IN SILICO MINING OF THE NAMIB DESERT HYPOLITH METAGENOMIC DATASET

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INTRODUCTION

The use of renewable sources of energy as alternatives to fossil fuels is the focus of world efforts at sustainable energy development. Consequently, lignocellulosic biomass has been harnessed as a renewable feedstock in the production of biofuels. However, lignocellulosic biomass is recalcitrant to complete bioconversion due to certain structural complexities such as acetylation. Acetyl xylan esterases (AcXEs) are Carbohydrate-Active Enzymes (CAZy) that hydrolyse ester bonds to liberate acetic acid in acetylated polymeric xylan and xylooligosaccharides. They belong to Carbohydrate Esterase 1-7 and 16 families and are known to act on a variety of substrates. Although, several AcXEs have been identified from a range of lignocellulose-degrading microorganisms, saccharification remains a major bottle-neck during industrial biomass-to-biofuel conversions. Metagenomic screening methods allow access to novel metabolites/gene products of the ≥90% uncultured microorganisms within any given environmental sample. This review provides comprehensive information on the phylogeny and ambiguity of bioinformatic annotations of AcXEs. Furthermore, it synthesizes available information on the classification, metagenomic bioprospecting of unique extremophilic environments, large-scale screening limitations and industrial applications of AcXEs. This will facilitate knowledge about this class of accessory enzymes for potential improvements in current and developing AcXE bioprospecting and/or engineering technologies.

Acetylated xylan

Many plants have evolved mechanisms for protection of their tissues from physical and biochemical attack (e.g., from insects, microorganisms and enzymes) [14,15]. Such mechanisms include acetylation and ‘feruloyation’ of plant cell polymers (xylan, man- nan, pectin, peptidoglycan and chitin), thereby reducing the efﬁciency of polysaccharide hydrolases [4,5]. Acetylation occurs mainly in hardwood (acetyl glucuronoxylan), typically at the C2 or C3 or both positions, depending on whether it is a mono- or di-O-acetylated xylopyranosyl unit (Fig. 3). On non-reducing xylopyranosyl residues of oligosaccharides or xylopyranosides, acetyl groups can migrate to position C4 [5,16]. The migration is accelerated by increased pH and temperature [17]. The migration between position 2 and 3 on internal xylopyranosyl residues in the polysaccharide is also anticipated, however it has not been proven and demonstrated experimentally. Other substitutions, such as C5 acetylation of the L-arabinofuranosyl side chain residues of xyloglucan, have also been reported [18].

Positional speciﬁcity of AcXEs

In addition to substrate speciﬁcity, AcXEs exhibit positional speciﬁcity (regioselectivity) where a speciﬁc carbon position is preferably deacetylated over others [95]. The positional speci- ﬁcities of AcXEs and other CEs have been investigated using techniques capable of monitoring the release of unique acetate groups from acetyl xylan: there include matrix assisted laser desorption/ionisation time of ﬂight (MALDI-TOF) mass spectrom- etry [62], enzyme coupled assays [96], proton-NMR (1H NMR) [66,67,78] and capillary electrophoresis with laser-induced ﬂuo- rescence (CE-LIF) [62].

Future prospects and conclusion

It is argued that the need for improvements in the efﬁciency of industrial scale biodegradation of plant biomass necessitates continuous bioprospecting for novel lignocellulose-hydrolysing enzymes [10]. One of the areas where signiﬁcant advances are possible is considered to be the exploitation of synergies derived from use of accessory enzymes (such as AcXEs) in conjunction with endo-acting cellulases and hemicellulases [2,29,45,107,167–169]. Options for identiﬁcation of novel (and possibly superior) variants of such enzymes include the use of new screening methodolo- gies, and the targeting of under-explored biological communities. Microbial communities within unique (and extremophilic) eco- logical niches, such as haloalkaline lacustrine habitats, acid-mine drainage, hot and cold deserts soils, etc., are valid targets for metagenomic bioprospecting for novel AcXEs.

