Non-productive binding of cellobiohydrolase i investigated by surface plasmon resonance spectroscopy

Future biorefineries are facing the challenge to separate and depolymerize biopolymers into their building blocks for the production of biofuels and basic molecules as chemical stock. Fungi have evolved lignocellulolytic enzymes to perform this task specifically and efficiently, but a detailed understanding of their heterogeneous reactions is a prerequisite for the optimization of large-scale enzymatic biomass degradation. Here, we investigate the binding of cellulolytic enzymes onto biopolymers by surface plasmon resonance (SPR) spectroscopy for the fast and precise characterization of enzyme adsorption processes. Using different sensor architectures, SPR probes modified with regenerated cellulose as well as with lignin films were prepared by spin-coating techniques. The modified SPR probes were analyzed by atomic force microscopy and static contact angle measurements to determine physical and surface molecular properties. SPR spectroscopy was used to study the activity and affinity of Trichoderma reesei cellobiohydrolase I (CBHI) glycoforms on the modified SPR probes. N-glycan removal led to no significant change in activity or cellulose binding, while a slightly higher tendency for non-productive binding to SPR probes modified with different lignin fractions was observed. The results suggest that the main role of the N-glycosylation in CBHI is not to prevent non-productive binding to lignin, but probably to increase its stability against proteolytic degradation. The work also demonstrates the suitability of SPR-based techniques for the characterization of the binding of lignocellulolytic enzymes to biomass-derived polymers. Graphic abstract

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

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.

Chimeric cellobiohydrolase I expression, activity, and biochemical properties in three oleaginous yeast

Consolidated bioprocessing using oleaginous yeast is a promising modality for the economic conversion of plant biomass to fuels and chemicals. However, yeast are not known to produce effective biomass degrading enzymes naturally and this trait is essential for efficient consolidated bioprocessing. We expressed a chimeric cellobiohydrolase I gene in three different oleaginous, industrially relevant yeast: Yarrowia lipolytica, Lipomyces starkeyi, and Saccharomyces cerevisiae to study the biochemical and catalytic properties and biomass deconstruction potential of these recombinant enzymes. Our results showed differences in glycosylation, surface charge, thermal and proteolytic stability, and efficacy of biomass digestion. L. starkeyi was shown to be an inferior active cellulase producer compared to both the Y. lipolytica and S. cerevisiae enzymes, whereas the cellulase expressed in S. cerevisiae displayed the lowest activity against dilute-acid-pretreated corn stover. Comparatively, the chimeric cellobiohydrolase I enzyme expressed in Y. lipolytica was found to have a lower extent of glycosylation, better protease stability, and higher activity against dilute-acid-pretreated corn stover.

NMR elucidation of nonproductive binding sites of lignin models with carbohydrate-binding module of cellobiohydrolase I

Background Highly efficient enzymatic saccharification of pretreated lignocellulose is a key step in achieving lignocellulosic biorefinery. Cellobiohydrolase I (Cel7A) secreted by Trichoderma reesei is an industrially used cellulase that possesses carbohydrate-binding module 1 (TrCBM1) at the C-terminal domain. The nonproductive binding of TrCBM1 to lignin significantly decreases the enzymatic saccharification efficiency and increases the cost of biomass conversion because of the additionally required enzymes. Understanding the interaction mechanism between lignin and TrCBM1 is essential for realizing a cost-effective biofuel production; however, the binding sites in lignin have not been clearly elucidated. Results Three types of 13C-labeled β-O-4 lignin oligomer models were synthesized and characterized. The 2D 1H–13C heteronuclear single-quantum correlation (HSQC) spectra of the 13C-labeled lignin models confirmed that the three types of the 13C labels were correctly incorporated in the (1) aromatic rings and β positions, (2) α positions, and (3) methoxy groups, respectively. The TrCBM1-binding sites in lignin were analyzed by observing NMR chemical shift perturbations (CSPs) using the synthetic 13C-labeled β-O-4 lignin oligomer models. Obvious CSPs were observed in signals from the aromatic regions in oligomers bound to TrCBM1, whereas perturbations in the signals from aliphatic regions and methoxy groups were insignificant. These findings indicated that hydrophobic interactions and π–π stacking were dominating factors in nonproductive binding. The synthetic lignin models have two configurations whose terminal units were differently aligned and donated C(I) and C(II). The C(I) ring showed remarkable perturbation compared with the C(II), which indicated that the binding of TrCBM1 was markedly affected by the configuration of the lignin models. The long-chain lignin models (degree of polymerization (DP) 4.16–4.70) clearly bound to TrCBM1. The interactions of TrCBM1 with the short-chain lignin models (DP 2.64–3.12) were insignificant, indicating that a DP greater than 4 was necessary for TrCBM1 binding. Conclusion The CSP analysis using 13C-labeled β-O-4 lignin oligomer models enabled the identification of the TrCBM1 binding sites in lignins at the atomic level. This specific interaction analysis will provide insights for new molecular designs of cellulase having a controlled affinity to cellulose and lignin for a cost-effective biorefinery process.

