Reverse-and forward-engineering specificity of carbohydrate-processing enzymes
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This document summarizes a project to engineer plant cell wall degrading enzymes from Dickeya bacteria for enhanced biofuel production. The project aims to compile a library of diverse enzyme specificities by exploiting natural diversity found in Dickeya genomes and sequences. The project plan involves identifying candidate enzyme families from literature and sequence analysis, determining additional enzyme structures, and applying techniques like gene shuffling and saturated mutagenesis to generate novel diversity. The goal is to understand structure-function relationships and epistasis to enable rational engineering of enzyme specificity for new waste processing applications and biofuel production.
Reverse-and forward-engineering specificity of carbohydrate-processing enzymes
- 1. Reverse- and Forward- Engineering Specificity of Carbohydrate Processing Enzymes The James Hutton Institute 23rd March 2016 Leighton Pritchard, Sean Chapman, Tracey Gloster, Eirini Xemantilotou Information and Computational Sciences The James Hutton Institute
- 2. Acceptable Use Policy Recording of this talk, taking photos, discussing the content using email, Twitter, blogs, etc. is permitted (and encouraged), providing distraction to others during the presentation is minimised. These slides will be made available on SlideShare.
- 3. Project Plan Project Start: October 2015 Public data: genomes (NCBI) Public data: sequences (CAZy) Public data: structures (RCSB) Identify set of candidate diverse enzyme families: literature sequence analysis dN/dS Determine additional structures Candidate enzyme set Determine assays Directed evolution/gene shuffling Saturating single-site mutagenesis Screen specificity/ activity Library of diverse specificity Sectors/ epistatic pathways Novel synthetic pathways New waste processing applications Structure- function relationships Rational engineering of specificity Engineering of plant cell wall degrading enzymes for enhanced biocatalysis in biofuel produc8on REV 0.1 Project flowchart 20/3/2016 LP
- 4. Table of Contents Challenge: Food or Fuel, or Both? Food or Fuel? Microbial Energy Production Insight: Dickeya Why Dickeya? Action: Compiling a Library Natural Diversity Outcome: Exploiting Diversity Protein Sectors and Epistasis Project Plan Acknowledgements Without Whom. . .
- 5. Food or Fuel? a a Mohr & Rahman et al. (2013) Energy Policy doi:10.1016/j.enpol.2013.08.033 • Biofuels: ”Riches to Rags” • 1st generation: fuel from food crops • 2nd generation: fuel from cellulosic crops, e.g. miscanthus, willow • Stealing food, or stealing land/water?
- 6. Food and Fuel? a a Mohr & Rahman et al. (2013) Energy Policy doi:10.1016/j.enpol.2013.08.033 • Waste material = carbon-neutral feedstock • Agricultural waste as feedstock? • 2nd generation fuel from food crops? • Maize stover, straw, sugarcane bagasse, etc.
- 7. What’s in waste? a a Miedes et al. (2014) Front. Plant Sci. doi:10.3389/fpls.2014.00358 • Plant primary cell walls: largely carbohydrate • cellulose, hemicellulose, pectin, O-glycoprotein • Plant secondary cell walls: lignocellulosic • cellulose, xylan, lignin • lignocellulosic biomass: only feasible renewable resource for fuel/feedstock
- 8. Microbial Energy Research a a Torto-Alalibo et al. (2014) Front. Microbiol. doi:10.3389/fpls.2014.00358 • Engineered multi-enzyme processes • Production of advanced biofuels/chemical precursors • alcohols (butanol, isopropanol, etc.) • processing of isoprenoids, terpenes, fatty acids, etc. • Supplements/substitute for gasoline, diesel, jet fuel • Manufactured/stored/distributed by existing infrastructure
- 9. Table of Contents Challenge: Food or Fuel, or Both? Food or Fuel? Microbial Energy Production Insight: Dickeya Why Dickeya? Action: Compiling a Library Natural Diversity Outcome: Exploiting Diversity Protein Sectors and Epistasis Project Plan Acknowledgements Without Whom. . .
