Abstract
BackgroundsEngineering yeast as a consolidated bioprocessing (CBP) microorganism by surface assembly of cellulosomes has been aggressively utilized for cellulosic ethanol production. However, most of the previous studies focused on Saccharomyces cerevisiae, achieving efficient conversion of phosphoric acid-swollen cellulose (PASC) or microcrystalline cellulose (Avicel) but not carboxymethyl cellulose (CMC) to ethanol, with an average titer below 2 g/L.ResultsHarnessing an ultra-high-affinity IM7/CL7 protein pair, here we describe a method to engineer Pichia pastoris with minicellulosomes by in vitro assembly of three recombinant cellulases including an endoglucanase (EG), an exoglucanase (CBH) and a β-glucosidase (BGL), as well as a carbohydrate-binding module (CBM) on the cell surface. For the first time, the engineered yeasts enable efficient and direct conversion of CMC to bioethanol, observing an impressive ethanol titer of 5.1 g/L.ConclusionsThe research promotes the application of P. pastoris as a CBP cell factory in cellulosic ethanol production and provides a promising platform for screening the cellulases from different species to construct surface-assembly celluosome.
Highlights
Cellulosic biomass derived from low-value agricultural and wood pulping wastes is likely the most abundant renewable resource in the world [1,2,3]
Displaying Immunity protein 7 (IM7) scaffoldins on the P. pastoris cell surface In conventional yeast cell surface-display methods, the dockerin–cohesin pairs from bacterial cellulosomes are adopted, in which the dockerin is roughly a 10-kDa calcium-binding module that non-covalently associates with the scaffoldin at affinity in the sub-nM (~ 10−6 M) range [19]
We believed that the ultra-strong protein–protein interaction between IM7 and CL7 would be helpful for cellulosome assembly
Summary
Cellulosic biomass derived from low-value agricultural and wood pulping wastes is likely the most abundant renewable resource in the world [1,2,3]. The works demonstrated that in vivo or in vitro assembly of multiple cellulases on the S. cerevisiae cell surface in a structure termed cellulosome [19, 20] can significantly increase the ethanol yields [21,22,23,24]. The cellulosome is a complicated multi-enzyme machine produced by many cellulolytic microorganisms [25, 26]. Those methods required displaying multiple components on the yeast surface, including heterogeneous dockerin–cohesin pairs, carbohydrate-binding modules (CBMs) and appropriated bacterial cellulases, leading to low displaying efficiency sometimes. More effort is needed to achieve the goal of industrial production of cellulosic ethanol using yeast as the CBP cell factory
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