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

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.

NMR Elucidation of Nonproductive Binding Sites of Lignin Models with Carbohydrate-Binding Module of Cellobiohydrolase I

Background : Highly efficient enzymatic saccharification of pretreated lignocellulose is a key step in achieving lignocellulosic biorefinery. Cellobiohydrolase I (Cel7A) secreted by Trichoderma reesei is an industrially used cellulase that possesses carbohydrate-binding module 1 (TrCBM1) at the C-terminal domain. The nonproductive binding of TrCBM1 to lignin significantly decreases the enzymatic saccharification efficiency and increases the cost of biomass conversion because of the additionally required enzymes. Understanding the interaction mechanism between lignin and TrCBM1 is essential for realizing a cost-effective biofuel production; however, the binding sites in lignin have not been clearly elucidated.Results: Three types of 13C-labeled β-O-4 lignin oligomer models were synthesized and characterized. The 2D 1H–13C heteronuclear single-quantum correlation (HSQC) spectra of the 13C-labeled lignin models confirmed that the three types of the 13C labels were correctly incorporated in the (1) aromatic rings and β positions, (2) α positions, and (3) methoxy groups, respectively. The TrCBM1 binding sites in lignin were analyzed by observing NMR chemical shift perturbations (CSPs) using the synthetic 13C-labeled β-O-4 lignin oligomer models. Obvious CSPs were observed in signals from the aromatic regions in oligomers bound to TrCBM1, whereas perturbations in the signals from aliphatic regions and methoxy groups were insignificant. These findings indicated that hydrophobic interactions and π–π stacking were dominating factors in nonproductive binding. The synthetic lignin models have two configurations whose terminal units were differently aligned and donated C(I) and C(II) . The C(I) ring showed remarkable perturbation compared with the C(II) , which indicated that the binding of TrCBM1 was markedly affected by the configuration of the lignin models. The long-chain lignin models (degree of polymerization (DP) 4.16–4.70) clearly bound to TrCBM1. The interactions of TrCBM1 with the short-chain lignin models (DP 2.64–3.12) were insignificant, indicating that a DP greater than 4 was necessary for TrCBM1 binding.Conclusion: The CSP analysis using 13C-labeled β-O-4 lignin oligomer models enabled the identification of the TrCBM1 binding sites in lignins at the atomic level. This specific interaction analysis will provide insights for new molecular designs of cellulase having a controlled affinity to cellulose and lignin for a cost-effective biorefinery process.

NMR Elucidation of Non-productive Binding Sites of 13C-Labeled Lignin Models with Carbohydrate Binding Module of Cellobiohydrolase I for Efficient Biomass Conversion

Background: Highly efficient enzymatic saccharification of pretreated lignocellulose is a primary key step in achieving lignocellulosic biorefinery. Cellobiohydrolase I (Cel7A) secreted by Trichoderma reesei is an industrially used cellulase possessing carbohydrate binding module 1 (TrCBM1) as the C-terminal domain. Non-productive binding of TrCBM1 to lignin significantly decreases enzymatic saccharification efficiency and enhance cost of biomass conversion due to required additional enzymes. Understanding of the interaction mechanism between lignin and TrCBM1 is essentially required to realize cost-effective biofuels production, but the binding sites in lignin have not been clearly elucidated. Results: Three types of 13C-labeled b-O-4 lignin oligomer models were synthesized and characterized. The 2D 1H-13C HSQC spectra of the 13C-labeled lignin models exhibited that 13C-labels were correctly incorporated in the (1) aromatic rings and b positions, (2) a positions, and (3) methoxy groups, respectively. The TrCBM1 binding sites in lignin were analyzed by observing NMR chemical shift perturbations (CSPs) using the synthetic 13C-labeled b-O-4 lignin oligomer models. Obvious CSPs were observed in signals from the aromatic regions in oligomers bound to TrCBM1, whereas perturbations in the signals from aliphatic regions and methoxy groups were insignificant. This indicated that hydrophobic interactions and p–p stacking were dominating factors in non-productive binding. The synthetic lignin models have two configurations whose terminal units were differently aligned and donated C(I) and C(II). The C(I) ring showed remarkable perturbation compared with C(II), which indicated that binding of TrCBM1 is evidently affected by configuration of lignin models. Long-chain lignins (DP 4.16–4.70) clearly bound to TrCBM1. Interactions with short-chain lignins (DP 2.64–3.12) were insignificant, indicating that a DP greater than 4 was necessary for TrCBM1 binding. Conclusion: The CSP analysis using 13C-labeled b-O-4 lignin oligomer models enabled us to identify TrCBM1 binding sites in lignin at the atomic level. This specific interaction analysis will lead to new molecular design of cellulase having controlled affinity to cellulose and lignin for cost-effective biorefinery process.