Geobacillus thermoglucosidasius a catabolically versatile host for metabolic engineering

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1 Geobacillus thermoglucosidasius a catabolically versatile host for metabolic engineering

2 Global warming & energy security

3 Biofuel Feedstock (Food vs Fuel) Sugar oligomers from edible biomass are easily digested to monomers. Non edible lignocellulosics are composed of lignin and difficult to breakdown sugars in the form of (hemi)cellulose.

4 Biofuels from lignocellulose Options Strategies Create pentose and oligosaccharide utilising ethanologens (S. cerevisiae, Z. mobilis) Transfer known ethanol fermentation pathways into catabolically versatile strains (Zymomonas pdc and adh in E. coli, K. oxytoca etc) Create novel ethanol fermentation pathways in catabolically versatile strains

5 Advantages Geobacillus spp for ethanol production Geobacillus spp Gm +ve, o C, spore forming reclassified 2001 (Nazina et al) Metabolically versatile (hexose, pentose, cellobiose, xylans) Rapid growth (eg μ 1 1 glc = 0.85 h 1, μ xyl = 0.60 h 1 aerobic 66 o C) Growth at ~70 o C allows ethanol removal by gas stripping Growth temperature compatible with typical process operations Facultative anaerobes allow 1) aerobic biomass 2) anaerobic fermentation Disadvantages Mixed acid fermentation pathway Mixed acid fermentation pathway Low ethanol tolerance (compared to S. cerevisiae) Limited genetic tools

6 Initial pre treatment studies mild Severity of pre treatment severe Sugars released by pre treatment

7 Following enzyme hydrolysis mild Severity of pre treatment severe Sugar monomers released by pre treatment Additional sugar monomers released by EH

8 After Fermentation mild Severity of pre treatment severe Sugar monomers released by pre treatment Additional sugar monomers released by EH Ethanol yield

9 Utilisation of oligomers/polymers IC: Pre treated DG or Fibre. No EH step Series of resolved complex polymers c.dp10 to DP30 Unresolved bump Polymers > DP30 7% Ethanol Mixed sugars Real feedstocks

10 Utilisation of oligomers/polymers IC: Post Fermentation Majority of polymeric sugar utilised 7% Ethanol Mixed sugars Real feedstocks

11 Saccharification strategy 1.20 High Energy High Enzymes Chart Title Low Energy Low Enzymes % 99% 97% Soluble su ugars % 44% % solubilisation Post PT Post EH Post F Post PT Post EH Post F monomers polymers % solubilisation EtOH Yield Not Commercially Viable High enzyme cost Commercially Viable 30 enzyme cost 11

12 Shuttle vectors and reporter genes p(bla) Ampr r Nde I p(bla) Amp r Nde I Kan r ORI p(lac) Eco RI Sac I Xba I Pst I Hind III Nde I pucg bp Kanamycin Marker (H Liao) G. stearothermophilus ORI p(lac) ORI Sac I G. stearotherm. pheb gene. Xba I pucg18 pheb 7490 bp Pst I ldh promoter NdeI G. stearotherm. ORI Exchange for the ORI from pub110 for T s (55 o C max) suicide vector for knockouts 1A 2A 1B 2B Expressing superfolder GFP Catechol C23O 2 hydroxymuconic semialdehyde

13 Ethanologenic Engineering Strategies Fermentation pathways in G. thermoglucosidasius DL33, DL62 and DL81 Lactate LDH Glucose Pyruvate PDH AcetylCoA AcDH (Acetaldehyde) PFL ADH Acetate Ethanol Formate

14 Ethanologenic Engineering Strategies Fermentation pathways in G. thermoglucosidasius DL62 Glucose Pyruvate PDH AcetylCoA AcDH (Acetaldehyde) ADH PDC (pyruvate decarboxylase) Acetate Ethanol Acetaldehyde ADH (alcohol dehydrogenase) Ethanol

15 Growth of TM242 on cellobiose Cellobiose breakdown product from biomass hydrolysis Typical Ethanol yield = 0.45 g/g sugar (90% of theoretical)

16 TMO Cellulosic Ethanol Demonstration Facility Cost: 7.8M Pipework: 5km Cbli Cabling: 50km I/O: >2000 Flexible Capability Multiple Feedstocks High Solids Handling 24/7 Operation

17 Lignocellulosics process diagram Typical Process Stream Size reduction Pretreatment Eg dilute acid, AFEX etc Liquid/solid separation. Solids wash Liquid Detoxification Solid Enzyme production Enzymatic treatment Hexose & Pentose Fermentation Downstream processing

