Carbohydrate active enzyme domains from extreme thermophiles

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Introduction

Biomaterials (materials derived from renewable and sustainable biological substrates) could provide an economically viable strategy to mitigate or ameliorate environmental challenges and potentially replace products derived from petrochemical feedstocks (Naik et al. 2010). However, to maximise efficiency of bioproduct synthesis, large sources of simple polysaccharides are required. Second generation feedstocks are a potential source of such material, as they are typically not food crops (such as many first generation bioproduct feedstocks) and, therefore, do not impact on human food security. Some plant species produce large amounts of dense lignocellulosic biomass and are able to grow in a wide range of conditions and environments (Hendriks and Zeeman 2009; Himmel et al. 2007). However, lignocellulosic biomass is highly recalcitrant (Himmel et al. 2007), requiring large investments of energy (with associated financial and waste costs) to access (Alvira et al. 2010). The integration of Carbohydrate Active Enzymes (CAZymes) in the processing of lignocellulosic biomass is a promising strategy for reducing the difficulty and cost of biopolymer extraction (Mir et al. 2014; Turumtay 2015). CAZymes are active on oligosaccharides, polysaccharides and glycoconjugates. They consist of protein domains that are classified into a hierarchy of families based on their structure and function, including Glycoside Hydrolases (GHs), GlycosylTransferases (GTs), Carbohydrate-Binding Module (CBMs), Carbohydrate Esterases (CEs), Polysaccharide Lyases (PLs) and Auxiliary Activity families (AAs) (Cantarel et al. 2009; Lombard et al. 2014). However, while CAZymes represent a useful tool for breaking down lignocellulosic biomass, they are generally not suited to the harsh conditions (especially extreme temperature) that form the basis of many industrial pre-treatments of lignocellulosic biomass (Blumer-Schuette et al. 2014). As one approach to overcoming the problem of enzyme functional stability, thermostable CAZymes have been identified in and isolated from hyperthermophilic and extremely thermophilic organisms [i.e., organisms that grow optimally at temperatures exceeding 700C (Leuschner and Antranikian 1995; Gerday and Glansdorff 2007)].
By mining genomes of extremely thermophilic organisms, it is possible to identify CAZymes that may operate effectively under the extreme conditions characteristic of industrial pre-treatments and other white biotechnological (i.e. industrial) applications. An added benefit of using extremely thermophilic CAZymes is that they may be expressed in planta with little fear of cytotoxicity, due to their very low activity at mesophilic temperatures (Mir et al. 2014). This approach can potentially reduce the need for exogenous enzyme loading, where accumulation of the expressed enzyme in the plant tissues produces ‘self-processing’ plants, potentially increasing the efficiency of lignocellulose degradation at high temperatures [without impacting the normal growth and development of the plant at mesophilic temperatures (Mir et al. 2014)]. The field of synthetic biology has a long history, and through innovations such as the iGEM competition and Biobricks foundation (Smolke 2009; Vilanova and Porcar 2014) has established a registry of ‘components’ (including promoters, ribosome binding sites, protein coding sequences and terminators, among others; http://parts.igem.org) for the rational design and programming of systems in living cells (Endy 2005; Hartwell et al. 1999; Purnick and Weiss 2009).
However, characterisation and incorporation of new individual components into the system remains an issue, and is a bottleneck to progress (Cameron et al. 2014). Most components are derived from wild-type systems and are limited in function. For example, a xylanase which binds to cellulose may be desirable, in order to digest xylan closely associated with cellulose to make it more accessible to enzymic degradation, but xylanase CBMs do not typically bind well to cellulose (McCartney et al. 2006). Nevertheless, the concept of engineering rationally designed multi-component CAZymes, potentially capable of targeting lignocellulose biopolymer backbones and accessory structures would appear to offer considerable promise for lignocellulosic biomass processing.
There have been a number of recent reviews summarising the industrial applications of extremely thermophilic CAZymes, either of specific enzyme classes (Atomi et al. 2011; Elleuche et al. 2015; Fatima and Hussain ; Nisha and Satyanarayana 2016) or with a focus on lignocellulose processing (Blumer-Schuette et al. 2014; Elleuche et al. 2014; Guerriero et al. 2015; Mir et al. 2014; Turumtay 2015; Urbieta et al. 2015). However, these reviews do not highlight the modular nature of hyperthermophilic CAZymes or how they might be used to tailor enzymes for lignocellulose deconstruction. In this review, we provide a survey of CAZyme modules with known lignocellulose degrading abilities derived from extremely thermophilic organisms, and explore how they might be applied in white biotechnology and enzyme engineering. We identify a list of predicted CAZyme domains (using publicly available proteomes from Ensemble Bacteria and HMMER protein domain prediction; Online Resource 1) from the proteomes of a selection of extremely thermophilic organisms (Figure 1.1, Online Resource 2). These domains cover a significant proportion of existing CAZyme families (Figure 1.2) and comprise a wide range of activities and substrate specificities, as summarised in the CAZy database [www.cazy.org (Lombard et al. 2014)].

