1 synergistic saccharification performance with CBH1 on high substrate 2 loading upon simultaneous overexpression 3 4 5 Abstract 20 Lytic polysaccharide monooxygenases (LPMOs) are crucial industrial enzymes required in 21 the biorefinery industry as well as in natural carbon cycle. These enzymes known to possess 22 auxiliary activity are produced by numerous bacterial and fungal species to assist in the 23 degradation of cellulosic biomass. In this study, we annotated and performed structural 24 analysis of an uncharacterized thermostable LPMO from Penicillium funiculosum 25 (PfLPMO9) in an attempt to understand nature of this enzyme in biomass degradation. 26 PfLPMO9 exhibited 75% and 36% structural identity to Thermoascus aurantiacus 27 (TaLPMO9A) and Lentinus similis (LsLPMO9A), respectively. Analysis of the molecular 28 interactions during substrate binding revealed that PfLPMO9 demonstrated a higher binding 29 affinity with a ∆G free energy of -46 k kcal/mol when compared with that of TaLPMO9A (-30 31 kcal/mol). The enzyme was further found to be highly thermostable at elevated 31 temperature with a half-life of ~88 h at 50 o C. Furthermore, multiple fungal genetic 32 manipulation tools were employed to simultaneously overexpress this LPMO and 33 Cellobiohydrolase I (CBH1) in catabolite derepressed strain of Penicillium funiculosum, 34PfMig1 88 , in order to improve its saccharification performance towards acid pretreated wheat 35 straw (PWS) at 20% substrate loading. The resulting transformants showed ~200% and ~66% 36 increase in LPMO and Avicelase activities, respectively. While the secretomes of 37 individually overexpressed LPMO and CBH1-strains increased saccharification of PWS by 38 6% and 13%, respectively, over PfMig1 88 at same enzyme concentration, the simultaneous 39 overexpression of these two genes led to 20% increase in saccharification efficiency over 40 PfMig1 88 , which accounted for 82% saccharification of PWS at 20% substrate loading. 41 42 43 44 3 Importance 45Enzymatic hydrolysis of cellulosic biomass by cellulases continues to be a significant 46 bottleneck in the development of second-generation bio-based industries. While efforts are 47 being intensified at how best to obtain indigenous cellulase for biomass hydrolysis, the high 48 production cost of this enzyme remains a crucial challenge confronting its wide availability 49 for efficient utilization of cellulosic materials. This is because it is challenging to get an 50 enzymatic cocktail with balanced activity from a single host. This report provides for the first 51 time the annotation and structural analysis of an uncharacterized thermostable lytic 52 polysaccharide monooxygenase (LPMO) gene in Penicillium funiculosum and its impact in 53 biomass deconstruction upon overexpression in catabolite derepressed strain of P. 54 funiculosum. Cellobiohydrolase I (CBH1) which is the most important enzyme produced by 55 many cellulolytic fungi for saccharification of crystalline cellulose was further overexpressed 56 simultaneous...