18 Lignocellulosics consolidated bioprocess Consolidated Process Stream Size reduction Pretreatment Eg dilute acid, AFEX etc Liquid/solid separation. Solids wash Liquid Neutralisation Solid Hydrolysis and Fermentation Downstream processing

19 Consolidated Bioprocessing with G. thermoglucosidasius Engineered G. thermoglucosidasius Biomass Enzyme CAZY GH Family Organism α L arabinofuranosidase1 GH51 Cellvibrio japonicus Ueda107 α L arabinofuranosidase GH43 Thermoanaerobacterium thermosaccharolyticum M0795 Xylanase GH10 Caldicellulosiruptor saccharolyticus DSM 8903 Xylanase GH11 Thermoanaerobacterium saccharolyticum JW/SL YS485 Acetylxylan esterase GH11 Clostridium thermocellum ATCC Exoglucanase GH48 Thermobifida fusca YX Exo&Endoglucanase GH48 &GH9 Caldicellulosiruptor saccharolyticus Endoglucanase GH5 Thermotoga maritima Endoglucanase GH5 Caldicellulosiruptor saccharolyticus Endoglucanase GH9 Clostridium thermocellum Fuel / Chemical

20 Improvements in GH activity 800 al protei in nmols sugar/mi in/mg tot pcex pcex pcex pcex CH 10 fold increase in CMCase activity in culture supernatant. Published activity of 616 U/mg on CMC.

21 Assembling pcex variants Golden Gate assembly using BsaI (Type IIs restriction enzyme)

22 Destination vectors Generate linearized product which BsaI will cut in the StuI and SacI sites. StuI AGGCCT SacI GAGCTC BsaI GGTCTC GGTCTCGAGCTC CCAGAGCTCGAG GGTCTCG...AGCTC CCAGAGCTCGA...G + BsaI AGGCCTGAGACC TCCGGACTCTGG A...GGCCTGAGACC TCCGG...ACTCTGG

23 Screening of signal sequences

24 Preliminary GH activities Optimised a cassette for GH expression Expressed and measured activities in culture supernatants Enzyme Organism Substrate Activity 60 C ph7.0 α L arabinofuranosidase1 T. thermosaccharolyticum Oat Spelt Xylan, o/p nitrophenylα l arabinofuranoside 20 μmol min 1 mg 1 M0795 α L arabinofuranosidase C. japonicus Ueda107T As above 3 μmol min 1 mg 1 Xylanase C. saccharolyticus DSM Beech/Oat spelt xylan 100 μmol min 1 mg o nitrophenyl β d xylobioside T. saccharolyticum As above JW/SL YS485 Acetylxylan esterase C. thermocellum ATCC Accelerant of xylanase activity N/D Exoglucanase T. fusca YX Filter paper, pp PASC, Avicel 0.3 μmol min 1 mg 1 Exo&Endoglucanase C. saccharolyticus As above N/D Endoglucanase T. maritima CMC, HEC 0.6 μmol min 1 mg 1 Endoglucanase C. saccharolyticus As above 0.1 μmol min 1 mg 1 Endoglucanase C. thermocellum As above μmoll min 1 1 mg 1 1

25 Assessing recombinant GH activity Hemicellulases Endoglucanase Exoglucanase H1 H2 H3 H4 End1 End2 End3 Exo1 Exo2 P2n28 SP3 h3 SP1 h1 SP1 endo3 SP2 exo2 Pβglu SP2 h2 SP1 endo3 SP3 endo2 SP1 exo1 SP2 h3

26 Evaluating CBP M. giganteus C BA α L arabinofuranosidase GH α L arabinofuranosidase T. thermosaccharolyticum enzymes were expressed in G. thermoglucosidasius. T. thermosaccharolyticum i Xylanase (GH11) Strains Xylanase T. thermosaccharolyticum (GH11) were grown in 2TY media to and T. thermosaccharolyticum Xl Xylanase OD above (GH10) 2.0 (OD600nm of 1 equates Xylanase C. saccharolyticus (GH10) to ~0.25g/L cells) C. saccharolyticus Saccharification Endoglucanase (GH9) measured as mass loss Endoglucanase T. maritima of insoluble (GH9) M. giganteus after C. T. maritima saccharolyticus Exoglucanase incubation (GH48) for 60 C (originally Exoglucanase T. fusca YX (GH48) 285mg). T. fusca YX Acetylxylan esterase C. thermocellum

27 CBP Mass loss % of M. available giganteus (hemi)cellulose total mass loss mass after loss CBP after CBP. Assumes lignin is not degraded A B C Control

