Bacterial Enzymes for Lignin Degradation: Generating Renewable Chemicals from Lignin

Prof. Tim Bugg Department of Chemistry, University of Warwick

OM e MeO OH OM e OH RO OH OMe HO OH O O O O OH OMe OMe HO O O OH OH OM e OM e OM e HO OM e

phenylcoumarane β- a r y l e t h e r p in or e si n o l di a ry l p ro p an e bi p h en yl R OH OH OH Me O CO 2 H HO HO O R O OM e HO OH OMe HO M eO OM e O OH O OMe OMe HO 2 C OM e OH OH OMe OM e OH OH 1 ) de me t hy l a se pe r ox i d as e 2 ) ex t r a d io l l ya s e H C H O d io xy g e na s e

CH O lignostilbene MeO CO H 2 d io xy g e n as e HO OH HO OM e Me O OH OM e C- C h y d ro l as e O HO C a ld eh y de CO 2 H 2 dehydrogenase MeO CO2H HO CO H de c a rb o xy l as e 2 OM e HO OH CO 2 H CO2H OH CO2H O d e me t hy l a s e h yd r a ta s e CO H HO CO H d ec a rb ox y l as e 2 2 CO 2 H CO H extradiol 2 CO H CO H aldolase 2 a ld e hy d e 2 di ox y g e na s e d e h yd r o ge na s e C O 2 O OH OH C OH HO O CO H 2 x HO 2 C 2 O O CO 2 H OH CO 2 H Lignocellulose-Based Bio-refinery Concept

2nd Generation Bioethanol Process

Thermochemical Biomass pretreatment

CO2H CHO Lignin Cellulose Hemi-cellulose

OMe OMe cellulases xylanases OH OH Glucose Xylose Vanillin Ferulic acid 14-15 $/kg 85-100 $/kg fermentation Polylactic acid Bio-based polymer $3-6/kg Ethanol

distillation Organic Lactic acid 1.3-1.6 $/kg Acids Succinic acid 6-9 $/kg Biofuel EtOH 0.5-1 $/litre Molecular Structure of Lignin

• High molecular weight aromatic polymer

• Heterogenous structure, C C C O highly cross-linked C C OCH3 C O • Contains aryl-C3 units: HOCH OCH3 HOCH 2 2 H2C CH HC O HC OCH3 HC CH HOCH HC O HC CH2 • Different substitution patterns O in different plant types: OCH OCH 3 3 OCH3 OH C C C OH OH C C C β-aryl ether phenylcoumarane pinoresinol C C C C C C C C OCH3 H3CO OCH3 C C C OH OH OH C C C guaiacyl unit (G) syringyl unit (S) p-hydroxyphenyl unit (H) C

OCH3 CH O OCH O 3 3 CH3O OH OH OH Potential source of aromatic products biphenyl diaryl ether Fungal Enzymes for Lignin Oxidation Lignin Manganese Versatile Laccase peroxidase peroxidase peroxidase P. chrysosporium P. chrysosporium Pleurotus eryngii Trametes versicolor

Heme Fe enzyme Heme Fe enzyme Heme Fe enzyme Multi-copper enzyme Oxidant H2O2 Oxidant H2O2 Oxidant H2O2 Oxidant O2

Able to oxidise Oxidises Mn(II) to Oxidises Mn(II) or Able to oxidise wide range Lignin model cpds Mn(III), oxidant for lignin of phenols, using redox Cα-Cβ cleavage lignin mediator Cα oxidation

Bacteria – Ramachandra et al., Appl. Env. Microbiol. 1988, 54, 3057 report that Streptomyces viridosporus contains extracellular lignin peroxidase activity, but no gene identified. Reports that strains of Nocardia, Rhodococcus have lignin oxidation ability. Possible advantages: easier protein expression, genetic tools available, thermophilic enzymes? Identification of Rhodococcus jostii Peroxidase DypB

HO HO CHO lignin C(NO2)4 degrader

or H3CO H3CO NO2 H3CO NO2 HNO OH 3 OH OH AcOH lignin nitrated lignin Increase in A430

M. Ahmad et al, Mol. Biosystems, 2010, 6, 815-821

2 unannotated peroxidase genes found in R. jostii genome, Recombinant DypB is active ΔdypB R. jostii mutant shows loss of activity: in nitrated MWL assay

RHA WT ∆dypB ∆dypA ∆mco

0.008

0.006

0.004

0.002

0

-0.002

-0.004

-0.006

!plus H2O 2 -0.008 !minus H2O 2 -0.01 Catalytic cycle for DypB • formation of compound I (397 nm) and compound II (417 nm) observed using stopped flow kinetics

