Plant cell wall architecture guided design of CBM3-GH11 chimeras with enhanced xylanase activity using a tandem repeat left-handed β-3-prism scaffold

Pinheiro, Matheus Pinto; Reis, Renata A.G.; Dupree, Paul; Ward, Richard J.

doi:10.1016/j.csbj.2021.01.011

Cited by 7 publications

(2 citation statements)

References 59 publications

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“…Extracts were concentrated using an Amicon Ultra‐15 centrifugal filter unit (MERCK) by centrifugation (4700 g , 4°C) until the final volume was 1 ml or less. The structure of regenerated cellulose in Amicon Ultra‐15 centrifugal filter unit is different from that of natural cellulose; therefore, cellulose‐binding proteins are not adsorbed by this filter unit (Duan et al., 2016; Pinheiro et al., 2021; Toyoda et al., 2009). The protein samples were washed and desalted using the Ready‐Prep 2‐D clean‐up kit (Bio‐Rad).…”

Section: Methodsmentioning

confidence: 99%

Plant secondary cell wall proteome analysis with an inducible system for xylem vessel cell differentiation

et al. 2021

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Plant cell walls are typically composed of polysaccharide polymers and cell wall proteins (CWPs). CWPs account for approximately 10% of the plant cell wall structure and perform a wide range of functions. Previous studies have identified approximately 1000 CWPs in the model plant Arabidopsis thaliana; however, the analyses mainly targeted primary cell walls, which are generated at cell division. In contrast, little is known about CWPs in secondary cell walls (SCWs), which are rigid and contain the phenolic polymer lignin. Here, we performed a cell wall proteome analysis to obtain novel insights into CWPs in SCWs. To this end, we tested multiple methods for cell wall extraction with cultured Arabidopsis cells carrying the VND7‐VP16‐GR system, with which cells can be transdifferentiated into xylem‐vessel‐like cells with lignified SCWs by dexamethasone treatment. We then subjected the protein samples to in‐gel trypsin digestion followed by LC‐MS/MS analysis. The different extraction methods resulted in the detection of different cell wall fraction proteins (CWFPs). In particular, centrifugation conditions had a strong impact on the extracted CWFP species, resulting in the increased number of identified CWFPs. We successfully identified 896 proteins as CWFPs in total, including proteases, expansins, purple phosphatase, well‐known lignin‐related enzymes (laccase and peroxidase), and 683 of 896 proteins were newly identified CWFPs. These results demonstrate the usefulness of our CWP analysis method. Further analyses of SCW‐related CWPs could be expected to produce information useful for understanding the roles of CWPs in plant cell functions.

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Section: Methodsmentioning

confidence: 99%

Plant secondary cell wall proteome analysis with an inducible system for xylem vessel cell differentiation

et al. 2021

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“…This has been demonstrated for chimeras combining a CAZyme with a CBM targeting another substrate than the substrate of the CAZyme. For example, cellulose-xylose proximity in the cell wall of sugarcane bagasse, maize stems and Miscanthus resulted in an elevated hydrolytic activity of the chimera of the cellulose-specific CBM3 of the CipA scaffoldin from C. thermocellum and the xylan specific GH11 endoxylanase from Bacillus subtilis (Hervé et al 2010;Pinheiro et al 2021;Zhang et al 2014). Moreover, insights into the effects of contaminants and enzyme inhibitors present in side streams is of crucial importance.…”

Section: Selection Of Cazymesmentioning

confidence: 99%

Current challenges in designer cellulosome engineering

Lamote

Fonseca

Vanderstraeten

et al. 2023

Appl Microbiol Biotechnol

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Designer cellulosomes (DCs) are engineered multi-enzyme complexes, comprising carbohydrate-active enzymes attached to a common backbone, the scaffoldin, via high-affinity cohesin-dockerin interactions. The use of DCs in the degradation of renewable biomass polymers is a promising approach for biorefineries. Indeed, DCs have shown significant hydrolytic activities due to the enhanced enzymesubstrate proximity and inter-enzyme synergies, but technical hurdles in DC engineering have hindered further progress towards industrial application. The challenge in DC engineering lies in the large diversity of possible building blocks and architectures, resulting in a multivariate and immense design space. Simultaneously, the precise DC composition affects many relevant parameters such as activity, stability and manufacturability. Since protein engineers face a lack of high-throughput approaches to explore this vast design space, DC engineering may result in an unsatisfying outcome. This review provides a roadmap to guide researchers through the process of DC engineering. Each step, starting from concept to evaluation, is described and provided with its challenges, along with possible solutions, both for DCs that are assembled in vitro or are displayed on the yeast cell surface. Key points Construction of designer cellulosomes is a multi-step process. Designer cellulosome research deals with multivariate construction challenges. Boosting designer cellulosome efficiency requires exploring a vast design space.

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Using mini-xylanosomes as a biotechnological tool for sisal fiber deconstruction and enzyme immobilization

Hamann

Gomes

Silva

et al. 2022

Biomass Conv. Bioref.

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Plant cell wall architecture guided design of CBM3-GH11 chimeras with enhanced xylanase activity using a tandem repeat left-handed β-3-prism scaffold

Abstract: Graphical abstract

Cited by 7 publications

References 59 publications

Plant secondary cell wall proteome analysis with an inducible system for xylem vessel cell differentiation

Plant secondary cell wall proteome analysis with an inducible system for xylem vessel cell differentiation

Current challenges in designer cellulosome engineering

Using mini-xylanosomes as a biotechnological tool for sisal fiber deconstruction and enzyme immobilization

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