Construction of thermostable cellobiohydrolase I from the fungus Talaromyces cellulolyticus by protein engineering

2019 ◽  
Vol 32 (1) ◽  
pp. 33-40
Author(s):  
Makoto Nakabayashi ◽  
Saori Kamachi ◽  
Dominggus Malle ◽  
Toshiaki Yanamoto ◽  
Seiichiro Kishishita ◽  
...  
Keyword(s):  
Protein Engineering ◽  
Structural Information ◽  
Expression System ◽  
Complex Structure ◽  
Hydrolytic Activity ◽  
Cellulosic Biomass ◽  
Crystalline Cellulose ◽  
Cellobiohydrolase I ◽  
Positive Effects ◽  
Talaromyces Cellulolyticus

Abstract Fungus-derived GH-7 family cellobiohydrolase I (CBHI, EC 3.2.1.91) is one of the most important industrial enzymes for cellulosic biomass saccharification. Talaromyces cellulolyticus is well known as a mesophilic fungus producing a high amount of CBHI. Thermostability enhances the economic value of enzymes by making them more robust. However, CBHI has proven difficult to engineer, a fact that stems in part from its low expression in heterozygous hosts and its complex structure. Here, we report the successful improvement of the thermostability of CBHI from T. cellulolyticus using our homologous expression system and protein engineering method. We examined the key structures that seem to contribute to its thermostability using the 3D structural information of CBHI. Some parts of the structure of the Talaromyces emersonii CBHI were grafted into T. cellulolyticus CBHI and thermostable mutant CBHIs were constructed. The thermostability was primarily because of the improvement in the loop structures, and the positive effects of the mutations for thermostability were additive. By combing the mutations, the constructed thermophilic CBHI exhibits high hydrolytic activity toward crystalline cellulose with an optimum temperature at over 70°C. In addition, the strategy can be applied to the construction of the other thermostable CBHIs.

scholarly journals Insights into structure of Penicillium funiculosum LPMO and its synergistic saccharification performance with CBH1 on high substrate loading upon simultaneous overexpression

2020 ◽  
Author(s):  
Olusola A. Ogunyewo ◽  
Anmoldeep Randhawa ◽  
Mayank Gupta ◽  
Vemula Chandra Kaladhar ◽  
Praveen Kumar Verma ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Genetic Manipulation ◽  
Cellulosic Biomass ◽  
Crystalline Cellulose ◽  
Minimal Amount ◽  
Penicillium Funiculosum ◽  
Cellobiohydrolase I ◽  
Biomass Hydrolysis ◽  
Lytic Polysaccharide Monooxygenase ◽  
High Substrate

AbstractLytic 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 from Penicillium funiculosum (PfLPMO9) in an attempt to understand nature of this enzyme in biomass degradation. PfLPMO9 exhibited 75% and 36% structural identity to Thermoascus aurantiacus (TaLPMO9A) and Lentinus 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 of Penicillium 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, over PfMig188 at same enzyme concentration, the simultaneous overexpression of these two genes led to 20% increase in saccharification efficiency over PfMig188, which accounted for 82% saccharification of PWS at 20% substrate loading.ImportanceEnzymatic 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 in Penicillium funiculosum and its impact in biomass deconstruction upon overexpression in 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 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.

scholarly journals Traffic Jams Reduce Hydrolytic Efficiency of Cellulase on Cellulose Surface

Science ◽  
2011 ◽  
Vol 333 (6047) ◽  
pp. 1279-1282 ◽  
Author(s):  
Kiyohiko Igarashi ◽  
Takayuki Uchihashi ◽  
Anu Koivula ◽  
Masahisa Wada ◽  
Satoshi Kimura ◽  
...  
Keyword(s):  
High Speed ◽  
Cellulose Degradation ◽  
Polymorphic Form ◽  
Cellulosic Biomass ◽  
Crystalline Cellulose ◽  
Cellobiohydrolase I ◽  
Force Microscopy ◽  
Real Time Visualization ◽  
Enzyme Molecules ◽  
Cellulose Surface

A deeper mechanistic understanding of the saccharification of cellulosic biomass could enhance the efficiency of biofuels development. We report here the real-time visualization of crystalline cellulose degradation by individual cellulase enzymes through use of an advanced version of high-speed atomic force microscopy. Trichoderma reesei cellobiohydrolase I (TrCel7A) molecules were observed to slide unidirectionally along the crystalline cellulose surface but at one point exhibited collective halting analogous to a traffic jam. Changing the crystalline polymorphic form of cellulose by means of an ammonia treatment increased the apparent number of accessible lanes on the crystalline surface and consequently the number of moving cellulase molecules. Treatment of this bulky crystalline cellulose simultaneously or separately with T. reesei cellobiohydrolase II (TrCel6A) resulted in a remarkable increase in the proportion of mobile enzyme molecules on the surface. Cellulose was completely degraded by the synergistic action between the two enzymes.

