Enhanced recombinant xylanase B expression in Komagataella phaffii through metabolic engineering of methanol utilization.

Aspergillus niger ATCC 1015 xylanase B is an important enzyme in food industry and agricultural lignocellulose utilization. Heterologous expression of xylanase B by microbial chassis cells attracted a lot of attentions, especially Komagataella phaffii. Co-expressing enzymes involved in methanol metabolism and cofactor recycling by recombinant K. phaffii increased recombinant xylanase B expression efficiently in this study. Firstly K. phaffii formaldehyde dehydrogenase (Fld) was co-expressed to promote formaldehyde dissimilation. Furtherly, Saccharomyces cerevisiae NADH kinase Pos5 promoting NADPH production and K. phaffii 2-oxoglutarate transporter (Odc1) involving in the regeneration of mitochondrial NAD + were co-expressed to maintain the NAD + /NADH balance by converting 2-oxoglutarate to malate. The volumetric productivity and specific productivity of xylanase B were 26.22 U/(mL·h) and 8.27 U/(g·h), which were 93.2% increment of volumetric productivity and 101.8% increment of specific productivity respectively. Furtherly, increasing the utilization of methanol allows more carbon flow to the metabolic pathway, thus increasing the production of recombinant proteins. Fld, Pos5, and Odc1 co-expression enhanced methanol consumption by 10%, without retardation of specific growth rate, provided an engineered K. phaffii to express recombinant xylanase B expression significantly. In this study, the multi-enzyme co-expression strategy provided the expression level of recombinant protein increment effectively for food and agricultural industry.