Mining saltmarsh sediment microbes for enzymes to degrade recalcitrant biomass

Juliana Sanchez Alponti

PhD

University of York

Biology
September 2019

Abstract

Abstract

The recalcitrance of biomass represents a major bottleneck for the efficient production of fermentable sugars from biomass. Cellulase cocktails are often only able to release 75-80% of the potential sugars from biomass and this adds to the overall costs of lignocellulosic processing. The high amounts of fresh water used in biomass processing also adds to the overall costs and environmental footprint of this process. A more sustainable approach could be the use of seawater during the process, saving the valuable fresh water for human consumption and agriculture. For such replacement to be viable, there is a need to identify salt tolerant lignocellulose-degrading enzymes. We have been prospecting for enzymes from the marine environment that attack the more recalcitrant components of lignocellulosic biomass. To achieve these ends, we have carried out selective culture enrichments using highly degraded biomass and inoculum taken from a saltmarsh. Saltmarshes are highly productive ecosystems, where most of the biomass is provided by land plants and is therefore rich in lignocellulose. Lignocellulose forms the major source of biomass to feed the large communities of heterotrophic organisms living in saltmarshes, which are likely to contain a range of microbial species specialised for the degradation of lignocellulosic biomass. We took biomass from the saltmarsh grass Spartina anglica that had been previously degraded by microbes over a 10-week period, losing 70% of its content in the process. This recalcitrant biomass was then used as the sole carbon source in a shake-flask culture inoculated with saltmarsh sediment. Cultures were grown for 8 weeks and then analysed using meta-omic approaches. Meta-genomics were used to investigate the microbial community present in the final recalcitrant biomass, while combined meta-proteomics and meta-transcriptomics were used to identify putative CAZymes (Carbohydrate active enzymes). Candidate enzymes have been cloned, heterologous expressed in E. coli and characterized according to their salt tolerance.
List of contents

List of contents

Abstract ............................................................................................................................................ 2 List of contents.................................................................................................................................. 3 List of tables ...................................................................................................................................... 6 List of figures..................................................................................................................................... 7 Acknowledgments............................................................................................................................. 9 Declaration...................................................................................................................................... 11 Chapter 1 General introduction ....................................................................................................... 12
1.1 Lignocellulose biomass........................................................................................................... 12 1.2 What makes lignocellulose biomass difficult to digest? ........................................................... 12
1.2.1 Cellulose.......................................................................................................................... 13 1.2.2 Hemicellulose.................................................................................................................. 14 1.2.3 Lignin .............................................................................................................................. 18 1.2.4 Pectins ............................................................................................................................ 19
1.3 CAZymes ................................................................................................................................ 21
1.3.1 Enzymatic cellulose degradation...................................................................................... 22 1.3.2 Enzymatic hemicellulose degradation .............................................................................. 24
1.4 Challenges for the use of seawater in biorefineries ................................................................. 27 1.5 Saltmarshes are source of salt tolerant enzymes..................................................................... 30 1.6 Aims of this project ................................................................................................................ 30
Chapter 2 Materials and Methods ................................................................................................... 32
2.1 2.2
Chemicals and reagents.................................................................................................... 32 Production of recalcitrant biomass................................................................................... 32
Initial recalcitrant biomass production ...................................................................... 32 Final recalcitrant biomass and weight loss................................................................. 32
Biomass composition analysis .......................................................................................... 33
Lignin content........................................................................................................... 33 Hemicellulose content .............................................................................................. 33 Crystalline cellulose content...................................................................................... 34
Meta-“omics” approaches................................................................................................ 34

Combined genomic DNA (gDNA) and total RNA extraction......................................... 34 DNA preparation for meta-genomics and DNA sequencing ........................................ 35 Bioinformatic analysis and microbial community profile pipeline ............................... 39 RNA preparation for meta-transcriptomics and RNA sequencing ............................... 41
2.2.1 2.2.2

2.3

2.3.1 2.3.2 2.3.3

2.4

2.4.1 2.4.2 2.4.3 2.4.4

• 2.4.5
• Protein extraction and extracellular protein purification............................................ 43

3
List of contents

2.4.6

Proteomic analysis.................................................................................................... 45
Molecular Biology techniques .......................................................................................... 46
Host organisms for cloning and protein expression.................................................... 46 Media....................................................................................................................... 47 Polymerase Chain reaction (PCR)............................................................................... 48 Agarose gel electrophoresis ...................................................................................... 53 DNA purification ....................................................................................................... 54 Gene cloning using StrataClone technology............................................................... 54 Colony screening by colony PCR ................................................................................ 56 Plasmid DNA extraction and Sanger sequencing ........................................................ 57 Nucleic acid quantification........................................................................................ 57
2.5

2.5.1 2.5.2 2.5.3 2.5.4 2.5.5 2.5.6 2.5.7 2.5.8 2.5.9

2.5.10 DNA sequencing (Sanger sequencing) ....................................................................... 57

2.5.11 Subcloning into the expression vector pet52b+ using In-Fusion HD cloning................ 58

• 2.6
• Recombinant protein expression...................................................................................... 61

