Production of a high-efficiency cellulase complex via β-glucosidase engineering in Penicillium oxalicum

Abstract

BackgroundTrichoderma reesei is a widely used model cellulolytic fungus, supplying a highly effective cellulase production system. Recently, the biofuel industry discovered filamentous fungi from the Penicillium genus as a promising alternative to T. reesei.ResultsIn our study, we present a systematic over-expression analysis of nine β-glucosidase encoding genes in the wild-type strain 114-2 of Penicillium oxalicum. We found that the over-expression of BGL1, BGL4, or BGL5 significantly enhanced both β-glucosidase activity and hydrolysis efficiency of the enzyme system on filter paper. We utilised two strategies to over-express β-glucosidase in the strain RE-10 that—although over-producing cellulase, does so at the cost of the cellulase mixture deficiency. The constitutive promoter of gene pde_02864 encoding 40S ribosomal protein S8 was used to over-express three β-glucosidases: BGL1, BGL4, and BGL5. We found that all mutants show significantly enhanced levels of β-glucosidase at transcriptional, protein, and activity levels. Furthermore, the inducible promoter from bgl2 was used to conditionally over-express the β-glucosidases BGL1 and BGL4. Surprisingly, this induced expression strategy enables significantly improved expression efficiency. The BGL1 over-expressing mutant I1-13 particularly improved the β-glucosidase activity at a factor of 65-folds, resulting in levels of up to 150 U/ml. All our BGL over-expression mutants displayed significant enhancement of cellulolytic ability on both microcrystalline cellulose and filter paper. In addition, they substantially reduced the enzyme loads in the saccharification of a natural lignocellulose material delignified corncob residue (DCCR). The mutant I4-32 with over-expression of BGL4 achieved the highest glucose yield in the saccharification of DCCR at only 25 % enzyme load compared to the parental strain RE-10.ConclusionsIn summary, genetically engineering P. oxalicum to significantly improve β-glucosidase activity is a potent strategy to substantially boost the hydrolytic efficiency of the cellulase cocktail, which will ultimately lead to a considerable reduction of cost for biomass-based biofuel.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0491-4) contains supplementary material, which is available to authorized users.

CAZyome and Sugar Transportome Unveil Singular Aspects of the Lignocellulolytic Enzyme System of Penicillium echinulatum 2HH

“…In the same way, extracellular β-glucosidase BGL3 (PDE 01277) of P. oxalicum 114-2 did not show activities on both pNPG and salicin in vitro and its role is not yet known [19]. In P. echinulatum 2HH we have not found the ortholog of this β-glucosidase of GH1 family.…”

“…This pathway acts when cellobiose or other cellodextrins accumulates into the cell, raising the secretion of cellulases. In this context, intracellular accumulation of cellodextrins can occur in two ways: i) by low activity of iBGLs, which reduces the hydrolysis of cellodextrins to D-glucose [19,34]; and ii) by the high expression of STs, which are able to transport cellodextrins from the medium into the cell [20,35]. In order to discover the features behind this cellodextrin induction system in P. echinulatum 2HH, we performed several genomic analyses including iBGLs and STs, as presented below.…”

“…The deletion of bgl2 facilitates the synergistic expression of cellulase genes. Lack of this iBGL facilitates the accumulation of intracellular cellodextrins, which can trigger signaling cascades that include expression of cellulase genes [42,19].…”

“…Along these lines, it has been shown that cellulolytic enzyme expression is induced by cellobiose in many species of fungi, regarding cellobiose is the primary end product generated from cellulose degradation by cellulases [18]. Recent research results support that accumulation of intracellular cellodextrins (mainly cellobiose) may raise cellulases secretion by a cascade signaling pathway in P. oxalicum [19] and Neurospora crassa [20].…”

Genetic modification: a tool for enhancing cellulase secretion

“…This makes further improvement of its β-glucosidase activity necessary. Yao et al (2016) have presented a systematic over-expression analysis of nine β-glucosidase encoding genes in the wild-type strain 114-2 of P. oxalicum. They overexpressed BGL1, BGL4, or BGL5 using both constitutive as well as inducible promoters.…”