Enhanced xylan degradation and utilisation by Pichia stipitis overproducing fungal xylanolytic enzymes

Abstract

β-Xylanase encoding genes of Trichoderma reesei ( xyn2) and Aspergillus kawachii ( xynC) were cloned as cDNA copies under transcriptional control of the inducible Pichia stipitis xylose reductase gene ( XYL1) promoter on episomal plasmids pRDH12 and pRDH16, respectively. A cDNA copy of the β-xylosidase encoding gene of Aspergillus niger ( xlnD) was cloned as an in-reading-frame fusion with the Saccharomyces cerevisiae MFα1 secretion signal under transcriptional control of the constitutive P. stipitis transketolase ( TKL) gene promoter on an episomal plasmid (pRDH21). Combinations of the individual β-xylanase encoding genes and β-xylosidase expression cassette were also cloned onto episomal plasmids (pRDH22 and pRDH26). All of the plasmids were subsequently transformed to P. stipitis TJ26 and the β-xylanase activity, β-xylosidase activity and growth of the recombinant strains on xylan as sole carbon source were monitored. The strains expressing the A. kawachii xynC gene reached the highest maximum levels of β-xylanase activity and the activity was sustained for a longer period than the T. reesei xyn2 expressing strains where the levels of activity declined rapidly after reaching a maximum. All recombinant strains as well as the control strain were shown to produce extracellular protease activity. All strains expressing the A. niger xlnD gene reached similar levels of β-xylosidase activity, markedly higher than the control strains. The recombinant xylanolytic enzymes, whether produced alone or simultaneously, lead to an increase in biomass production of the recombinant strains when grown on medium containing xylan as sole carbon source. Simultaneous expression of the A. kawachii xynC gene and the A. niger xlnD gene gave the highest level of biomass production of any of the recombinant strains.