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 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.

scholarly journals Blocking drug efflux mechanisms facilitate genome engineering process in hypercellulolytic fungus, Penicillium funiculosum NCIM1228

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Anmoldeep Randhawa ◽  
Nandita Pasari ◽  
Tulika Sinha ◽  
Mayank Gupta ◽  
Anju M. Nair ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Engineering ◽  
Efflux Pump ◽  
Genetic Manipulation ◽  
Function Analysis ◽  
Drug Tolerance ◽  
Growth Media ◽  
Penicillium Funiculosum ◽  
Cellobiohydrolase I ◽  
Drug Tolerability

Abstract Background Penicillium funiculosum NCIM1228 is a non-model filamentous fungus that produces high-quality secretome for lignocellulosic biomass saccharification. Despite having desirable traits to be an industrial workhorse, P. funiculosum has been underestimated due to a lack of reliable genetic engineering tools. Tolerance towards common fungal antibiotics had been one of the major hindrances towards development of reliable transformation tools against the non-model fungi. In this study, we sought to understand the mechanism of drug tolerance of P. funiculosum and the provision to counter it. We then attempted to identify a robust method of transformation for genome engineering of this fungus. Results Penicillium funiculosum showed a high degree of drug tolerance towards hygromycin, zeocin and nourseothricin, thereby hindering their use as selectable markers to obtain recombinant transformants. Transcriptome analysis suggested a high level expression of efflux pumps belonging to ABC and MFS family, especially when complex carbon was used in growth media. Antibiotic selection medium was optimized using a combination of efflux pump inhibitors and suitable carbon source to prevent drug tolerability. Protoplast-mediated and Agrobacterium-mediated transformation were attempted for identifying efficiencies of linear and circular DNA in performing genetic manipulation. After finding Ti-plasmid-based Agrobacterium-mediated transformation more suitable for P. funiculosum, we improvised the system to achieve random and homologous recombination-based gene integration and deletion, respectively. We found single-copy random integration of the T-DNA cassette and could achieve 60% efficiency in homologous recombination-based gene deletions. A faster, plasmid-free, and protoplast-based CRISPR/Cas9 gene-editing system was also developed for P. funiculosum. To show its utility in P. funiculosum, we deleted the gene coding for the most abundant cellulase Cellobiohydrolase I (CBH1) using a pair of sgRNA directed towards both ends of cbh1 open reading frame. Functional analysis of ∆cbh1 strain revealed its essentiality for the cellulolytic trait of P. funiculosum secretome. Conclusions In this study, we addressed drug tolerability of P. funiculosum and developed an optimized toolkit for its genome modification. Hence, we set the foundation for gene function analysis and further genetic improvements of P. funiculosum using both traditional and advanced methods.

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.

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.

Enzymes for Cellulosic Biomass Hydrolysis and Saccharification

2020 ◽  
pp. 283-326 ◽  
Author(s):  
Elmar M. Villota ◽  
Ziyu Dai ◽  
Yanpin Lu ◽  
Bin Yang
Keyword(s):  
Cellulosic Biomass ◽  
Biomass Hydrolysis

scholarly journals Microbial Beta Glucosidase Enzymes: Recent Advances in Biomass Conversation for Biofuels Application

Biomolecules ◽  
2019 ◽  
Vol 9 (6) ◽  
pp. 220 ◽  
Author(s):  
Neha Srivastava ◽  
Rishabh Rathour ◽  
Sonam Jha ◽  
Karan Pandey ◽  
Manish Srivastava ◽  
...  
Keyword(s):  
Fossil Fuels ◽  
Biomass Conversion ◽  
Cost Effective ◽  
Cellulosic Biomass ◽  
Biofuel Production ◽  
Biomass Hydrolysis ◽  
Advanced Technique ◽  
Recent Developments ◽  
Beta Glucosidase ◽  
Made In

The biomass to biofuels production process is green, sustainable, and an advanced technique to resolve the current environmental issues generated from fossil fuels. The production of biofuels from biomass is an enzyme mediated process, wherein β-glucosidase (BGL) enzymes play a key role in biomass hydrolysis by producing monomeric sugars from cellulose-based oligosaccharides. However, the production and availability of these enzymes realize their major role to increase the overall production cost of biomass to biofuels production technology. Therefore, the present review is focused on evaluating the production and efficiency of β-glucosidase enzymes in the bioconversion of cellulosic biomass for biofuel production at an industrial scale, providing its mechanism and classification. The application of BGL enzymes in the biomass conversion process has been discussed along with the recent developments and existing issues. Moreover, the production and development of microbial BGL enzymes have been explained in detail, along with the recent advancements made in the field. Finally, current hurdles and future suggestions have been provided for the future developments. This review is likely to set a benchmark in the area of cost effective BGL enzyme production, specifically in the biorefinery area.

