Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris

Abstract

Objective Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries. This study evaluated a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research. This strategy consisted of a constitutive and methanol-free enzyme production step coupled with a two-step filtration process. The aim was to determine if this strategy can yield copious amounts of GE, by secretion into the extracellular medium with an acceptable purity that could allow its direct application. This approach was further validated for cellobiose dehydrogenase, another emerging lignocellulose degrading enzyme which is scarcely available at high cost. Results The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489–2780 mg L−1), and were secreted at moderate to high percentages of the total extracellular protein (51–94%). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5–6-fold, reaching enzyme activity levels (1020–202 U L−1) that could allow their direct application.

Exploring class III cellobiose dehydrogenase: sequence analysis and optimized recombinant expression

“…From the clones with the methanol-inducible AOX promoter, 69% showed an activity higher than the limit of quantification of the assay, whereas no clone transformed with the GAP-promoter-containing construct produced enzyme activity higher than this limit. While the production of cdh genes under the control of AOX promoter has been well documented [ 33 – 35 ], there is only limited available data regarding CDH expression under the control of the GAP promoter [ 36 ], despite multiple reports on the use of this expression system for various other lignocellulolytic enzymes [ 37 – 42 ]. Our analysis shows that the expression system utilizing the AOX promoter was more successful than the GAP promoter to obtain detectable activities of Fs CDH.…”

The Model System Saccharomyces cerevisiae Versus Emerging Non-Model Yeasts for the Production of Biofuels

“…The removal of branches of glycuronic acid from hemicellulose can improve the release of fermentable sugars. Conacher et al [108] reported the production and secretion of GE at high yields through constitutive expression in P. pastoris. Li et al [109] expressed Rhizomucor miehei-derived β-mannanase (mRmMan5A), reaching a high level of expression (79,680 U m/L).…”