Construction of expression system of phosphite dehydrogenase gene for cofactor regeneration in Candida tropicalis

Abstract

To enhance the efficiency of xylitol production in Candida tropicalis, we developed XYL2-disrupted mutant, BSXDH-3. We tried to express phosphite dehydrogenase (PTDH) in XYL2-disrupted C. tropicalis to increase economical efficiency and productivity of process of xyltiol production by efficient cofactor regeneration. Firstly, we developed the protein expression system containing the strong constitutive GAP promoter from Pichia pastoris. To confirm the availability of this expression system in C. tropicalis, the expression plasmid harboring XYL2 gene which encodes xylitol dehydrogenase (XDH) of C. tropicalis was constructed and expressed in XYL2-disrupted C. tropicalis. The expression of XDH was verified by XDH activity assay. While the XYL2-disrupted C. tropicalis showed no XDH activity, the recombinant strain showed the specific activity of XDH at the level of 87.91 mU/mg protein in xylose minimal media. This result suggested that th e GAP promoter-based expression system was effective to express the protein in C. tropicalis. Using this expression system, phosphite dehydrogenase (PTDH) from Pseudomonas stutzeri was expressed in the XYL2-disrupted C. tropicalis. The transcription of PTDH gene which was confirmed by RT-PCR was succeeded, whereas the recombinant strain showed no PTDH activity. The codon bias of C. tropicalis was assumed to cause no expression of PTDH. The codon-optimized PTDH gene was synthesized and this gene was also expressed in the XYL2-disrupted C. tropicalis. The recombinant C. tropicalis exhibited the specific activity of PTDH at the level of 14.67 mU/mg protein. The optimization of expression of PTDH will be need to increase the xylitol production afterwards.