Insights into structure ofPenicillium funiculosumLPMO and its synergistic saccharification performance with CBH1 on high substrate loading upon simultaneous overexpression
Authors: Ogunyewo Olusola A. , Randhawa Anmoldeep , Gupta Mayank , Kaladhar Vemula Chandra , Verma Praveen Kumar , Yazdani Syed Shams 
Published: April 2020

Lytic polysaccharide monooxygenases (LPMOs) are crucial industrial enzymes required in the biorefinery industry as well as in natural carbon cycle. These enzymes known to possess auxiliary activity 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 thermostable LPMO fromPenicillium funiculosum(PfLPMO9) in an attempt to understand nature of this enzyme in biomass degradation. PfLPMO9 exhibited 75% and 36% structural identity toThermoascus aurantiacus(TaLPMO9A) andLentinus similis(LsLPMO9A), respectively. Analysis of the molecular interactions during substrate binding revealed that PfLPMO9 demonstrated a higher binding affinity with a ΔG free energy of -46 k kcal/mol when compared with that of TaLPMO9A (−31 kcal/mol). The enzyme was further found to be highly thermostable at elevated temperature with a half-life of ∼88 h at 50 °C. Furthermore, multiple fungal genetic manipulation tools were employed to simultaneously overexpress this LPMO and Cellobiohydrolase I (CBH1) in catabolite derepressed strain ofPenicillium funiculosum, PfMig188, in order to improve its saccharification performance towards acid pretreated wheat straw (PWS) at 20% substrate loading. The resulting transformants showed ∼200% and ∼66% increase in LPMO and Avicelase activities, respectively. While the secretomes of individually overexpressed LPMO and CBH1-strains increased saccharification of PWS by 6% and 13%, respectively, overPfMig188at same enzyme concentration, the simultaneous overexpression of these two genes led to 20% increase in saccharification efficiency overPfMig188, which accounted for 82% saccharification of PWS at 20% substrate loading.

Importance

Enzymatic hydrolysis of cellulosic biomass by cellulases continues to be a significant bottleneck in the development of second-generation bio-based industries. While efforts are being intensified at how best to obtain indigenous cellulase for biomass hydrolysis, the high production cost of this enzyme remains a crucial challenge confronting its wide availability for efficient utilization of cellulosic materials. This is because it is challenging to get an enzymatic cocktail with balanced activity from a single host. This report provides for the first time the annotation and structural analysis of an uncharacterized thermostable lytic polysaccharide monooxygenase (LPMO) gene inPenicillium funiculosumand its impact in biomass deconstruction upon overexpression in catabolite derepressed strain ofP. 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 the LPMO. The resulting secretome was analyzed for enhanced LPMO and exocellulase activities with the corresponding improvement in its saccharification performance at high substrate loading by ∼20% using a minimal amount of protein.