Publications
23 April 2024
Abstract
The properties of Cas12a nucleases constrict the range of accessible targets and their applications. In this study, we applied ancestral sequence reconstruction (ASR) to a set of Cas12a orthologs from hydrobacteria to reconstruct a common ancestor, ReChb, characterized by near-PAMless targeting and the recognition of diverse nucleic acid activators and collateral substrates. ReChb shares 53% sequence identity with the closest Cas12a ortholog but no longer requires a T-rich PAM and can achieve genome editing in human cells at sites inaccessible to the natural FnCas12a or the engineered and PAM-flexible enAsCas12a. Furthermore, ReChb can be triggered not only by double-stranded DNA but also by single-stranded RNA and DNA targets, leading to non-specific collateral cleavage of all three nucleic acid substrates with similar efficiencies. Finally, tertiary and quaternary structures of ReChb obtained by cryogenic electron microscopy reveal the molecular details underlying its expanded biophysical activities. Overall, ReChb expands the application space of Cas12a nucleases and underscores the potential of ASR for enhancing CRISPR technologies.
https://doi.org/10.1038/s41587-024-02461-323 April 2024
Microbial expansins with different modular structure were shown to increase interfibrillar spacing of cellulose microfibers and performance of enzymes that act on fiber surfaces. This study uncovers new possibilities for biocatalysts to create value-added materials from cellulosic biomass
Abstract
Microbial expansins (EXLXs) are non-lytic proteins homologous to plant expansins involved in plant cell wall formation. Due to their non-lytic cell wall loosening properties and potential to disaggregate cellulosic structures, there is considerable interest in exploring the ability of microbial expansins (EXLX) to assist the processing of cellulosic biomass for broader biotechnological applications. Herein, EXLXs with different modular structure and from diverse phylogenetic origin were compared in terms of ability to bind cellulosic, xylosic, and chitinous substrates, to structurally modify cellulosic fibrils, and to boost enzymatic deconstruction of hardwood pulp.
Results Five heterogeneously produced EXLXs (Clavibacter michiganensis; CmiEXLX2, Dickeya aquatica; DaqEXLX1, Xanthomonas sacchari; XsaEXLX1, Nothophytophthora sp.; NspEXLX1 and Phytophthora cactorum; PcaEXLX1) were shown to bind xylan and hardwood pulp at pH 5.5 and CmiEXLX2 (harboring a family-2 carbohydrate-binding module) also bound well to crystalline cellulose. Small-angle X-ray scattering revealed a 20–25% increase in interfibrillar distance between neighboring cellulose microfibrils following treatment with CmiEXLX2, DaqEXLX1, or NspEXLX1. Correspondingly, combining xylanase with CmiEXLX2 and DaqEXLX1 increased product yield from hardwood pulp by ~ 25%, while supplementing the TrAA9A LPMO from Trichoderma reesei with CmiEXLX2, DaqEXLX1, and NspEXLX1 increased total product yield by over 35%. Conclusion This direct comparison of diverse EXLXs revealed consistent impacts on interfibrillar spacing of cellulose microfibers and performance of carbohydrate-active enzymes predicted to act on fiber surfaces. These findings uncover new possibilities to employ EXLXs in the creation of value-added materials from cellulosic biomass. https://doi.org/10.1186/s13068-024-02500-w15 December 2023
Here we show the potential of loosenin proteins to enhance the enzymatic deconstruction of wood fiber. This work can aid the development of cost-effective technologies for biofuel and biochemical production from renewable resources
Abstract
Microbial expansin-related proteins, including loosenins, can disrupt cellulose networks and increase enzyme accessibility to cellulosic substrates. Herein, four loosenins from Phanerochaete carnosa (PcaLOOLs), and a PcaLOOL fused to a family 63 carbohydrate-binding module, were compared for ability to boost the cellulolytic deconstruction of steam pretreated softwood (SSW) and kraft pulps from softwood (ND-BSKP) and hardwood (ND-BHKP). Amending the Cellic® CTec-2 cellulase cocktail with PcaLOOLs increased reducing products from SSW by up to 40 %, corresponding to 28 % higher glucose yield. Amending Cellic® CTec-2 with PcaLOOLs also increased the release of glucose from ND-BSKP and ND-BHKP by 82 % and 28 %, respectively. Xylose release from ND-BSKP and ND-BHKP increased by 47 % and 57 %, respectively, highlighting the potential of PcaLOOLs to enhance hemicellulose recovery. Scanning electron microscopy and fiber image analysis revealed fibrillation and curlation of ND-BSKP after PcaLOOL treatment, consistent with increasing enzyme accessibility to targeted substrates.
https://doi.org/10.1016/j.biortech.2023.13018813 December 2023
Impacts of expansin-related proteins on lignocellulosic materials and prospective applications in lignocellulose prosessing
Abstract
Cellulose activation is a necessary step in many industrial processes including production of cellulose derivatives, regenerated cellulose, biofuels and biochemicals. Expansins and expansin-related proteins have been shown to disrupt the fibrillar aggregation and loosen the structure of lignocellulosic materials but typically lack lytic activity. Therefore, they offer a new but rather unexploited possibility for biomass to obtain better accessibility and reactivity. From an applied perspective, expansin-related proteins have been investigated for their potential to promote enzymatic hydrolysis of cellulosic substrates for the purpose of producing biofuels. The aim of this review is to compare conventional and emerging technologies relevant to cellulose activation, and critically evaluate the potential of expansin-related proteins for this purpose. As part of this assessment, methods to evaluate the action of expansin-related proteins on cellulosic substrates are summarized, and reported impacts are discussed in relation to source of the cellulosic substrate and treatment conditions. An outlook on prospective applications of expansin-related proteins is presented.