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

2020 ◽  
Vol 86 (23) ◽  
Author(s):  
Olusola A. Ogunyewo ◽  
Anmoldeep Randhawa ◽  
Mayank Gupta ◽  
Vemula Chandra Kaladhar ◽  
Praveen Kumar Verma ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Cellulosic Biomass ◽  
Crystalline Cellulose ◽  
Minimal Amount ◽  
Penicillium Funiculosum ◽  
Cellobiohydrolase I ◽  
Lytic Polysaccharide Monooxygenase ◽  
Content Type ◽  
High Level ◽  
Polysaccharide Monooxygenase

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 the 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 identities 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 the 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 The 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 the 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 the 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-level substrate loading using a minimal amount of protein.

Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

2019 ◽  
Vol 476 (21) ◽  
pp. 3227-3240 ◽  
Author(s):  
Shanshan Wang ◽  
Yanxiang Zhao ◽  
Long Yi ◽  
Minghe Shen ◽  
Chao Wang ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Magnaporthe Oryzae ◽  
Structural Information ◽  
Complex Structure ◽  
Critical Role ◽  
Catalytic Process ◽  
Structural Basis ◽  
Salt Bridges ◽  
Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

scholarly journals Crystal Structure of African Swine Fever Virus pS273R Protease and Implications for Inhibitor Design

2020 ◽  
Vol 94 (10) ◽  
Author(s):  
Guobang Li ◽  
Xiaoxia Liu ◽  
Mengyuan Yang ◽  
Guangshun Zhang ◽  
Zhengyang Wang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Structural Information ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Hydrolytic Activity ◽  
Viral Disease ◽  
Fever Virus ◽  
Dna Virus ◽  
Core Domain ◽  
The Core

ABSTRACT African swine fever (ASF) is a highly contagious hemorrhagic viral disease of domestic and wild pigs that is responsible for serious economic and production losses. It is caused by the African swine fever virus (ASFV), a large and complex icosahedral DNA virus of the Asfarviridae family. Currently, there is no effective treatment or approved vaccine against the ASFV. pS273R, a specific SUMO-1 cysteine protease, catalyzes the maturation of the pp220 and pp62 polyprotein precursors into core-shell proteins. Here, we present the crystal structure of the ASFV pS273R protease at a resolution of 2.3 Å. The overall structure of the pS273R protease is represented by two domains named the “core domain” and the N-terminal “arm domain.” The “arm domain” contains the residues from M1 to N83, and the “core domain” contains the residues from N84 to A273. A structure analysis reveals that the “core domain” shares a high degree of structural similarity with chlamydial deubiquitinating enzyme, sentrin-specific protease, and adenovirus protease, while the “arm domain” is unique to ASFV. Further, experiments indicated that the “arm domain” plays an important role in maintaining the enzyme activity of ASFV pS273R. Moreover, based on the structural information of pS273R, we designed and synthesized several peptidomimetic aldehyde compounds at a submolar 50% inhibitory concentration, which paves the way for the design of inhibitors to target this severe pathogen. IMPORTANCE African swine fever virus, a large and complex icosahedral DNA virus, causes a deadly infection in domestic pigs. In addition to Africa and Europe, countries in Asia, including China, Vietnam, and Mongolia, were negatively affected by the hazards posed by ASFV outbreaks in 2018 and 2019, at which time more than 30 million pigs were culled. Until now, there has been no vaccine for protection against ASFV infection or effective treatments to cure ASF. Here, we solved the high-resolution crystal structure of the ASFV pS273R protease. The pS273R protease has a two-domain structure that distinguishes it from other members of the SUMO protease family, while the unique “arm domain” has been proven to be essential for its hydrolytic activity. Moreover, the peptidomimetic aldehyde compounds designed to target the substrate binding pocket exert prominent inhibitory effects and can thus be used in a potential lead for anti-ASFV drug development.

scholarly journals Supercharged Cellulases Show Reduced Non-Productive Binding, But Enhanced Activity, on Pretreated Lignocellulosic Biomass

2021 ◽  
Author(s):  
Bhargava Nemmaru ◽  
Jenna Douglass ◽  
John M Yarbrough ◽  
Antonio De Chellis ◽  
Srivatsan Shankar ◽  
...  
Keyword(s):  
Cell Wall ◽  
Corn Stover ◽  
Lignocellulosic Biomass ◽  
Hydrolytic Activity ◽  
Fine Tuning ◽  
Cellulolytic Enzymes ◽  
Crystalline Cellulose ◽  
Cell Wall Components ◽  
Carbohydrate Binding Modules ◽  
Wall Components