- 10. Why Dickeya a b a Ma et al. (2007) Phytopath. doi:10.1094/PHYTO-97-9-1150 b Toth et al. (2011) Plant Path. doi:10.1111/j.1365-3059.2011.02427.x • Dickeya spp.: group of significant Soft Rot Enterobacterial pathogens • Attacks ornamental and crop plants • Blackleg and stem rot • Diverse genus, wide host range • Diverse set of Plant Cell Wall Degrading Enzymes (PCWDEs)
- 11. Processing Waste a b c a Beall & Ingram (1993) J. Indust. Micro. 11:151-155 b Zhou et al. (1999) Appl. Environ. Microbiol. 65:2439-2445 c Edwards et al. (2011) Appl. Environ. Microbiol. doi:10.1128/AEM.05700-11 • Soft rot pathogens • Plant Cell Wall Degrading Enzymes (PCWDEs): hydrolases and lyases • Engineer pathogens for ethanol production? • Beall & Ingram, 1993 • Express PCWDEs in ethanologenic E. coli? • Zhou et al. 1999, Edwards et al. 2011 • PCWDE libraries for synthetic biology? • SynBio is a platform technology • automated platforms for engineered microbial pathways (e.g. Cellulect, UoEdinburgh) • understanding enzyme structure-function relationships
- 12. Function Space Functional space theoretically available to enzyme family structure/ sequence Functional space explored in nature
- 13. Table of Contents Challenge: Food or Fuel, or Both? Food or Fuel? Microbial Energy Production Insight: Dickeya Why Dickeya? Action: Compiling a Library Natural Diversity Outcome: Exploiting Diversity Protein Sectors and Epistasis Project Plan Acknowledgements Without Whom. . .
- 14. CAZy a a Lombard et al. (2014) Nucl. Acids Res. doi:10.1093/nar/gkt1178 • CAZy: Carbohydrate-Active Enzymes database (http://www.cazy.org/) • 5 Dickeya, 14 Erwinia, 9 Pectobacterium genomes • 63 Dickeya, 66 Erwinia, 74 Pectobacterium families • Survey/mine natural diversity of CAZymes
- 15. Pathogen Diversity a a Pritchard et al. (2016) Anal. Methods doi:10.1039/C5AY02550H • 48 Dickeya, 38 Erwinia, 57 Pectobacteria genomes • Survey/mine natural diversity of PCWDEs Pectobacterium_atrosepticum_SCRI1043_uid57957Pectobacterium_atrosepticum_NCPPB8549Pectobacterium_atrosepticum_NCPPB3404Pectobacterium_atrosepticum_21APectobacterium_atrosepticum_JG10-08Pectobacterium_carotovorum_PC1_uid59295Pectobacterium_carotovorum_subsp_carotovorum_NCPPB312Pectobacterium_carotovorum_subsp_oderiferum_NCPPB3841Pectobacterium_carotovorum_subsp_oderiferum_NCPPB3839Pectobacterium_carotovorum_subsp_carotovorum_NCPPB3395Pectobacterium_carotovorum_PCC21_uid174335Pectobacterium_carotovorum_subsp_brasiliensis_B5Pectobacterium_carotovorum_subsp_brasiliensis_B4Pectobacterium_betavasculorum_NCPPB2293Pectobacterium_betavasculorum_NCPPB2795Pectobacterium_wasabiae_NCPPB3702Pectobacterium_wasabiae_NCPPB3701Pectobacterium_wasabiae_WPP163_uid41297Pectobacterium_SCC3193_uid193707Dickeya_solani_AMYI01Dickeya_solani_AMWE01Dickeya_solani_GBBC2040Dickeya_solani_IPO2222Dickeya_solani_MK16Dickeya_solani_MK10Dickeya_dianthicola_NCPPB_3534Dickeya_dianthicola_GBBC2039Dickeya_dianthicola_NCPPB_453Dickeya_dianthicola_IPO980Dickeya_spp_NCPPB_3274Dickeya_spp_MK7Dickeya_dadantii_NCPPB_2976Dickeya_dadantii_NCPPB_898Dickeya_dadantii_NCPPB_3537Dickeya_dadantii_3937_uid52537Pantoea_ananatis_AJ13355_uid162073Pantoea_ananatis_LMG_20103_uid46807Pantoea_ananatis_PA13_uid162181Pantoea_ananatis_uid86861Erwinia_amylovora_CFBP1430_uid46839Erwinia_amylovora_ATCC_49946_uid46943Erwinia_Ejp617_uid159955Erwinia_pyrifoliae_Ep1_96_uid40659Erwinia_pyrifoliae_DSM_12163_uid159693Dickeya_dadantii_Ech703_uid59363Dickeya_paradisiaca_NCPPB_2511Dickeya_aquatica_DW_0440Dickeya_aquatica_CSL_RW240Erwinia_tasmaniensis_Et1_99_uid59029Pantoea_At_9b_uid55845Pantoea_vagans_C9_1_uid49871Erwinia_billingiae_Eb661_uid50547Dickeya_zeae_APMV01Dickeya_zeae_AJVN01Dickeya_zeae_CSL_RW192Dickeya_zeae_NCPPB_3531Dickeya_dadantii_Ech586_uid42519Dickeya_zeae_APWM01Dickeya_zeae_NCPPB_2538Dickeya_zeae_MK19Dickeya_zeae_NCPPB_3532Dickeya_spp_NCPPB_569Dickeya_chrysanthami_NCPPB_402Dickeya_chrysanthami_NCPPB_516Dickeya_zeae_Ech1591_uid59297Dickeya_chrysanthami_NCPPB_3533 Pectobacterium_atrosepticum_SCRI1043_uid57957Pectobacterium_atrosepticum_NCPPB8549Pectobacterium_atrosepticum_NCPPB3404Pectobacterium_atrosepticum_21APectobacterium_atrosepticum_JG10-08Pectobacterium_carotovorum_PC1_uid59295Pectobacterium_carotovorum_subsp_carotovorum_NCPPB312Pectobacterium_carotovorum_subsp_oderiferum_NCPPB3841Pectobacterium_carotovorum_subsp_oderiferum_NCPPB3839Pectobacterium_carotovorum_subsp_carotovorum_NCPPB3395Pectobacterium_carotovorum_PCC21_uid174335Pectobacterium_carotovorum_subsp_brasiliensis_B5Pectobacterium_carotovorum_subsp_brasiliensis_B4Pectobacterium_betavasculorum_NCPPB2293Pectobacterium_betavasculorum_NCPPB2795Pectobacterium_wasabiae_NCPPB3702Pectobacterium_wasabiae_NCPPB3701Pectobacterium_wasabiae_WPP163_uid41297Pectobacterium_SCC3193_uid193707Dickeya_solani_AMYI01Dickeya_solani_AMWE01Dickeya_solani_GBBC2040Dickeya_solani_IPO2222Dickeya_solani_MK16Dickeya_solani_MK10Dickeya_dianthicola_NCPPB_3534Dickeya_dianthicola_GBBC2039Dickeya_dianthicola_NCPPB_453Dickeya_dianthicola_IPO980Dickeya_spp_NCPPB_3274Dickeya_spp_MK7Dickeya_dadantii_NCPPB_2976Dickeya_dadantii_NCPPB_898Dickeya_dadantii_NCPPB_3537Dickeya_dadantii_3937_uid52537Pantoea_ananatis_AJ13355_uid162073Pantoea_ananatis_LMG_20103_uid46807Pantoea_ananatis_PA13_uid162181Pantoea_ananatis_uid86861Erwinia_amylovora_CFBP1430_uid46839Erwinia_amylovora_ATCC_49946_uid46943Erwinia_Ejp617_uid159955Erwinia_pyrifoliae_Ep1_96_uid40659Erwinia_pyrifoliae_DSM_12163_uid159693Dickeya_dadantii_Ech703_uid59363Dickeya_paradisiaca_NCPPB_2511Dickeya_aquatica_DW_0440Dickeya_aquatica_CSL_RW240Erwinia_tasmaniensis_Et1_99_uid59029Pantoea_At_9b_uid55845Pantoea_vagans_C9_1_uid49871Erwinia_billingiae_Eb661_uid50547Dickeya_zeae_APMV01Dickeya_zeae_AJVN01Dickeya_zeae_CSL_RW192Dickeya_zeae_NCPPB_3531Dickeya_dadantii_Ech586_uid42519Dickeya_zeae_APWM01Dickeya_zeae_NCPPB_2538Dickeya_zeae_MK19Dickeya_zeae_NCPPB_3532Dickeya_spp_NCPPB_569Dickeya_chrysanthami_NCPPB_402Dickeya_chrysanthami_NCPPB_516Dickeya_zeae_Ech1591_uid59297Dickeya_chrysanthami_NCPPB_3533 0.00 0.25 0.50 0.75 1.00 ANIm_percentage_identity
- 16. Protein Structures a b a Chapon et al. (2001) J. Mol. Biol. doi:10.1006/jmbi.2001.4787 b Larson et al. (2003) Biochem. doi:10.1021/bi034144c • Several landmark enzyme structures from Dickeya • First GH5 xylanase structure (1NOF) • Cel5 (1EGZ) • Obtain novel structures for Dickeya CAZymes
