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Biocatalysis for sustainable solutions Lene Lange, Professor, dr.scient. Science Director, Molecular Biotechnology, Novozymes A/S

Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

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Page 1: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Biocatalysis for sustainable solutions

Lene Lange, Professor, dr.scient. Science Director, Molecular Biotechnology, Novozymes

A/S

Page 2: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Unlocking The Magic of Nature.

Natural microorganisms

Molecular Biotechnology

Large-scaleproduction

Industrialapplications

Page 3: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Novozyme’sNovozyme’s ToolboxToolbox

FungalExpression

New gene expression New gene expression Fungal developmentFungal development

Fermentation opt.Fermentation opt.

DirectedEvolutionRational designRational design

RegioRegio--mutagenesismutagenesisRandomRandom mutagenesismutagenesis

ScreeningScreening

Protein expression and comparisonProtein expression and comparison

Proteomics

Bioinformatics

Homologue identification Homologue identification & analysis& analysis

Microarrays

mRNAsmRNAs expression expression profiling, strain profiling, strain

comparisoncomparison

Protein purification, characterization, and testingProtein purification, characterization, and testing

BiochemicalCharacterization

Page 4: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Ethanol market drivers

• Reduction of oil dependence• Energy diversification• Rural economic development• Agribusiness development• MTBE and lead replacement• Kyoto protocol• Sustainable transport fuel

Page 5: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Enzymes are key factor in Bioethanol:

• 1st generation biofuel, from starch (or sugar)• starch: only two enzymes needed • enzymes have easy access to the substrate; • fungal amylases: eg from Talaromyces, produced in

Aspergillus oryzae

• 2nd generation biofuel, from lignocellulose agricultural waste• Challenge: more efficient blends; and better access• Numerous enzymes needed; fungal enzymes are superior• Several cellulases and numerous hemicellulases • Mostly used genes are from Trichoderma, Aspergillus,

Humicola and wood-degrading Basidiomycetes

Page 6: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

1.gen: Just two enzymes needed!But starch binding domains (SBD`s) play a significant role!

• The enzymes attach on the surface via the SBD

• SBD`s dissolve the starch structure

• Efficient enzymes drills holes in the starch granule

• Fungi are the main SBD donor organisms

GA AA

GA

GA

Catalyticalcore

SBD

Page 7: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Success of 1st Success of 1st gengen biofuelbiofuel enzymes due to enzymes due to significantly improved significantly improved (e.g. faster reduction of (e.g. faster reduction of viscocityviscocity))

0

5

10

15

20

25

30

5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8

pH

Visc

osity

, sec

B.licheniformisamylase

The novel B.stearothermophilus amylase

Method used:Falling ball timed in 85ºC mash.

Page 8: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Pectic Substances

Cellullose fibres

Xyloglucan

Plant cell walls are complex =>many enzymes needed for 2nd generation biofuel!

Page 9: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Novozymes R&D: 2nd generation biofuel

• Novozymes in US, Davis & Franklinton: • corn stover (lignocellulose) • NREL preprocessing • Abengoa collaboration

• Novozymes, DK:• wheat straw• wet oxidation or IBUS pre-processing• DTU, Risø and KVL university collaborations • Biogasol and Elsam collaboration (DONG Energy)

Page 10: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

DANIS

H B

IOET

HANOL

CONCE

PTThe technology

Wet OxidationIn: Biomass

SSF FermentationIn: Enzyme

Xylose FermentationOut: Ethanol

Anaerobic TreatmentIn: ManureOut: Biogas

DTU: Birgitte Ahring

Risø:BelindaThomsen

Page 11: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

At least 4 types of enzymes to break down the cellulose!

CBH I

CBH II ~15%

R

BG

?

CBH I ~60%

CBH II ~15%

EG III

EG I, II ~20%

NR

BG

???

In T. reesei:CBH I: 60%CBH II: 15%EG: 20%BG: 2% and many more hemicellulases!

