Discovery and Expression of Thermostable LPMOs from Thermophilic Fungi for Producing Efficient Lignocellulolytic Enzyme Cocktails

Agrawal, Dhruv; Basotra, Neha; Balan, Venkatesh; Tsang, Adrian; Chadha, Bhupinder Singh

doi:10.1007/s12010-019-03198-5

Cited by 24 publications

(14 citation statements)

References 49 publications

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“…The results showed that 96% of its original activity was retained at 50 °C after 300 min (5 h) of incubation while ∼40% residual activity was obtained at 70 °C after this time of incubation. At 80 °C also significant activity was observed with T 1/2 of 1.1 h. Interestingly, we found this PfLPMO9 to have comparable thermostability property with that reported from Scytalidium thermophilum (PMO9D_SCYTH) and Malbranchea cinnamomea (PMO9D_MALCI) with half-life of 23 h at 60 °C (40). Similarly, it exhibited higher thermostability than that reported for different Penicillium strains as well as some other fungal species such as Trichoderma reesei, Pestalotiopsis sp and Aspergillus fumigatus whose half-lives were in the range of 8 – 13 h at 50 oC (41–43).…”

Section: Resultssupporting

confidence: 69%

Insights into structure ofPenicillium funiculosumLPMO and its synergistic saccharification performance with CBH1 on high substrate loading upon simultaneous overexpression

Ogunyewo

Randhawa

Gupta

et al. 2020

Preprint

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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...

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Section: Resultssupporting

confidence: 69%

Insights into structure ofPenicillium funiculosumLPMO and its synergistic saccharification performance with CBH1 on high substrate loading upon simultaneous overexpression

Ogunyewo

Randhawa

Gupta

et al. 2020

Preprint

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“…Again, the only exceptions to this pattern among characterized AA10 members are TtLP-MO10A and KpLPMO10A [73,107]. There has recently been significant interest in the identification of thermostable LPMOs, with a particular focus on fungal AA9 members [109,110]. Interestingly, the cellulolytic LPMOs from the mesophiles C. fimi and C. flavigena studied here proved to be only moderately thermostable, with pH-dependent T m values in the range of 40-60 °C (Additional file 3: Figure S6), yet difficult to inactivate by boiling.…”

Section: Discussionmentioning

confidence: 99%

Four cellulose-active lytic polysaccharide monooxygenases from Cellulomonas species

Solhi

Goddard‐Borger

et al. 2021

Biotechnol Biofuels

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Background The discovery of lytic polysaccharide monooxygenases (LPMOs) has fundamentally changed our understanding of microbial lignocellulose degradation. Cellulomonas bacteria have a rich history of study due to their ability to degrade recalcitrant cellulose, yet little is known about the predicted LPMOs that they encode from Auxiliary Activity Family 10 (AA10). Results Here, we present the comprehensive biochemical characterization of three AA10 LPMOs from Cellulomonas flavigena (CflaLPMO10A, CflaLPMO10B, and CflaLPMO10C) and one LPMO from Cellulomonas fimi (CfiLPMO10). We demonstrate that these four enzymes oxidize insoluble cellulose with C1 regioselectivity and show a preference for substrates with high surface area. In addition, CflaLPMO10B, CflaLPMO10C, and CfiLPMO10 exhibit limited capacity to perform mixed C1/C4 regioselective oxidative cleavage. Thermostability analysis indicates that these LPMOs can refold spontaneously following denaturation dependent on the presence of copper coordination. Scanning and transmission electron microscopy revealed substrate-specific surface and structural morphological changes following LPMO action on Avicel and phosphoric acid-swollen cellulose (PASC). Further, we demonstrate that the LPMOs encoded by Cellulomonas flavigena exhibit synergy in cellulose degradation, which is due in part to decreased autoinactivation. Conclusions Together, these results advance understanding of the cellulose utilization machinery of historically important Cellulomonas species beyond hydrolytic enzymes to include lytic cleavage. This work also contributes to the broader mapping of enzyme activity in Auxiliary Activity Family 10 and provides new biocatalysts for potential applications in biomass modification.

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“…The most recent commercial cellulase cocktails have also been subjected to spiking-type of studies. As an example, Agrawal et al have shown that the performance of Cellic CTec2 on acid or alkali pretreated bagasse and rice straw can be boosted by addition of two AA9 LPMOs from the thermophilic fungi Scytalidium thermophilum and Malbranchea cinnamomea [8]. Very recently, von Freiesleben et al have reported that supplementation with GH5 and GH26 mannanases leads to improved saccharification of pretreated lodgepole pine by Cellic CTec3 [367], confirming previous indications concerning suboptimal levels of mannanase activities in Cellic CTec3 for softwood saccharification [61].…”

Section: Spiking Studies To Highlight Enzyme Activities Lacking In Comentioning

confidence: 99%

Enzymatic processing of lignocellulosic biomass: principles, recent advances and perspectives

Østby

Hansen

Horn

et al. 2020

Journal of Industrial Microbiology and Biotechnology

131

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Efficient saccharification of lignocellulosic biomass requires concerted development of a pretreatment method, an enzyme cocktail and an enzymatic process, all of which are adapted to the feedstock. Recent years have shown great progress in most aspects of the overall process. In particular, increased insights into the contributions of a wide variety of cellulolytic and hemicellulolytic enzymes have improved the enzymatic processing step and brought down costs. Here, we review major pretreatment technologies and different enzyme process setups and present an in-depth discussion of the various enzyme types that are currently in use. We pay ample attention to the role of the recently discovered lytic polysaccharide monooxygenases (LPMOs), which have led to renewed interest in the role of redox enzyme systems in lignocellulose processing. Better understanding of the interplay between the various enzyme types, as they may occur in a commercial enzyme cocktail, is likely key to further process improvements.

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Discovery and Expression of Thermostable LPMOs from Thermophilic Fungi for Producing Efficient Lignocellulolytic Enzyme Cocktails

Cited by 24 publications

References 49 publications

Insights into structure ofPenicillium funiculosumLPMO and its synergistic saccharification performance with CBH1 on high substrate loading upon simultaneous overexpression

Insights into structure ofPenicillium funiculosumLPMO and its synergistic saccharification performance with CBH1 on high substrate loading upon simultaneous overexpression

Four cellulose-active lytic polysaccharide monooxygenases from Cellulomonas species

Enzymatic processing of lignocellulosic biomass: principles, recent advances and perspectives

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