• rate constants measured for oxidation of β-aryl ether and Mn(II)

• Crystal structure of R. jostii DypB determined in collaboration with Prof. L. Eltis (UBC, Canada)

M. Ahmad, E.M. Hardiman et al., Biochemistry, 2011, 50, 5096 J.N. Roberts et al., Biochemistry, 2011, 50, 5108. Screening for Novel Lignin Degrading Strains

Plating

Culturing – selective medium Nitrated lignin Sampling spray assay

Pure cultures

Plate assay • Identification bacterial species (e.g.16S rRNA) • Purify and identify extracellular enzymes • Recombinant expression Identification of Isolates Obtained from Screening

9 mesophilic isolates identified from soil enrichment 2 thermotolerant isolates from screening of composted wheat straw at 45 oC

Strain Identification from 16S rRNA Bacterial Activity in nitrated sequencing family lignin assay (mAU)

+ H2O2 -H2O2 A1.1 Microbacterium phyllosphaerae Actinobacteria 1.9 2.7 A1.2 Microbacterium marinilacus Actinobacteria 1.7 0.6 A2.1 Microbacterium marinilacus Actinobacteria 0.9 1.4 A4.3 Ochrobactrum pseudogrignonense α-Proteobacteria 1.2 4.7 A5.1 Rhodococcus erythropolis Nocardioform 1.6 1.9 A5.2 Microbacterium oxydans Actinobacteria 1.1 5.4 B5.3 Micrococcus luteus Actinobacteria 1.9 1.1 C4.1 Ochrobactrum rhizosphaerae α-Proteobacteria 2.0 1.9 E1.1 Micrococcus luteus Actinobacteria 0.9 1.1

T1 Rhizobiales sp. α-Proteobacteria 0.2 2.0 T2 Sphingobacterium sp. Bacteroides 9.0 30

C.R. Taylor, E.M. Hardiman et al., J. Appl. Microbiol., 2012, 113, 521. Possible Enzyme Activities from Sphingobacterium sp. T2 Genome Sequence 3.2 Mb genome No dyp-type peroxidase! Predicted peroxidases: • Cytochrome c peroxidase • Glutathione peroxidase • Thiol peroxidase • Catalase/peroxidase • Catalase Proteomic data from extracellular fracons acve in nitrated lignin assay:

1000,00 Mr (kDa) Predicted funcon Source organism 900,00 nitrated MWL without hydrogen peroxide 800,00 ABTS without hydrogen peroxide 52.9 700,00 OmpA/MotB outer membrane protein Sphingobacterium spirivorum 57.1 -1 Oligoendopepdasenitrated MWL with hydrogen F peroxide Sphingobacterium spirivorum 600,00mg 42.9 -1 Idh/MocAABTS periplasmicwith hydrogen peroxide oxidoreductase Sphingobacterium spirivorum

500,00min 25.5 Binuclear metallohydrolase Sphingobacterium spirivorum 23.7 400,00 Peroxiredoxin Sphingobacterium spirivorum 50.6 300,00 Possible M20 pepdase Sphingobacterium spirivorum 22.3 200,00 Superoxide dismutase (Mn) Sphingobacterium spirivorum 26.0 100,00 Thioredoxin Sphingobacterium spirivorum 0,00 32.0 mAU / activity Specific Thrombospondin repeat protein Pedobacter saltans 25.3 Superoxide dismutase (Fe/Mn) Chryseobacterium gleum Fractions of purified enzyme Reaction of Sphingobacterium MnSOD with Organosolv lignin

Reaction of MnSOD with Organosolv Lignin + Enzyme -Enzyme Aromatic products detected by LC-MS:

CO2H CO2H CO2H CO2H CHO

OMe OH HO OMe OMe OH OH OH OH OH 1 2 3 4 5

HO OH *DAD1 A, Sig=310,16 Ref =600,100 (GORAN\SOD20034.D) O O *DAD1 A, Sig=310,16 Ref =600,100 (GORAN\SOD20038.D) *DAD1 A, Sig=310,16 Ref =600,100 (GORAN\SOD20042.D) *DAD1 A, Sig=310,16 Ref =600,100 (GORAN\SOD20046.D) OMe OMe OMe OH HO OH mAU OH OH OH OH 6 7 8 9 10

20

OH

HO 15 + enz 5hr O OH OMe SpMnSOD1 + KO2 OMe OMe HO 10 + enz 1hr OH OH OMe

5

0

0 5 10 15 20 25 min

G.M.M. Rashid et al, ACS Chem Biol 2015, 10, 2286-2294 How could Superoxide Dismutase Attack Lignin?

