Producing Industrial Enzyme from Cellulosic Biomass—Towards Engineering Xylose Utilizing Ability into Protein Expression Systems

Growing energy crisis and environmental pressures led to renewed interest in cellulosic biomass as a renewable feedstock for the production of fuels, chemicals and bulk enzymes, which demands xylose utilization from microbial cell factories. However, many good industrial enzyme producers cannot effectively metabolize xylose.

In this study, using Pichia pastoris, one of the most widely used expression systems as an example, we demonstrated successful engineering of xylose metabolizing ability into protein expression systems. A heterologous XI (xylose isomerase) pathway was introduced into P. pastoris GS115 by overexpressing the Orpinomyces spp. XI or/and the endogenous XK (xylulokinase) gene, and evolutionary engineering strategies were also applied. Results showed that the XI pathway could be functionally expressed in P. pastoris. After 50generation of sequential batch cultivation, a set of domesticated recombinant P. pastoris strains with different performance metrics on xylose were obtained. One evolved strain showed the highest xylose assimilation ability, whose cell yield on xylose can even be comparable to that on glucose or glycerol. This strain also showed significantly increased β-mannanase production when cultured on xylose medium. Furthermore, transcription analysis of xylose pathway genes suggested that overexpression of XI and XK might be the key factors affecting effective xylose assimilation.

This study is the first work demonstrating the construction of efficient xylose utilizing P. pastoris strains, thus providing a basis for using cellulosic biomass for bulk industrial enzyme production.