations was better at 22 °C than at 30 °C. Both the yield of protein of expected molecular mass and the XET activity improved at the lower temperature. Under all cultivation conditions studied, higher amounts of XET from B. pendula (BXET) were expressed than XET from G. hybrida (GXET). Both XET enzymes were produced in 16 l fed-batch bioreactor cultures. GXET was produced in methanol-limited fed-batch cultivation in minimal medium, and BXET in temperature-limited fed-batch (TLFB) in minimal or complex medium. Production was highest in TLFB in complex medium. BXET was purified from the culture filtrate and characterized. Based on the specific activity of the purified protein, 60–70 mg l −1 BXET was produced in the TLFB in complex medium.