Page 12: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

ProteomicProteomic Analysis of Analysis of CellulolyticCellulolytic FungiFungiCharge

Mass

CBH I

CBH II

CBH I

EG

CBH I

EGCBH I

T. reesei

Page 13: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Now much stronger discovery technology!TAST signal trapping:= the most efficient method for discovery(*) of secreted enzymes

TAST advantages:-positive selection of secreted hits-independent of available assay-independent of known sequences

*) from species with no available whole genomesequence dataOBS secreted proteins only ca 4% of mRNA

Page 14: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Transposon assisted Signal Trapping = TAST

OUT !OUT ! Gene 1

Gene 2

active

no secretion => inactive

signal

signal

Gene for secreted protein

Gene for non-secreted protein

+ transposon (b-lactamase /no signal) + Ampicillin

gene library

secreted protein

Page 15: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Many fungal sources of CBH1 genesND000575

ND001182ND000548ND000571

ND000543ND000577

ND000549ND000534

ND000574ND000562

ND001181ND000536

ND000538ND000544

ND001264ND001269

ND000551ND001175ND001339ND001349

* Acremonium thermophilum

* Scytalidium thermophilum

* Chaetomium thermophilum

* Thermoascus aurantiacus

* Trichophaea saccata

* Sporotrichum pruinosum

*Thermophiles

Page 16: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Discovery of a fungal Cyanovirin!

• Citrinovirin (11kda) was foundby Signal Trapping of a cDNAlibrary of Penicillium citrinum(Lange & Schnorr, 2003)

• Citrinovirin has a signal peptide and is a secretedprotein both in the wild type P.citrinum and in heterolog expression in Aspergillusoryzae

• heterolog expression wassuccessful from both gDNA and cDNA

Citrinovirin fromPencillium citrinum

Cyanovirin

conserved S-bridges onboth!

Page 17: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

TAST is done in E.coli but works for all groups ofEukaryotes (signals processedcorrectly!)

TAST-screening of cDNA libraries made successfully on:• Ascomycetes• Basidiomycetes• Zygomycetes• Plants• Invertebrates• Mammalsand• G+ bacteria• G- bacteria• Archaea

=> signal peptides are recognized by E.coli across all the biologicalkingdoms

Page 18: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Interesting break through for biocatalysisof arabino xylan

• TAST gene discovery combined with• improved understanding of enzyme / substrate

interaction

• (acknowledgement: Hanne Sørensen, post doc, Novozymes; published: PhD thesis, DTU, 2006)

Page 19: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Arabinoxylan degrading enzyme activities

EC 3.2.1.37

EC 3.1.1.73EC 3.2.1.55

EC 3.2.1.8EC 3.1.1.72

Page 20: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Mode of action of α-L-arabinofuranosidase

O

OH

OO

OH

OO O

OOOH OO

OOO

OH

OH

αAra1,3mono

αAra1,3di

αAra1,2di

Submitted to Appl. Microbiol. Biotechnol., may, 2006

GH43 AraF

αAra1,3mono

αAra1,2di

αAra1,3di

O

OH

OO O

OOOH OO

OOO

OH

OH

Studied by 1H NMR

Page 21: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Minimal enzyme cocktails

Water soluble and water insoluble wheat arabinoxylan

• GH51 α-L-arabinofuranosidase from M. giganteus

• GH43 α-L-arabinofuranosidase II from H. insolens

• GH10 Endo-1,4-β-Xylanase III from H. insolens

• GH3 β-xylosidase from T. reesei

In press: Biotechnol. Progr. 2006

Page 22: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Addition of ferulic acid esterases

Addition of ferulic acid esterases:Feruloyl esterase from A. nigerFeruloyl esterase from H. insolens

Total enzyme dosage: 0.5 g enzyme protein · kg-1

DMReaction parameters: 24 hours at pH 5, 50ºC

Release of 69% arabinose and 76% xylose

= Additional release of 12% arabinose and 12% xylose

Submitted to J. Biotechnol., may, 2006.

Page 23: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Why not also use plant genes?

Page 24: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Heterologue Expression of non-fungal genes in filamentous fungi is a bottle neck

• The commercialized enzymesorginate almost exclusively from fungi and bacteria

• Ample new and unexploitedenzyme diversity is availablefrom plants

• Heterologue expression of plant genes in filamentous fungi hasproven to be difficult (pooryields)

• A msc thesis at Novozymes studied the reason why

• Results published:

• Hamann T & Lange L., 2006:”Discovery, cloning and heterologous expression ofsecreted potato proteinsreveal erroneous pre-mRNAsplicing in Aspergillus oryzae”

• J. Biotechnol. 2006 June 28

Page 25: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

mRNA of plant gene expressed in Aspergillus oryzaefound to be of several lenghts

M        1           2           3           4          5        M    BECh2      ‐ empty     +

1000 bp800 bp

600 bp

lane

Page 26: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

What happened with the plant gene in A.oryzae?

