Abstract
Filamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes). This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in P. pastoris. We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users’ community.
Highlights
Lignocellulosic biomass is recognized as a sustainable source of mixed sugars for producing second generation biofuels by fermentation, and for the synthesis of biomaterials (Lynd et al, 2008)
We have found that heterologous expression of fungal enzymes in P. pastoris remained timeconsuming, and reasoned that protocols for transformation, screening of transformants, cultures, purification of recombinant proteins could benefit from streamlining to try to increase protein production-throughput
We have observed that expressing three reference carbohydrate-active enzymes (CAZymes) was plasmid-dependent. Since testing both constitutive and inducible expression in parallel might prove impractical for large projects, a serial approach is more realistic
Summary
Lignocellulosic biomass is recognized as a sustainable source of mixed sugars for producing second generation biofuels by fermentation, and for the synthesis of biomaterials (Lynd et al, 2008). Historically filamentous fungi (e.g., Trichoderma reesei, Aspergillus niger) and other yeast (e.g., Saccharomyces cerevisiae, Yarrowia lipolytica) have been used for expressing native or recombinant fungal enzymes (Dashtban et al, 2009), the yeast Pichia pastoris has become the premier example of yeast species used for the production of recombinant proteins (Cregg et al, 2000; Damasceno et al, 2012). Advantages of this yeast include the use of efficient and tightly regulated promoters, higher expression levels for lower cost than insect and mammalian cells. It secretes very low levels of endogenous proteins none of which has been reported to be active on lignocellulosic biomass, which simplifies protein purification
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