Competent cells transformation................................................................................ 61 Bacterial protein expression...................................................................................... 61

Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis (SDS-PAGE) ............... 62

Western Blot (WB) analysis ....................................................................................... 63 Protein quantification by Bradford ............................................................................ 63
Protein purification .......................................................................................................... 64
Affinity chromatography ........................................................................................... 64 Protein concentration............................................................................................... 64 Gel filtration chromatography................................................................................... 64
Characterization of soluble targets................................................................................... 64
Reagents and substrates ........................................................................................... 65 Determination of optimum pH.................................................................................. 66 Determination of optimum temperature................................................................... 66 Influence of seawater in the optimum temperature .................................................. 67 Salt tolerance against NaCl........................................................................................ 67

2.6.1 2.6.2 2.6.3 2.6.4 2.6.5

2.7

2.7.1 2.7.2 2.7.3

2.8

2.8.1 2.8.2 2.8.3 2.8.4 2.8.5

Chapter 3 Production of the recalcitrant biomass and its compositional analysis............................. 68
3.1 Introduction........................................................................................................................... 68 3.2 Aims of the chapter ............................................................................................................... 69 3.3 Results and discussion ........................................................................................................... 69
Chapter 4 Selection of putative CAZymes through combined proteomic and transcriptomic analysis informed by microbial community profiling..................................................................................... 75

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List of contents
4.1 Introduction........................................................................................................................... 75 4.2 Aims of the chapter ............................................................................................................... 76 4.3 Results and discussion ........................................................................................................... 76
4.3.1 Combined genomic DNA and total RNA extraction ........................................................... 76

4.3.2 DNA preparation for meta-genomics and DNA sequencing............................................... 79 4.3.3 RNA preparation for meta-transcriptomics and RNA sequencing ...................................... 80 4.3.4 Protein extraction and extracellular protein purification................................................... 82

4.3.5 Protein annotation .......................................................................................................... 83 4.3.6 Bacterial community profile............................................................................................. 97 4.3.7 Selection of putative CAZymes for further study............................................................. 102
Chapter 5 Cloning and heterologous protein production of selected putative CAZymes................. 109
5.1 Introduction......................................................................................................................... 109 5.2 Aims of the chapter ............................................................................................................. 110 5.3 Results and discussion ......................................................................................................... 110
5.3.1 Sequence analysis and preparation for cloning............................................................... 110 5.3.2 Cloning.......................................................................................................................... 111 5.3.3 Subcloning into expression vector pet52b+.................................................................... 114 5.3.4 Recombinant protein production................................................................................... 117 5.3.5 Protein purification........................................................................................................ 119
Chapter 6 Enzyme characterisation, influence of seawater and influence of salt concentration ..... 122
6.1 Introduction......................................................................................................................... 122 6.2 Aims of the chapter ............................................................................................................. 123 6.3 Results and discussion ......................................................................................................... 123
6.3.1 Characterisation of a putative GH51 - clone 8GH51........................................................ 124 6.3.2 Characterisation of a putative GH3 - clone 14GH3.......................................................... 128 6.3.2 Characterisation of a putative CE1 - clone 34CE1 ........................................................... 131
Chapter 7 Final discussion ............................................................................................................. 138
7.1 General discussion............................................................................................................... 138 7.2 Future work ......................................................................................................................... 144
Appendices.................................................................................................................................... 146 List of abbreviations ...................................................................................................................... 149 References .................................................................................................................................... 152

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List of tables

List of tables

TABLE 1.1 AVERAGE COMPOSITION OF SEAWATER...................................................................................... 29 TABLE 2.1 PRIMERS USED FOR THE PCR AMPLIFICATION OF 16S RRNA. ......................................................... 37 TABLE 2.2 PCR CONDITIONS FOR 16S RRNA AMPLIFICATION USING 515F-Y AND 806R PRIMERS. ....................... 37

TABLE 2.3 ILLUMINA INDEX PRIMER ADAPTERS USED FOR EACH ONE OF THE 16S RRNA AMPLICONS...................... 38 TABLE 2.4 PCR CONDITIONS FOR THE INCLUSION OF ILLUMINA ADAPTERS TO 16S RRNA AMPLICONS.................... 38

TABLE 2.5 COMMANDS USED FOR THE ANALYSIS OF 16S RRNA AMPLICON DATABASE........................................ 40

TABLE 2.6 LIST OF PRIMERS USED FOR THE AMPLIFICATION OF THE GENES TARGETS............................................ 49

TABLE 2.7 PCR CONDITIONS FOR THE AMPLIFICATION OF THE GENES TARGETS.................................................. 53 TABLE 2.8 PCR CONDITIONS FOR THE COLONY PCR.................................................................................... 56 TABLE 2.9 PRIMERS USED FOR COLONY PCR REACTIONS. ............................................................................. 56 TABLE 2.10 PCR CONDITIONS FOR THE PLASMID LINEARIZATION.................................................................... 59