Declaration
Acknowledgements
ABSTRACT
CHAPTER ONE: LITERATURE REVIEW METAGENOMIC BIOPROSPECTING OF MICROBIAL ACETYL XYLAN ESTERASES
1.0 INTRODUCTION
1.1 Article: Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases
1.2 SUMMARY AND CONCLUSIONS
CHAPTER TWO IN SILICO MINING OF THE NAMIB DESERT HYPOLITH METAGENOMIC DATASET FOR NOVEL AcXE-ENCODING GENES
2.0 INTRODUCTION
2.1 MATERIALS AND METHODS
2.1.1 Retrieval of AcXE protein sequence homologs
2.1.2 Interrogation of the Namib hypolith metagenomic dataset for AcXE-encoding genes
2.1.2.1 E-values and sequence length
2.1.2.2 Contig analysis for protein coding regions
2.1.3 Conserved domain search and filter
2.1.4 Confirmation of selected domain function with NCBI curated models
2.1.5 PCR mining of the Namib Desert soil metagenomic libraries and sequencing
2.1.6 Gene synthesis and primer design
2.2 RESULTS AND DISCUSSION2.2.1 AcXE protein sequence homologs per CE family
2.2.2 AcXE-encoding gene hits within the Namib hypolith metagenomic dataset
2.2.3 E-value and sequence length filter outputs
2.2.4 Protein coding regions hits with full open reading frames (ORFs)
2.2.5 AcXE conserved domain hits
2.2.6 NCBI curated models and confirmation of AcXE domain function
2.2.7 Nucleotide and protein sequence analyses
2.2.8 PCR mining and sequencing of putative novel AcXE-encoding genes
2.2.9 Synthesis and amplification of putative AcXE-encoding genes
2.3 CONCLUSION
2.4 REFERENCES
CHAPTER THREE EXPRESSION, PURIFICATION AND FUNCTIONAL CHARACTERISATION OF NOVEL AcXEs
3.0 INTRODUCTION
3.1 MATERIALS AND METHODS
3.1.1 Materials
3.1.2 Sub-cloning and transformation of putative novel AcXEs
3.1.3 Gene expression and protein production
3.1.4 Protein isolation and purification
3.1.5 Polyacrylamide gel electrophoresis and Western blotting
3.1.6 Preparation of acetylated xylan
3.1.7 Functional characterization of NaM1
3.1.7.1 Determination of molar absorption coefficients (ε)
3.1.7.2 Effects of temperature and pH
3.1.7.3 Effects of stabilizing solutes
3.1.7.4 Effects of metal ions and chemical agents
3.1.7.5 Substrate specificity studies
3.2 RESULTS AND DISCUSSION
3.2.1 Cloning, expression and purification
3.2.2 Functional Characterization of NaM1
3.2.2.1 pH ‘optimum’ and stability
3.2.2.2 Temperature ‘optimum’ and stability
3.2.2.3 Thermal inactivation profile
3.2.2.4 Effects of stabilizing solutes on activity and thermal stability
3.2.2.5 Effects of di-valent metal ions and chemical agents
3.2.2.6 Substrate specificity and enzyme kinetics
3.2.3 Database accession numbers
3.3 CONCLUSION
3.4 REFERENCES
CHAPTER FOUR STRUCTURAL CHARACTERISATION OF NaM1
4.0 INTRODUCTION
4.1 MATERIALS AND METHODS
4.1.1 Crystallization and data collection
4.2 RESULTS AND DISCUSSION
4.2.1 Crystallization
4.2.2 Data collection and processing
4.2.4 Structure of NaM1
4.2.4.1 Active site and oxyanion hole
4.2.4.2 Inter-subunit interactions
4.2.4.3 Non-physiological interactions
4.2.4.4 Comparison with other CE7 esterase structures
4.2.4.5 Substrate binding site
4.2.5 Structural basis for substrate specificity
4.2.6 Structural basis for thermostability
4.2.7 Database accession number
4.3 CONCLUSION
4.4 REFERENCES
CHAPTER FIVE PROTEIN ENGINEERING: DIRECTED EVOLUTION AND SITE DIRECTED MUTAGENESIS OF NaM1
5.0 INTRODUCTION
5.1 MATERIALS AND METHODS
5.1.1 Error-prone and site-directed mutagenesis PCR conditions
5.1.2 Cloning, transformation and mutant library construction
5.1.3 EpPCR mutant library screening
5.1.3.1 Cultivation, pre-culture and expression
5.1.3.2 Thermal stability screen
5.1.4 Confirmation of thermal stability of selected mutants
5.1.5 Purification and functional characterization of selected NaM1 variants
5.1.6 Kinetics of NaM1 wild type and selected variants
5.1.7 Fluorescence and Circular Dichroism Spectroscopy
5.1.8 Sequence analyses and structural modelling of mutants
5.2 RESULTS AND DISCUSSION
5.2.1 Error-prone PCR, cloning, transformation and library construction
5.2.2 Library analyses and screening
5.2.3 Thermal stability and inactivation assays
5.2.3.1 Directed evolution variants
5.2.3.2 Site-directed mutagenesis variants
5.2.4 Optimal activity assays of H2
5.2.5 Kinetics of NaM1 and thermostable variants
5.2.6 Thermostability and acetylated xylan specificity
5.2.7 Fluorescence spectra of NaM1 and H2
5.2.8 Thermal-induced unfolding of NaM1 and H2
5.2.9 Sequence and structure to function analyses of mutants
5.2.9.1 F210L
5.2.9.2 N96S
5.2.9.3 T94A
5.2.9.4 N228D
5.2.9.5 T306P
5.2.9.6 E11A
5.3 CONCLUSION
5.4 REFERENCES
CHAPTER SIX
6.0 GENERAL CONCLUSIONS AND RECOMMENDATIONS
6.1 REFERENCES