Synergistic Action of a Lytic Polysaccharide Monooxygenase and a Cellobiohydrolase from Penicillium funiculosum in Cellulose Saccharification under High-Level Substrate Loading

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are crucial industrial enzymes required in the biorefinery industry as well as in the natural carbon cycle. These enzymes, known to catalyze oxidative cleavage of glycosidic bonds, are produced by numerous bacterial and fungal species to assist in the degradation of cellulosic biomass. In this study, we annotated and performed structural analysis of an uncharacterized LPMO from Penicillium funiculosum (PfLPMO9) based on computational methods in an attempt to understand the behavior of this enzyme in biomass degradation. PfLPMO9 exhibited 75% and 36% sequence identity with LPMOs from Thermoascus aurantiacus (TaLPMO9A) and Lentinus similis (LsLPMO9A), respectively. Furthermore, multiple fungal genetic manipulation tools were employed to simultaneously overexpress LPMO and cellobiohydrolase I (CBH1) in a catabolite-derepressed strain of Penicillium funiculosum, PfMig188 (an engineered variant of P. funiculosum), to improve its saccharification performance toward acid-pretreated wheat straw (PWS) at 20% substrate loading. The resulting transformants showed improved LPMO and CBH1 expression at both the transcriptional and translational levels, with ∼200% and ∼66% increases in ascorbate-induced LPMO and Avicelase activities, respectively. While the secretome of PfMig88 overexpressing LPMO or CBH1 increased saccharification of PWS by 6% or 13%, respectively, over the secretome of PfMig188 at the same protein concentration, the simultaneous overexpression of these two genes led to a 20% increase in saccharification efficiency over that observed with PfMig188, which accounted for 82% saccharification of PWS under 20% substrate loading. IMPORTANCE Enzymatic hydrolysis of cellulosic biomass by cellulases continues to be a significant bottleneck in the development of second-generation biobased industries. While increasing efforts are being made to obtain indigenous cellulases for biomass hydrolysis, the high production cost of this enzyme remains a crucial challenge affecting its wide availability for efficient utilization of cellulosic materials. This is because it is challenging to obtain an enzymatic cocktail with balanced activity from a single host. This report describes the annotation and structural analysis of an uncharacterized lytic polysaccharide monooxygenase (LPMO) gene in Penicillium funiculosum and its impact on biomass deconstruction upon overexpression in a catabolite-derepressed strain of P. funiculosum. Cellobiohydrolase I (CBH1), which is the most important enzyme produced by many cellulolytic fungi for saccharification of crystalline cellulose, was further overexpressed simultaneously with LPMO. The resulting secretome was analyzed for enhanced LPMO and exocellulase activities and the corresponding improvement in saccharification performance (by ∼20%) under high substrate loading using a minimal amount of protein.

Accessory enzymes of hypercellulolytic Penicillium funiculosum facilitate complete saccharification of sugarcane bagasse

“…The concentration of each monosaccharide was calculated from calibration curves of external standards (xylose and glucose) purchased from Absolute Standards Inc. The theoretical conversions of cellulose and hemicellulose (in percentage) into monomeric sugars were calculated using the equations provided in NREL's LAP TP-510-43630 as earlier reported [ 12 , 49 ].…”

Blocking drug efflux mechanisms facilitate genome engineering process in hypercellulolytic fungus, Penicillium funiculosum NCIM1228

“…Therefore, we suggest screening of large number of transformants to achieve the recombinant strain with high expression of the gene of interest. We have recently used AMT-based random integration to over-express two important cellulases to enhance the cellulolytic capability of NCIM1228 secretome [ 29 ].…”

“…cbh1 deletion cassette: A DNA fragment of 4314 bp having cbh1 gene along with flanking regions at either side was PCR amplified from genomic DNA using primers CBH1 1 kb up F and CBH1 1.5 kb dn R (Additional file 1 : Table S1) and cloned in pCambia1302 replacing T-DNA at Bst BI and Aat II restriction sites. A cbh1 deletion construct was generated by removing 1558-bp cbh1 ORF region from pOAO2 [ 29 ] by restriction digestion and replacing it with 1916-bp hygromycin resistance cassette at Eam 1105I/ Oli I restriction sites. Chemically synthesized 1916-bp hygromycin cassette (Genscript) was PCR amplified using Hph cas F and Hph cas R primers and cloned at Eam 1105I/ Oli I restriction sites.…”

A sensitive, accurate, and high-throughput gluco-oligosaccharide oxidase based HRP colorimetric method for lytic polysaccharide monooxygenase activity assay

“…Currently, there are two main approaches for LPMO activity assay. One approach is to determine the amount of cello-oligosaccharides or derivates released from LPMO reaction through HPAEC-PID or HPLS-MS analysis [24][25][26]. This approach is time-consuming and has low-throughput.…”