https://doi.org/10.1007/s10570-023-05637-323 June 2023
Abstract
Fast growing hardwoods are one of the major renewable resources available to produce bio-based materials, platform chemicals and biofuels. However, the industrial processing of lignocellulosic biomass is hindered by the complex molecular structure of the cell wall components and their supramolecular organization. This highlights the necessity of improving green processing strategies to enhance biomass conversion to valuable products from industrial wood production species. In the present study, we implemented a hydrothermal step by sequential subcritical water (SW) in aspen wood prior to saccharifi- cation and validated the process for trees grown in greenhouse and field conditions. Subcritical water enables extraction of non-cellulosic cell wall polysaccharides in native polymeric form. A major part of the pectic fraction was easily extracted within the first 10 min, while acetylated xylan was enriched in the subsequent extracts after 20- and 30-min rounds. Prolonged extraction (above 60 min) resulted in partial deacetylation and a reduction of the molar mass of xylan. The analysis of the residues enriched with cell- ulose and lignin showed several micromorphological changes caused by subcritical water treatment, such as an increased porosity, a loosening of the fibre matrix and a decrease in the macrofibrillar dimensions. These morphological and molecular changes in the organization of cell wall polymers after SW treatment significantly enhanced saccharification yields compared to those of non-treated aspen wood chips from both field and greenhouse conditions. Our study demonstrates that SW can be implemented as pretreat- ment prior to saccharification reducing the requirements for chemical acid pretreatments. This process enables the extraction of native non-cellulosic cell wall polymers for potential material applications and promotes the subsequent biochemical conversion of the residual biomass into fermentable sugars and platform chemicals in future biorefineries.
https://doi.org/10.1039/D3GC01020A26 January 2023
Loosenins are expansin-related proteins. These proteins show potential to loosenin cellulose networks. This property can be relevant to sustainable manufacturing of dissolving pulps used in textiles.
Abstract
Microbial expansin-related proteins are ubiquitous across bacterial and fungal organisms and reportedly play a role in the modification and deconstruction of cell wall polysaccharides, including lignocellulose. So far, very few microbial expansin-related proteins, including loosenins and loosenin-like (LOOL) proteins, have been functionally characterized. Herein, four LOOLs encoded by Phanerochaete carnosa and belonging to different subfamilies (i.e., PcaLOOL7 and PcaLOOL9 from subfamily A and PcaLOOL2 and PcaLOOL12 from subfamily B) were recombinantly produced and the purified proteins were characterized using diverse cellulose and chitin substrates. The purified PcaLOOLs weakened cellulose filter paper and cellulose nanofibril networks (CNF); however, none significantly boosted cellulase activity on the selected cellulose substrates (Avicel and Whatman paper). Although fusing the family 63 carbohydrate-binding module (CBM63) of BsEXLX1 encoded by Bacillus subtilis to PcaLOOLs increased their binding to cellulose, the CBM63 fusion appeared to reduce the cellulose filter paper weakening observed using wild-type proteins. Binding of PcaLOOLs to alpha-chitin was considerably higher than that to cellulose (Avicel) and was pH dependent, with the highest binding at pH 5.0. Amendment of certain PcaLOOLs in fungal liquid cultivations also impacted the density of the cultivated mycelia. The present study reveals the potential of fungal expansin-related proteins to impact both cellulose and chitin networks and points to a possible biological role in fungal cell wall processing.
https://doi.org/10.1128/aem.01863-2212 January 2023
Abstract
Ultrafast folding proteins have become an important paradigm in the study of protein folding dynamics. Due to their low energetic barriers and fast kinetics, they are amenable for study by both experiment and simulation. However, single molecule force spectroscopy experiments on these systems are challenging as these proteins do not provide the mechanical fingerprints characteristic of more mechanically stable proteins, which makes it difficult to extract information about the folding dynamics of the molecule. Here, we investigate the unfolding of the ultrafast protein Engrailed Homeodomain (EnHD) by single-molecule atomic force microscopy experiments. Constant speed experiments on EnHD result in featureless transitions typical of compliant proteins. However, in the force-ramp mode we recover sigmoidal curves that we interpret as a very compliant protein that folds and unfolds many times over a marginal barrier. This is supported by a simple theoretical model and coarse-grained molecular simulations. Our results show the ability of force to modulate the unfolding dynamics of ultrafast folding proteins.
https://doi.org/10.1038/s42005-022-01125-52 January 2023
Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR–Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes.
https://doi.org/10.1038/s41564-022-01265-yAbstract
Numerous enzymes have the potential to upgrade biomass, converting it into high-tech materials for new applications. However, the features of natural enzymes often limit their use beyond chemical conversion of the substrate. The development of strategies for the enzymatic conversion of biomass into high-value materials may broaden the range of applications of enzymes and enzyme design techniques. A relevant case is lytic polysaccharide monooxygenase (LPMO), a class of enzymes that catalyzes the oxidative cleavage of glycosidic bonds. Here, we show that an ancestral LPMO can generate chitin nanocrystals. Physicochemical characterization of the chitin nanocrystals demonstrates modifications that make it superior compared to chitin obtained by chemical treatments. We show that the nanocrystals are suitable for controlled 2D and 3D cell cultures, as well as for engineering a biomatrix that combines with graphene oxide, forming a hybrid conductive bioink.
https://doi.org/10.1038/s43246-022-00277-9