superoxide peroxide - 2- O2 O2

Catalytic cycle 2+ Mn3+ attacks lignin? of MnSOD Mn

- O2 O2 dioxygen superoxide

superoxide peroxide peroxide hydroxyl radical - 2- 2- O2 O2 O2 HO attacks lignin? Possible modification: Mn2+ Mn3+ Mn2+ Mn3+

- - O2 O2 O2 O2 dioxygen superoxide dioxygen superoxide A mulcopper oxidase CueO from Ochrobactrum sp. acve towards lignin model compounds and lignosulfonate

Dr Fabio Squina (Univ Sorocaba)

R.S. Granja-Travez, R.C. Wilkinson, G.F. Persinoti, F.M. Squina, V. Fulop, T.D.H. Bugg, FEBS J. 2018, 285, 1684-1700 Discovery of New Biocatalysts from Metagenomics

Dr Fabio Squina (Univ Sorocaba)

E.C. Moraes; T.M. Alvarez ; G.F. Persinoti; G. Tomazetto; L. Brenelli; D. Paixão; C. Caldana; N. Dixon; T.D.H. Bugg; F.M. Squina. Biotechnology for Biofuels, 11, 2018, 75. OMe MeO OH Lignin Breakdown OMe OH RO OH OMe Pathways HO OH O O deduced from O O OH OMe observed metabolites & OMe HO O O OH catabolic pathways in S.paucimobilis OH OMe OMe OMe HO OMe

phenylcoumarane Initial products β-aryl ether pinoresinol diarylpropane R biphenyl OH of oxidative OH OH cleavage MeO CO2H (by lignin HO O R O OMe HO OH peroxidases, HO OMe laccases, etc) HO MeO OMe O OH O OMe OMe HO2C OMe OH OH OMe OMe OH 1) demethylase OH lyase HCHO 2) extradiol peroxidase dioxygenase HO O OH OH MeO CO2H CH OH lignostilbene MeO 2 dioxygenase OMe CHO C-C hydrolase HO BphD OMe O OH CHO CO H aldehyde 2 MeO CO H HO C ketone 1 dehydrogenase decarboxylase 2 2 CO H OH 2 HO CO H OMe HO 2 OMe CO H OH CO H 2 OH 2 CO2H vanillin vanillic acid diacid 2 CO H vanillate 2 demethylase oxalic acid CO2H protocatechuic acid oxidative ring cleavage (intradiol or extradiol) OH OH Can these pathways be manipulated via gene knockouts or enzyme inhibition? Gene Knockouts in Vanillin Catabolic Pathway in Rhodococcus jostii RHA1

vanillin vanillic acid protocatechuic acid

CHO aldehyde CO H vanillate CO H CO2H dehydrogenase 2 demethylase 2 protocatechuate 3,4-dioxygenase β−aryl ether vdh, ro02986 vanA, ro04165 lignin fragments NAD+ CO2H OCH 3 OCH3 OH CO2H OH OH OH

β−ketoadipate Gene deletion strains pathway

GC-MS Analysis of organic extract from R. jostii Δvdh mutant grown on 1 g/l wheat straw lignocellulose

CO2H protocatechuic Increase of vanillin peak vs time p-hydroxy acid benzaldehyde using LC-MS vanillin OH OH CHO CHO

p-coumaric

OCH3 acid

OH OH CO2H CO2H

ferulic acid

OCH3 OH OH 96 mg/L vanillin Yields of metabolites from RHA045 mutant:

Minimal M9 medium supplemented with 2.5% wheat straw lignocellulose Yields from LC-MS analysis. VAN vanillin; 4HBA 4-hydroxybenzaldehyde FA ferulic acid

Other carbon sources: 0.1% beech xylan: no vanillin observed 0.5% industrial Kraft lignin: 13 mg/L VAN (120 hr)

Hemi-

cellulose (Xyl)n O O Ara CO2H Rationalisation CHO of catabolic pathways: β-oxidation OCH OCH 3 3 OH OH OH OH Growth on M9/0.1% ferulic acid G type lignin CHO CO2H produces >100 mg/L vanillic acid CO2H vanillate demethylase via β-oxidation, but not vanillin X OCH3 OCH3 OCH OH OH vanillin 3 OH dehydrogenase OH OH H type lignin vdh, ro02986 CHO CO2H NAD+ p-hydroxy- benzoate X hydroxylase oxidative P.D. Sainsbury et al, ring cleavage OH (intradiol) ACS Chem Biol 2013, 8, 2151-2156. OH OH Generaon of Aromac Dicarboxylic Acids from Microbial Lignin Breakdown

