Expression and secretion of a xylanase from the extreme thermophile, thermotoga strain FjSS3B.1, in Kluyveromyces lactis.
The yeast Kluyveromyces lactis has been developed as a host for extracellular production of thermophilic hemicellulases by employing expression vectors based on the 2 mu-like plasmid pKD1 of Kluyveromyces drosophilarium. A beta-1,4-xylanase gene (xynA) from the extreme thermophile Thermotoga sp. strain FjSS3B.1 was fused inframe with a synthetic secretion signal derived from the K. lactis killer toxin and expressed under control of the K. lactis LAC4 (beta-galactosidase) promoter. Correctly processed xylanase enzyme with full biological activity on oat spelts xylan was secreted during shake-flask cultivation of K. lactis transformants. The transcriptional activity of the LAC4 promoter dramatically affected mitotic stability of the expression vector under nonselective conditions. However, one combination of host strain and expression plasmid showed higher stability and good yield and has been employed for scaled-up production of XynA and other thermostable hemicellulases in chemostat culture. XynA secreted by K. lactis is as thermostable as the native enzyme, having a half-life of 48 h at 90 degrees C.
Walsh
D J
DJ
Centre for Gene Technology, University of Auckland, New Zealand.
Gibbs
M D
MD
Bergquist
P L
PL
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D003470:Culture Media; D005798:Genes, Bacterial; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D007716:Kluyveromyces / Q000235:genetics* / Q000254:growth & development; D010446:Peptide Fragments / Q000737:chemistry; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
D003470:Culture Media; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase