Expression and Secretion of Bacterial Thermophilic Hemicellulases in Kluyveromyces lactis

Abstract

The yeast Kluyveromyces lactis has been developed as a host for extracellular production of thermophilic hemicellulases employing expression vectors based on the 2μ-like plasmid pKD1 of Kluyveromyces drosophilarium, Α β-1,4-xylanase gene (xynA) from Dictyoglomus thermophilum strain Rt46B.1 was fused in-frame with a synthetic secretion signal derived from the K. lactis killer toxin and expressed under control of the Κ. lactis LAC4 (β-galactosidase) promoter. Correctly processed xylanase enzyme with full biological activity on Oat Spelt Xylan was secreted during shake-flask cultivation of Κ. lactis transformants. Yield was found to be dependent on the strain and the composition of the growth medium. The transcriptional activity of the LAC4 promoter dramatically affected mitotic stability of the expression vector under non-selective conditions. However, one isolate combined higher plasmid stability and good yield and has been employed for scaled-up production of XynA and other thermostable hemicellulases in chemostat culture. Similar results have been obtained for expression of a fusion of the xynA gene of Thermotoga strain FjSS3.B1 cloned into the same secretion vectors.