Identification of a thermostable fungal lytic polysaccharide monooxygenase and evaluation of its effect on lignocellulosic degradation

Zhang, Ruiqin; Liu, Yucui; Zhang, Yi; Feng, Dingqing; Hou, Shaoli; Guo, Wei; Niu, Kangle; Jiang, Yi; Han, Lijuan; Sindhu, Lara; Xu, Fang

doi:10.1007/s00253-019-09928-3

Cited by 56 publications

(32 citation statements)

References 64 publications

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“…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). Utilization of thermostable enzymes, including LPMOs, are of interest industrially as saccharification performed at high temperature decreases the risk of microbial contamination and enables enhanced saccharification rates at higher substrate loading due to reduced slurry viscosity (44).…”

Section: Resultsmentioning

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: Resultsmentioning

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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“…But more important is that it can enhance the biomass converion efficiency of the Cellulase. So far highest synergistic effect was reported by AA9 which is less than two fold (Zhang et al 2019). AaAA16, the recent addition of AA16 family of LPMO in the CAZY database showed synergism with CBH1 for the degradation of cellulose.…”

Section: Discussionmentioning

confidence: 98%

“…This result indicates that the exo-glucanase activity of the AfLPMO16 is absent which is required for depolymerization of the crystalline structure of the Avicel (Väljamäe et al 1999;Asztalos et al 2012). This particular family of enzyme has earned so much research interest due to its synergistic effect or boosting effect on cellulase enzyme (Zhang et al 2019).…”

Section: Discussionmentioning

confidence: 99%

Biochemical, structural insights of newly isolated AA16 family of Lytic Polysaccharide Monooxygenase (LPMO) fromAspergillus fumigatusand investigation of its synergistic effect using biomass

Hossain

Dodda

Kapoor

et al. 2020

Preprint

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Efficient conversion of lignocellulosic biomass into fermentable sugar is a bottleneck for cheap production of bio-ethanol. Recently identified enzyme Lytic Polysaccharide Monooxygenase (LPMO) family has brought new hope because of its boosting capabilities of cellulose hydrolysis. Cellulase enzymes of Aspergillus origin are important for their thermostable nature. Here we have identified and characterized a new class of auxiliary (AA16) oxidative enzyme LPMO from the genome of a locally isolated thermophilic fungus Aspergillus fumigatus (NITDGPKA3). The biochemical, structural, and biomass conversion efficiency of AfLPMO16 have been explored. The AfLPMO16 is an intronless gene and encodes the protein of about 29kDa of molecular weight. Sequence-wise it is close to C1 type and structurally it is similar to AA11 family of LPMOs. The structure exclusively consists of loops and sheets. The gene was expressed under inducible promoter (AOX1) with C-terminal His tag in the Pichia pastoris. The protein was purified, and enzyme kinetics was studied with 2, 6-DMP. Polysaccharides hydrolysis activity was observed with Carboxymethyl cellulose (CMC) and Phosphoric acid swollen cellulose (PASC). Further, the lignocellulosic biomass conversion enhancement with Cellulase cocktail gives 2-fold enhancement in reducing sugar release suggests the importance of AfLPMO16 in the bio-ethanol industry.

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“…Effect of light and temperature on the stability of WSCP-Chl a versus free Chl a For application of photobiocatalytic LPMO reactions, the photosensitizer should ideally be stable under a broad range of temperatures. For example, several fungal LPMOs (AA9) have been shown to have the highest activity levels at temperatures ranging between 40-50 °C 34 . Therefore, photostability was tested at 25 °C and 50 °C.…”

Section: Stability Of Wscp-chla Versus Free Chl Amentioning

confidence: 99%

Water-soluble chlorophyll-binding proteins from Brassica oleracea allow for stable photobiocatalytic oxidation of cellulose by a lytic polysaccharide monooxygenase

Dodge

Russo

Blossom

et al. 2020

Preprint

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Abstract Background Lytic polysaccharide monooxygenases (LPMOs) are indispensable redox enzymes used in industry for the saccharification of plant biomass. LPMO-driven cellulose oxidation can be enhanced considerably through photobiocatalysis using chlorophyll derivatives and light. Water soluble chlorophyll binding proteins (WSCPs) make it is possible to stabilize and solubilize chlorophyll in aqueous solution, allowing for in vitro studies on photostability and ROS production. Here we aim apply a WSCP-Chl a as a photosensitizing complex for photobiocatalysis with the LPMO, TtAA9. Results We have in this study demonstrated how WSCP reconstituted with chlorophyll a (WSCP-Chl a) can create a stable photosensitizing complex which produces controlled amounts of H2O2 in the presence of ascorbic acid and light. WSCP-Chl a is highly reactive and allows for tightly controlled formation of H2O2 by regulating light intensity. TtAA9 together with WSCP-Chl a shows increased cellulose oxidation under low light conditions, and the WSCP-Chl a complex remains stable after 24 hours of light exposure. Additionally, the WSCP-Chl a complex demonstrates stability over a range of temperatures and pH conditions relevant for enzyme activity in industrial settings. Conclusion With WSCP-Chl a as the photosensitizer, the need to replenish Chl is greatly reduced, enhancing the catalytic lifetime of light-driven LPMOs and increasing the efficiency of cellulose depolymerization. WSCP-Chl a allows for stable photobiocatalysis providing a sustainable solution for biomass processing.

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Identification of a thermostable fungal lytic polysaccharide monooxygenase and evaluation of its effect on lignocellulosic degradation

Cited by 56 publications

References 64 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

Biochemical, structural insights of newly isolated AA16 family of Lytic Polysaccharide Monooxygenase (LPMO) fromAspergillus fumigatusand investigation of its synergistic effect using biomass

Water-soluble chlorophyll-binding proteins from Brassica oleracea allow for stable photobiocatalytic oxidation of cellulose by a lytic polysaccharide monooxygenase

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