- 17. Project Plan Project Start: October 2015 Public data: genomes (NCBI) Public data: sequences (CAZy) Public data: structures (RCSB) Identify set of candidate diverse enzyme families: literature sequence analysis dN/dS Determine additional structures Candidate enzyme set Determine assays Directed evolution/gene shuffling Saturating single-site mutagenesis Screen specificity/ activity Library of diverse specificity Sectors/ epistatic pathways Novel synthetic pathways New waste processing applications Structure- function relationships Rational engineering of specificity Engineering of plant cell wall degrading enzymes for enhanced biocatalysis in biofuel produc8on REV 0.1 Project flowchart 20/3/2016 LP
- 18. Table of Contents Challenge: Food or Fuel, or Both? Food or Fuel? Microbial Energy Production Insight: Dickeya Why Dickeya? Action: Compiling a Library Natural Diversity Outcome: Exploiting Diversity Protein Sectors and Epistasis Project Plan Acknowledgements Without Whom. . .
- 19. Generate Diversity • Gene shuffling/directed evolution • Saturating site-directed mutagenesis Functional space theoretically available to enzyme family structure/sequence Functional space explored in nature
- 20. Gene Shuffling a a Crameri et al. (1998) Nature doi:10.1038/34663 • Generate novel diversity and select for substrate specificity
- 21. Positional epistasis a a McLaughlin et al. (2012) Nature doi:10.1038/nature11500 • Context-dependence of mutation and function: epistasis • “hotspots”/pathways for control of substrate specificity • Saturated single substitutions, with substrate assay screens
- 22. Protein Sectors a b a Pritchard & Dufton (2000) J. Theor. Biol. doi:10.1006/jtbi.1999.1043 b Halabi et al. (2009) Cell doi:10.1016/j.cell.2009.07.038 • Sequence diversity and protein structure enables: • Correlated mutation/conservation analysis • Decomposition of structure into ”sectors” • Sectors: subdomain structural/functional organisation of proteins
- 23. Project Plan Project Start: October 2015 Public data: genomes (NCBI) Public data: sequences (CAZy) Public data: structures (RCSB) Identify set of candidate diverse enzyme families: literature sequence analysis dN/dS Determine additional structures Candidate enzyme set Determine assays Directed evolution/gene shuffling Saturating single-site mutagenesis Screen specificity/ activity Library of diverse specificity Sectors/ epistatic pathways Novel synthetic pathways New waste processing applications Structure- function relationships Rational engineering of specificity Engineering of plant cell wall degrading enzymes for enhanced biocatalysis in biofuel produc8on REV 0.1 Project flowchart 20/3/2016 LP
- 24. Anticipated Outputs • Libraries of carbohydrate-processing enzymes for SynBio • Survey of natural PCWDE diversity • Novel specificity variants from gene shuffling • Saturated site-specific mutagenesis libraries for screening against novel substrates • IP? • Structure-function insight • New PCWDE enzyme structures • Sector and epistasis maps for PCWDEs • Reverse-engineering of carbohydrate-processing enzymes • Empirically-improved enzymes • Gene-shuffling targeted to novel specificity • Forward-engineering of carbohydrate-processing enzymes • IP?
- 25. Table of Contents Challenge: Food or Fuel, or Both? Food or Fuel? Microbial Energy Production Insight: Dickeya Why Dickeya? Action: Compiling a Library Natural Diversity Outcome: Exploiting Diversity Protein Sectors and Epistasis Project Plan Acknowledgements Without Whom. . .
- 26. Acknowledgements Project Eirini Xemantilotou (UoStA/JHI) Sean Chapman (JHI) Tracey Gloster (UoStA) Judith Huggan (IBioIC) JHI Sonia Humphris Emma Campbell Ian Toth