Non-productive adsorption of cellulolytic enzymes to various plant cell wall components, such as lignin and cellulose, necessitates high enzyme loadings to achieve efficient conversion of pretreated lignocellulosic biomass to fermentable sugars. Carbohydrate-binding modules (CBMs), appended to various catalytic domains (CDs), promote lignocellulose deconstruction by increasing targeted substrate-bound CD concentration but often at the cost of increased non-productive enzyme binding. Here, we demonstrate how a computational protein design strategy can be applied to a model endocellulase enzyme (Cel5A) from Thermobifida fusca to allow fine-tuning its CBM surface charge, which led to increased hydrolytic activity towards pretreated lignocellulosic biomass (e.g., corn stover) by up to ~330% versus the wild-type Cel5A control. We established that the mechanistic basis for this improvement arises from reduced non-productive binding of supercharged Cel5A mutants to cell wall components such as crystalline cellulose (up to 1.7-fold) and lignin (up to 1.8-fold). Interestingly, supercharged Cel5A mutants that showed improved activity on various forms of pretreated corn stover showed increased reversible binding to lignin (up to 2.2-fold) while showing no change in overall thermal stability remarkably. In general, negative supercharging led to increase hydrolytic activity towards both pretreated lignocellulosic biomass and crystalline cellulose whereas positive supercharging led to a reduction of hydrolytic activity. Overall, selective supercharging of protein surfaces was shown to be an effective strategy for improving hydrolytic performance of cellulolytic enzymes for saccharification of real-world pretreated lignocellulosic biomass substrates. Future work should address the implications of supercharging cellulases from various families on inter-enzyme interactions and synergism.

scholarly journals Self-Supervised Representation Learning of Protein Tertiary Structures (PtsRep): Protein Engineering as A Case Study

2020 ◽  
Author(s):  
Junwen Luo ◽  
Yi Cai ◽  
Jialin Wu ◽  
Hongmin Cai ◽  
Xiaofeng Yang ◽  
...  
Keyword(s):  
Deep Learning ◽  
Protein Engineering ◽  
Structural Information ◽  
Representation Learning ◽  
Sequence Information ◽  
Structural Representation ◽  
Tertiary Structures ◽  
Structural Space ◽  
General Protein ◽  
And Function

AbstractIn recent years, deep learning has been increasingly used to decipher the relationships among protein sequence, structure, and function. Thus far deep learning of proteins has mostly utilized protein primary sequence information, while the vast amount of protein tertiary structural information remains unused. In this study, we devised a self-supervised representation learning framework to extract the fundamental features of unlabeled protein tertiary structures (PtsRep), and the embedded representations were transferred to two commonly recognized protein engineering tasks, protein stability and GFP fluorescence prediction. On both tasks, PtsRep significantly outperformed the two benchmark methods (UniRep and TAPE-BERT), which are based on protein primary sequences. Protein clustering analyses demonstrated that PtsRep can capture the structural signals in proteins. PtsRep reveals an avenue for general protein structural representation learning, and for exploring protein structural space for protein engineering and drug design.

scholarly journals A Thermolabile Phospholipase B from Talaromyces marneffei GD-0079: Biochemical Characterization and Structure Dynamics Study

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 231 ◽  
Author(s):  
Rabia Durrani ◽  
Faez Iqbal Khan ◽  
Shahid Ali ◽  
Yonghua Wang ◽  
Bo Yang
Keyword(s):  
Fatty Acids ◽  
Conformational Changes ◽  
Catalytic Mechanism ◽  
Biochemical Characterization ◽  
Structural Information ◽  
Expression System ◽  
Talaromyces Marneffei ◽  
Structure Dynamics ◽  
Phospholipase B ◽  
Hydrolysis Of

Phospholipase B (EC 3.1.1.5) are a distinctive group of enzymes that catalyzes the hydrolysis of fatty acids esterified at the sn-1 and sn-2 positions forming free fatty acids and lysophospholipids. The structural information and catalytic mechanism of phospholipase B are still not clear. Herein, we reported a putative phospholipase B (TmPLB1) from Talaromyces marneffei GD-0079 synthesized by genome mining library. The gene (TmPlb1) was expressed and the TmPLB1 was purified using E. coli shuffle T7 expression system. The putative TmPLB1 was purified by affinity chromatography with a yield of 13.5%. The TmPLB1 showed optimum activity at 35 °C and pH 7.0. The TmPLB1 showed enzymatic activity using Lecithin (soybean > 98% pure), and the hydrolysis of TmPLB1 by 31P NMR showed phosphatidylcholine (PC) as a major phospholipid along with lyso-phospholipids (1-LPC and 2-LPC) and some minor phospholipids. The molecular modeling studies indicate that its active site pocket contains Ser125, Asp183 and His215 as the catalytic triad. The structure dynamics and simulations results explained the conformational changes associated with different environmental conditions. This is the first report on biochemical characterization and structure dynamics of TmPLB1 enzyme. The present study could be helpful to utilize TmPLB1 in food industry for the determination of food components containing phosphorus. Additionally, such enzyme could also be useful in Industry for the modifications of phospholipids.

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