Presentation for James Hutton Institute Synthetic Biology Workshop, 23/3/2016

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