• Aspergillus oryzae recognizescertain parts of the codingregion as splicing sites

• => erroneous pre-mRNAsplicing

• => several lengths mRNA

• => no full length and nofunctional protein produced

• Aspergillus orzyzae, the major production organism for Novozymes fungal enzymes

Page 27: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Details of splicing sites and branch points

CI-2CI-1

5’ splice site 3’ splice site↓ 61.7% AU content ↓

...A GUCAGU......UGCUAAU....UAG Gbranch point

60 nucleotides

StXTH1 mRNA

5’ splice site 3’ splice site↓ 58.2% AU content ↓

...G GUACGA......UGCUAAU....AAG Gbranch point

67 nucleotides

Hamann & LangeJ.Biotech. 2006

Page 28: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Enzymes are eco-friendly solutions, used in numerous market segments => need for broad spectrum discoveryReplacingchemicals

Saving energy Diminishingpollution

Better use ofraw materials

Saving water

Enzymesreplacebromate in breadproduction

Detergents with enzymeswash theclothes at lowertemperatures

Enzymes in animal feedreduce theamount ofphosphorusin the dung

Enzymessqeeze out all of thejuice from the fruits

With enzymes, tanneries useless rinsewater in leatherproduction

Page 29: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Novozymes A/S•• Largest Industrial Enzymes Producer (45% market Largest Industrial Enzymes Producer (45% market

share)share)•• More than 600 products sold in 130 countriesMore than 600 products sold in 130 countries•• Revenues (2005): ~$1 billion Revenues (2005): ~$1 billion •• Employees: ~4,000 (> 600 in R&D)Employees: ~4,000 (> 600 in R&D)•• Subsidiaries in the U.S. Subsidiaries in the U.S.

-- NovozymesNovozymes, Inc., Davis, CA, Inc., Davis, CA-- NovozymesNovozymes North America, Franklinton, N.C. North America, Franklinton, N.C.

•• Other Major Subsidiaries in Japan, China, Brazil, Other Major Subsidiaries in Japan, China, Brazil, France, SwitzerlandFrance, Switzerland

•• Production sites in Denmark, U.S., China, BrazilProduction sites in Denmark, U.S., China, Brazil•• Over 4300 active patentsOver 4300 active patents•• Largest number of recombinant protein products Largest number of recombinant protein products

in the worldin the world•• Additional business areas in Additional business areas in NovozymesNovozymes: :

BiologicalsBiologicals and Biopolymersand Biopolymers

Page 30: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Directed Evolution Process

Rational DesignRandom

Mutagenesis

Hit Shuffling

Prepare Enzyme Samples

Robotic Screening

Application Testing

Page 31: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Conventional Screening

New Technology

Volume per assay

Protein per assay

Cells needed per assay

Assays per time period

5-50 μL

10 - 100 ng

500-5000

1000 per hour

0.000001 μL

0.000001 ng

1-500

1000 per second

· Multiple target screening for simultaneous specificity and efficacy· Rare targets · Primary cells from Bacterial or Fungal libraries

What if?What is!

Future needs for Novozymes, in relation to - HTScreening with Nanoapproach.

Page 32: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Emission of green house gases from production and use of one kg enzyme product from Novozymes

Enzymeproduction

1-10 kg CO2

Oil and fats Leather

Cereal products

Textile BrewingDetergent

Animal feed

Cosmetics

Forest products

Food

…kg CO2

…kg CO2

-1300 kg CO2

-3800 kg CO2

- 100kg CO2

… kg CO2

-30 kg CO2

… kg CO2

-150 kg CO2

… kg CO2

Page 33: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Novozymes’ vision

”We imagine a future where our biological solutions create the necessary balance between better business, cleaner environment and better lives”.

Page 34: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Acknowledgements

• Colleagues in Novozymes R&D in Davis, California especially Joel Cherry and Paul Harris

• Colleagues in Novozymes R&D Bagsværd, Denmark• especially the TAST group (Fiona Duffner et al), and Hanne

Sørensen, Mads Bjørnvad, Thomas Callisen & Allan Svendsen

• Life cycle analysis by Per Henning Nielsen, Novozymes

• Thomas Hamann, KVL (phd student)

• Birgitte Ahring and Claus Hviid, DTU

Page 35: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Conclusion:

• Biological production and Biocatalysis are cornerstones for development of products, processes and solutions, building a knowledge-based bioeconomy

• Biology is back in focus, for products and processes(genomics and nanotech are tools. not the goals)

Page 36: Biocatalysis for sustainable solutions - University of York Lange... · Biocatalysis for sustainable solutions Lene Lange, Professor, ... post doc, Novozymes; published: PhD thesis,

Thanks for your attention!