Lignocellulose degrading extremozymes produced by Pichia pastoris: current status and future prospects

Abstract

In this review article, extremophilic lignocellulosic enzymes with special interest on xylanases, β-mannanases, laccases and finally cellulases, namely, endoglucanases, exoglucanases and β-glucosidases produced by Pichia pastoris are reviewed for the first time. Recombinant lignocellulosic extremozymes are discussed from the perspectives of their potential application areas; characteristics of recombinant and native enzymes; the effects of P. pastoris expression system on recombinant extremozymes; and their expression levels and applied strategies to increase the enzyme expression yield. Further, effects of enzyme domains on activity and stability, protein engineering via molecular dynamics simulation and computational prediction, and site-directed mutagenesis and amino acid modifications done are also focused. Superior enzyme characteristics and improved stability due to the proper post-translational modifications and better protein folding performed by P. pastoris make this host favourable for extremozyme production. Especially, glycosylation contributes to the structure, function and stability of enzymes, as generally glycosylated enzymes produced by P. pastoris exhibit better thermostability than non-glycosylated enzymes. However, there has been limited study on enzyme engineering to improve catalytic efficiency and stability of lignocellulosic enzymes. Thus, in the future, studies should focus on protein engineering to improve stability and catalytic efficiency via computational modelling, mutations, domain replacements and fusion enzyme technology. Also metagenomic data need to be used more extensively to produce novel enzymes with extreme characteristics and stability.

Laccases: Production, Expression Regulation, and Applications in Pharmaceutical Biodegradation

“…The most common heterologous host is the methylotrophic yeast Pichia pastoris with its inducible alcohol oxidase ( AOX1 ) promoter (Antošovǎ and Sychrová, 2016; Ergün and Çalık, 2016). Other hosts, such as prokaryotes (e.g., E. coli and B. subtilis ), yeasts (e.g., Saccharomyces cerevisiae and Yarrowia lipolytica ), filamentous fungi (e.g., Trichoderma reesei and Aspergillus niger ), and plants (e.g., Nicotiana tabacum and O. sativa ), are also used (Piscitelli et al, 2010; Kittl et al, 2012; Liebeton et al, 2014; Mate and Alcalde, 2015; Antošovǎ and Sychrová, 2016).…”

“…Expression systems like P. pastoris are used to produce enzymes at the industrial scale, but ligninolytic enzymes like laccases are notoriously difficult to express heterologously (Gu et al, 2014; Ergün and Çalık, 2016). Summaries of heterologously expressed laccases can be found in recent publications (Kittl et al, 2012; Mate and Alcalde, 2015; Antošovǎ and Sychrová, 2016; Ergün and Çalık, 2016).…”

Trends in recombinant protein use in animal production

“…Considering that, in many cases, glycosylation is necessary to obtain functional and stable xylanases [125, 126], yeasts appear as the most promising heterologous expression host for their production as an alternative to fungi. Besides, some yeast have been accredited with the generally recognized as safe (GRAS) status by the American Food and Drug Administration (FDA), which brings additional value to this expression system.…”

“…Besides, some yeast have been accredited with the generally recognized as safe (GRAS) status by the American Food and Drug Administration (FDA), which brings additional value to this expression system. Altogether these advantages make yeast, and more specifically K. pastoris , the most widely used microorganism for xylanase production (extensively reviewed by [126]). Briefly, K. pastoris has been explored for the production of xylanases from T. reesei [127], Aspergillus sulphureus [128], A. niger [129, 130], and Streptomyces sp.…”

“…Briefly, K. pastoris has been explored for the production of xylanases from T. reesei [127], Aspergillus sulphureus [128], A. niger [129, 130], and Streptomyces sp. S38 [131], among others [126]. Albeit at a lesser extent, S. cerevisiae has also been studied for the production of fungal xylanases [112, 132, 133].…”