PET (polyethylene terephthalate) PBAT (polybutyrate adipate terephthalate) with Biome O O O Bioplastics Ltd O O O O O n O O O O m n

RO LIGNIN OMe OH OMe OH O O O O O OH O OMe RO Identification by MeO Intens . 2,4, pyridine dca M9_23_01_13936.d: EIC 168.0 +All MS OMe 7 MeO OH OH OMe x10 LC-MS from M9/1%

4 wheat straw:

CO H 3 CO2H CO2H CHO CO2H 2 protocatechuate NH Cl 2,3-dioxygenase 2 CHO 4

1 CO2H N OMe OMe OH PraA 0 OH OH OH Intens . OH CO2H LigAb 5d_30_01_13911.d: EIC 168.0 +All MS x105 vanillin vanillic 2,5-PDCA acid protocatechuate 6 3,4-dioxygenase protocatechuate CO2H CO2H 4 4,5-dioxygenase NH4Cl CO H HO C CO H 2 2 CO2H 2 LigAB O2 OH N CO H HO2C 2 O O CO H 0 2,4-PDCA 2 Intens . wt 5d_28_01_13909.d: EIC 168.0 +All MS CO2H CO2H MODIFIED PATHWAYSx105 O β-ketoadipate 6 β-KETOADIPATE PATHWAY Insert S. paucimobilis4 ligAB genes or

Paenibacillus praA2 gene into Rhodococcus jostii RHA1 using inducible pTipQ2 expression vector 0 5 10 15 20 25 Time [min]

Intens . +MS, 9.5-10.1min #(1429-1532) [%] 213.9 100 172.9

80

60 187.9 236.0 145.9 150.9 199.9 40 202.8 140.9 196.9 226.9 161.9 177.9 20 182.9 241.0 283.2 157.9 230.9 244.9 255.0 269.1 125.0 131.9 279.0 299.2 303.0 0 120 140 160 180 200 220 240 260 280 300 m/z Production of Pyridine Dicarboxylic Acids by Fermentation 6 5 2,4 PDCA 4 2,5 PDCA 3 2 1 Peak size (relave units) 0 72 120 168 240 288 Fermentaon me (hr)

Construct Product Scale Carbon source for M9 minimal media

0.1% vanillic 1% (w/v) chopped 0.5% Kra lignin acid wheat straw R. josi 2,4-PDCA 50 mL 112 mg/L 90 mg/L NT (7 days)* (7 days)* pTipQC2-ligAB 2.5 L NT 125 mg/L (9d)* 53 mg/L bioreactor 102 mg/L (9d)# (9 days)#

R. josi 2,5-PDCA 50 mL 80 mg/L 79 mg/L NT (5 days)* (5 days)* pTipQC2-praA 2.5 L NT 106 mg/L (9d)* NT bioreactor 65 mg/L (9d)# Yield estimated by *LC-MS analysis #product isolated by chromatography (UV-vis)

Z. Mycroft et al, Green Chemistry 2015, 17, 4974-4979. Lignin valorizaon in cellulosic ethanol plants: biocatalyc conversion via ferulic acid to high value chemicals (BBSRC/FAPESP Project) Project leaders: Prof T.D.H. Bugg (Univ Warwick), Dr. Fabio Squina (Univ Sorocaba)

LIGNIN OMe (Xyl)n OH O OH ferulic acid Ara O O O OH Lignin oxidation, OH O OMe O biocatalyst discovery biocatalysis O O O LIGNIN

OMe ferulate-Ara OH ester linkage OMe O pharmaceutical ferulate-lignin metabolic ether linkage LIGNIN engineering chemicals

Sugar cane bagasse

Meeting in Campinas fragrance chemicals 24th/25th July 2019 Acknowledgements

Mark Ahmad (PhD 2007-2010) Dr. Elizabeth Hardiman (postdoc 2009-2012) Dr. Zoe Mycroft (postdoc 2013-2014) Charles Taylor (PhD 2009-2012) Paul Sainsbury (PhD 2010-2014) Rahman Rahmanpour (PhD 2011-2014, pdoc 2014-2018) Goran Rashid (PhD 2011-2015, postdoc 2015-present)

Prof. Lindsay Eltis (UBC, Canada) Dr Fabio Squina (Univ Sorocaba, Brazil) Paul Mines, Paul Law (Biome Bioplastics)