Heterogeneous Acid-Catalyzed Hydrolysis of Cellulose

2012 ◽  
Vol 512-515 ◽  
pp. 421-425
Author(s):  
Jia Xin Liu ◽  
Yu Dong Huang
Keyword(s):  
Fossil Fuel ◽  
Solid Acid ◽  
Cellulosic Biomass ◽  
Biomass Hydrolysis ◽  
Key Technology ◽  
Selective Transformation ◽  
Hydrolysis Of Cellulose ◽  
Acid Catalyzed ◽  
Effective Use ◽  
Hydrolysis Of

With the world’s focus on reducing our dependency on fossil fuel resources, one of the challenges will be the development of efficient catalysts for selective transformation of cellulosic biomass. Hydrolysis of cellulose to glucose is a key technology for effective use of lignocellulose because glucose can be efficiently converted into various chemicals, biofuels, foods, and medicines. Thus far, substantial efforts have been devoted to the degradation of cellulose but these processes have significant drawbacks. Some of these problems can potentially be overcome with the application of solid acid catalysts. In this paper, recent studies on heterogeneous acid-catalyzed hydrolysis of cellulose are summarized.

Preliminary results of neutron and X-ray diffraction data collection on a lytic polysaccharide monooxygenase under reduced and acidic conditions

2021 ◽  
Vol 77 (4) ◽  
Author(s):  
Gabriela C. Schröder ◽  
William B. O'Dell ◽  
Paul D. Swartz ◽  
Flora Meilleur
Keyword(s):  
Reaction Mechanism ◽  
Neutron Diffraction ◽  
Diffraction Data ◽  
Crystalline Cellulose ◽  
Neutron Diffraction Data ◽  
Lytic Polysaccharide Monooxygenase ◽  
Nondestructive Technique ◽  
Data Set ◽  
Polysaccharide Monooxygenases ◽  
Acidic Conditions

Lytic polysaccharide monooxygenases (LPMOs) are copper-center enzymes that are involved in the oxidative cleavage of the glycosidic bond in crystalline cellulose and other polysaccharides. The LPMO reaction is initiated by the addition of a reductant and oxygen to ultimately form an unknown activated copper–oxygen species that is responsible for polysaccharide-substrate H-atom abstraction. Given the sensitivity of metalloproteins to radiation damage, neutron protein crystallography provides a nondestructive technique for structural characterization while also informing on the positions of H atoms. Neutron cryo-crystallography permits the trapping of catalytic intermediates, thereby providing insight into the protonation states and chemical nature of otherwise short-lived species in the reaction mechanism. To characterize the reaction-mechanism intermediates of LPMO9D from Neurospora crassa, a cryo-neutron diffraction data set was collected from an ascorbate-reduced crystal. A second neutron diffraction data set was collected at room temperature from an LPMO9D crystal exposed to low-pH conditions to probe the protonation states of ionizable groups involved in catalysis under acidic conditions.

scholarly journals Inactivation of Cellobiose Dehydrogenases Modifies the Cellulose Degradation Mechanism of Podospora anserina

2016 ◽  
Vol 83 (2) ◽  
Author(s):  
Narumon Tangthirasunun ◽  
David Navarro ◽  
Sona Garajova ◽  
Didier Chevret ◽  
Laetitia Chan Ho Tong ◽  
...  
Keyword(s):  
Plant Biomass ◽  
Cellulose Degradation ◽  
Degradation Mechanism ◽  
Podospora Anserina ◽  
Cellulosic Biomass ◽  
Striking Difference ◽  
Crystalline Cellulose ◽  
Minor Role ◽  
Wild Type ◽  
Content Type

ABSTRACT Conversion of biomass into high-value products, including biofuels, is of great interest to developing sustainable biorefineries. Fungi are an inexhaustible source of enzymes to degrade plant biomass. Cellobiose dehydrogenases (CDHs) play an important role in the breakdown through synergistic action with fungal lytic polysaccharide monooxygenases (LPMOs). The three CDH genes of the model fungus Podospora anserina were inactivated, resulting in single and multiple CDH mutants. We detected almost no difference in growth and fertility of the mutants on various lignocellulose sources, except on crystalline cellulose, on which a 2-fold decrease in fertility of the mutants lacking P. anserina CDH1 (PaCDH1) and PaCDH2 was observed. A striking difference between wild-type and mutant secretomes was observed. The secretome of the mutant lacking all CDHs contained five beta-glucosidases, whereas the wild type had only one. P. anserina seems to compensate for the lack of CDH with secretion of beta-glucosidases. The addition of P. anserina LPMO to either the wild-type or mutant secretome resulted in improvement of cellulose degradation in both cases, suggesting that other redox partners present in the mutant secretome provided electrons to LPMOs. Overall, the data showed that oxidative degradation of cellulosic biomass relies on different types of mechanisms in fungi. IMPORTANCE Plant biomass degradation by fungi is a complex process involving dozens of enzymes. The roles of each enzyme or enzyme class are not fully understood, and utilization of a model amenable to genetic analysis should increase the comprehension of how fungi cope with highly recalcitrant biomass. Here, we report that the cellobiose dehydrogenases of the model fungus Podospora anserina enable it to consume crystalline cellulose yet seem to play a minor role on actual substrates, such as wood shavings or miscanthus. Analysis of secreted proteins suggests that Podospora anserina compensates for the lack of cellobiose dehydrogenase by increasing beta-glucosidase expression and using an alternate electron donor for LPMO.

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