TABLE OF CONTENTS :

• DECLARATION
• TABLE OF CONTENTS
• PREFACE
• ACKNOWLEDGEMENTS
• THESIS SUMMARY
• LIST OF TABLES
• CHAPTER LITERATURE REVIEW: Carbohydrate active enzyme domains from extreme thermophiles – components of a modular toolbox for lignocellulose degradation
  • 1.1 Abstract
  • 1.2 Keywords
  • 1.3 Introduction
  • 1.4 CAZyme categories
  • 1.5 Engineering CAZyme domains
  • 1.6 Applying the toolbox: Extremely thermophilic CAZymes for in planta lignocellulose degradation
  • 1.7 Conclusions
  • 1.8 Acknowledgements
  • 1.9 Conflict of interest
  • 1.10 Electronic Supplementary Material captions
  • 1.11 References
• CHAPTER Comparative analyses of hyperthermophile genomes reveals multiple lignocellulose degrading CAZyme domains
  • 2.1 Abstract
  • 2.2 Introduction
  • 2.3 Materials and Methods
    • 2.3.1 Acquisition of organism proteomes
    • 2.3.2 CAZyme domain identification and analysis
    • 2.3.3 Rarefaction curve
  • 2.4 Results
    • 2.4.1 Extremely thermophilic organisms have abundant GT and GH CAZyme domains
    • 2.4.2 Extremely thermophilic bacteria and archaea have unique CAZyme domain composition
    • 2.4.3 Lignocellulose binding and degrading CAZyme domains are prevalent in hyperthermophilic proteomes
      • 2.4.4 Low frequency CAZyme domains
      • 2.4.5 Overall coverage of CAZymes
  • 2.5 Discussion
    • 2.5.1 CAZyme abundance and diversity in archaea and bacteria
    • 2.5.2 Lignocellulose degrading capacity
    • 2.5.3 Future discovery
  • 2.6 Conclusion
  • 2.7 Acknowledgements
  • 2.8 References
• CHAPTER In planta expression of a chimeric xylanase in Arabidopsis shows targeting to the secondary cell wall but increases recalcitrance to enzymic degradation
  • 3.1 Abstract
  • 3.2 Introduction
  • 3.3 Materials and Methods
    • 3.3.1 Synthesis and cloning of Xyl22L
    • 3.3.2 Generation of transgenic Arabidopsis thaliana plants
    • 3.3.3 RT-qPCR analysis
    • 3.3.4 Plant phenotyping and microscopy
    • 3.3.5 Immunostaining and confocal microscopy
    • 3.3.6 Enzyme assays
    • 3.3.7 Dry weight measurements and biomass sugar release assays
    • 3.3.8 Plant protein extractions
    • 3.3.9 Western blotting
  • 3.3.10 Statistical tests
  • 3.4 Results
  • 3.4.1 Design of enzyme and generation transgenic plants
  • 3.4.2 Protein expression/accumulation in plants
  • 3.4.3 Phenotyping of plants
  • 3.4.4 Immunolocalisation
  • 3.4.5 Sugar release assays
  • 3.4.6 Synthesis of proteins and protein characterisation
  • 3.5 Discussion
  • 3.5.1 In planta expression of Xyl22L has no negative effect on plant growth
  • 3.5.2 Xyl22L accumulates in the plant and binds to the secondary cell wall
  • 3.5.3 Xyl22L increases recalcitrance of the secondary cell wall
  • 3.6 Conclusion
  • 3.7 Acknowledgements
  • 3.8 References
  • 3.8 Supplementary Tables and Figures
• CHAPTER Concluding Remarks
  • 4.1 Summary of findings
  • 4.2 Contributions of the thesis to current knowledge
  • 4.2 Limitations of the thesis
  • 4.3 Future work
  • 4.4 References

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