28 Carbohydrate composition of PKC Glucose 11.60g 22% Mannose 38.20g 72% Arabinose 1.19g 2% Galactose 0.90g 2% Xylose 0.90g 2% Mainly Mannose and Cellulose (mannan and glucan) in PKC Possibility of producing biofuels from this biomass Enzyme required for hydrolysis are cellulase, mannanase + mannosidase

29 Steam Pre treatment of PKC (5% DS) 30 Sugar (g/l) bar 5 20 minutes 6.5 bar 5 20 minutes Autoclaved Mannose Manno Oligo Galactose Galact Oligo Arabinose Arab Oligo Xylose Xylo Oligo Glucose Gluco Oligo Various pre treatments (PT) were carried out for 5 20 minutes released mainly oligomeric sugars (soluble sugars shown). Increasing sugar release with increasing time of PT.

30 Enzyme hydrolysis of 5% DS PKC bar 5 20 minutes 6.5 bar 5 20 minutes Autoclaved 30 Sugar (g g/l) Mannose Manno Oligo 15 Galactose 10 Galact Oligo Arabinose 5 Arab Oligo Xylose 0 EH 4.5 BAR 5 EH 4.5 BAR 10 EH 4.5 BAR 15 EH 4.5 BAR 20 EH 6.5 BAR 5 EH 6.5 BAR 10 EH 6.5 BAR 15 EH 6.5 BAR 20 EH PKC AUTOCLAVE Xylo Oligo Glucose Gluco Oligo This graph suggests 4.5 bar 15 as optimum (ph5, 50 C, 72hr), while sugar degradation at higher temperature, pressure and time.

31 Production of ethanol from 5% PKC Optimal ethanol yield (on total PKC sugars) from 5% PKC (10.38 FPU/g glucan+1.66% Mannanase/g mannan) Gluco Oligo Glucose Xylo Oligo Xylose Arab Oligo Arabinose manno Oligo Mannose Ethanol (g/l) % sugars Pre treatment Post hydrolysis Fermentation at 4.1% DS 4.5 bar 5 20 minutes 92.4% r (g/l) 20 8 Suga % 35.37% 6 4 ) Ethanol (g/l PKC PT EH TM242 Yeast (NCYC 3466) Turbo yeast 0 Post enzyme hydrolysis, 100% solubilisation of sugars (glucan and mannan) after 72hrs, but only 50% monomerisation of mannan (100% for glucan). Geobacillus ferments most of the sugars including the oligomers, while yeast only ferments the monomeric sugars

32 Mannanase production by TM242 Mannanase gene PCR extracted from Bacillus subtilis Engineered into E.coli with a holding vector (and colony PCR checked) Cloned into Geobacillus TM242 (constitutive expression vector) and confirmed by PCR Clones tested on mannan agar plates for expression (24hr). Clones currently being used for CBP and SSF of PKC TM242 mannanase clones Original TM242 Production of mannanase by clones of Geobacillus compared to the original ii organism on mannan agar plates

33 Thank you Jeremy Bartosiak Jentys & Ali Hussein 1 CBP and SigP Chris Ibenegbu, Charlie Bennett 1, Marisa Raita 1,2 PKC Steve Martin & Kirstin Eley 3 1 Dept of Biology & Biochemistry, University of Bath 2 Joint Graduate School of Energy and Environment, King Mongkut's University of Technology Thonburi 3 TMO Renewables Any Questions?

34 Palm Kernel Cake (PKC) Production Palm Tree Fresh Fruit Bunches (FFB) Sterilization and stripping Empty Palm Fruit Bunch (EPFB) 22% Palm Oil Milled Effluent (POME) Palm Fresh Fruit Digestion, clarification, pressing Oilpalmtree (25 years) To start bearing bunches 2½ 3 years after field planting After harvesting round days per month Crude Palm Oil Palm Nut Cracking Palm Fiber Palm Kernel Cake (PKC) 34 6% Palm Kernel Shell (PKS) Crude Palm Kernel Oil

35 High Solids Loading CBP Effective CBP requires solids loadings of ~30% (w/w) Particle size distribution changes during process. Monosaccharide release was rapid over first 18 Changing hours and rheological then linear properties over remaining of the 5 slurry days. during CBP are likely to have a significant effect on its Compositional analysis indicated extent of operation. degradation. Recommendation is to run a fed batch process.

36 Modelling gas stripping requirement for continuous culture 70 Steady state substrate concen ntration (g/l) Dilution rate (L/h) G=0L/h G=20 L/h G=40 L/h G=60 L/h G=80 L/h G=100 L/h G=150 L/h G=200 L/h G=300 L/h G=400 L/h G=500 L/h G=600 L/h G=800 L/h G=1000 L/h

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