In-Cell Enzymatic Glycosylation: a Way to Improve Productivity of Heterologous Biosynthesis Pathways in Micro-Organism Esben Hansen Evolva

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Metabolic Engineering IX Proceedings

Summer 6-7-2012 In-cell Enzymatic Glycosylation: A Way to Improve Productivity of Heterologous Biosynthesis Pathways in Micro-Organism Esben Hansen Evolva

Follow this and additional works at: http://dc.engconfintl.org/metabolic_ix Part of the Biomedical Engineering and Bioengineering Commons

Recommended Citation Esben Hansen, "In-cell Enzymatic Glycosylation: A Way to Improve Productivity of Heterologous Biosynthesis Pathways in Micro- Organism" in "Metabolic Engineering IX", E. Heinzle, Saarland Univ.; P. Soucaille, INSA; G. Whited, Danisco Eds, ECI Symposium Series, (2013). http://dc.engconfintl.org/metabolic_ix/19

This Conference Proceeding is brought to you for free and open access by the Proceedings at ECI Digital Archives. It has been accepted for inclusion in Metabolic Engineering IX by an authorized administrator of ECI Digital Archives. For more information, please contact [email protected]. In-cell enzymatic glycosylation

A way to improve productivity of heterologous biosynthesis pathways in micro-organisms

Biarritz June 7 th 2012 Disclaimer

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Evolva Confidential Material Slid Evolva Snapshot

Unique, proven, widely applicable, biosynthesis technology □ Creates novel products and production methods in yeast □ More than 50 patent families filed to date Product On Addressab Family Market Market Product focus in 3 ”health & wellness” areas □ Natural flavorants and sweeteners : Vanilla, Stevia Vanilla 2014 $ 0.4 bn □ Protection against microbial pathogens : Pomecins □ Diabetes & obesity: EV -077, Stevia Pomecin 2015 $ 2 bn

B2B model focused on core competencies □ Provide innovative ingredients to other companies Stevia 2015 $ 4 bn □ Partnerships with Roche, IFF, BASF, Roquette, US DoD □ 2010 revenues CHF 18.6 mn (1H 2011 CHF 6.9mn) EV-077 2017 $ 10 bn

Many pharma & food majors want to grow in space □ But have limited partnering or acquisition options □ Evolva is well positioned with differentiated assets and a highly experienced team

Evolva Confidential Material Slid This Presentation

Glycosylation and the effects of glycosylation HO O O O OH HO Glycosyltransferases OH

Examples □ Vanillin biosynthesis in yeast □ Saffron biosynthesis in yeast □ Stevia biosynthesis in yeast

Evolva Confidential Material Slid Glycosylation Glycosylation

A glycoside is a molecule in which a sugar group is bonded through its anomeric carbon to another carbon group via an O-glycosidic, S-glycosidic, or N-glycosidic bond

“Small molecule” glycosides play important roles in especially micro-organisms and plants, e.g. in defence Dhurrin Some toxic defence compounds are stored as non -toxic glycosides □ Cyanogenic glucoside □ E.g. dhurrin, vanillin-glucoside and glucosinolates Aglycon is unstable and degradation results in In animals, small compound glycosylation is used as a mean release of cyanide for detoxifying and excreting unwanted lipophilic compounds □ Glycosylation is performed by promiscuous glucuronosyltransferases in the liver □ Water solubility is increased, facilitating excretion via the kidneys

Typical effects of glycosylating a scaffold Glucosinolate □ Decreases the compound’s cellular toxicity Hydrolyzed by myrosinases upon □ Increases the compound’s aqueous solubility tissue damage, resulting in release of biologically active □ Increases the compound’s biosynthesis rate and yield compounds □ In some cases: Stabilization of the product molecule

Evolva Confidential Material Slid Glycosylation Increases Solubility of Curcumin

Principal curcuminoid of the popular Indian curry spice turmeric

Curcumin

Increased solubility : Insoluble curcumin gains solubility by glucosylation (Kaminaga et al .) O O

HO OH O O Solubility increase

Glc Glc Glc Glc 22.000.000 X Glc Glc Glc 15.000.000 X Glc Glc 12.000.000 X Use : 2.300.000 X Several experimental (cancer, Glc Glc anti-inflammation, etc.) Glc 230 X et al 1 X Ref.: Kaminaga . (2003) FEBS Lett. 555: 311

Curcumin

Evolva Confidential Material Slid Amplification of Microbial Production of Toxic Compounds

☺☺☺ ☺☺☺ ☺☺☺ Product glucoside

Natural Natural Precursor product Precursor Product

Production microorganism dies when high Production microorganism survives when concentration of toxic compound is formed toxic compound formed is glycosylated

Toxic aglycon Less toxic glycoside

Evolva Confidential Material Slid Glycosyltransferases

“Family 1” Family 1 Glycosyltransferases – The UGTs

A GT is an enzyme that can transfer a sugar from one molecule to another

Family 1 UDP-glycosyltransferases (UGTs) □ 1 out of 91 GT families – a large family present in both animals, plants and microorganisms □ Small hydrophobic molecules as natural substrates □ Involved in synthesis of many different secondary plant metabolites: Pigments, defence compounds, flavour and more □ Involved in detoxification of xenobiotic compounds

O O N HO N O O O N N OH O O O O O O HO O P O PO O P O PO O O OH O + + O O O O UDP-glycosyltransferase O Resveratrol UDP-glucose UDP Resveratrol-β-glucoside (Piceid)

□ Use uridine diphosphate-activated sugars as sugar donors □ The catalytic mechanism is inverting, resulting in a β-configuration of the glycosidic linkage

Evolva Confidential Material Slide Family 1 UGTs

Most uses UDP-glucose as sugar donor Many plant UGTs have shown to be very promiscuous towards acceptor substrate Arabidopsis thaliana contains 122 different UGTs 70% of 87 small molecule drugs were glucosylated (Kristensen et al., 2008)

Picture: Universität Karlsruhe Botanisches Institut

Rooted phylogenetic tree – Arabidopsis thaliana UGTs

Evolva Confidential Material Slide Evolva’s Glycosylation Platform

Catharanthus Gram+ bacteria Drosophila Sorghum Neisseria meningitidis Bos taurus Stevia rebaudiana roseus Vanilla planifolia Crocus sativus Arabidopsis melanogaster bicolor thaliana

Present platform : 220 GT enzymes Expressed in E.coli, P. pastoris, S. cerevisiae and/or in vitro

Ipomoea sp. Oryza sativa Chimeric UGTs Petunia hybrida Phaseolus vulgaris Rauwolfia Citrus maxima serpentina Bellis perennis Lycopersicon esculentum Nicotiana tabacum

Evolva Confidential Material Slide Possible Carbohydrate Species

CH2OH CH2OH COOH CH2OH O OH HO O OH O OH O OH O OH OH OH OH OH OH HO HO HO OH OH OH OH OH NH D-Glucose D-Galactose D-Xylose D-Glucuronic acid CO CH 3 N-Acetyl -glucosamine

COCH3 NH OH OH O OH O CH 3 COOH OH OH OH H C OH L-Rhamnose H C OH

CH 2OH N-Acetyl -neuraminic acid (one type of sialic acid)

Evolva Confidential Material Slide In-cell enzymatic glycosylation The Process of Making an In Cell Glycosylation for a Biosynthetic Process

1. Prerequisites: R1 OH □ Product needs to have a suitable hydroxy , thio or amino group that can be OH glycosylated R2 N □ The added sugar moiety is to some extent “wasted” – product therefore needs H2N R3 to be a high or medium priced product O

Evolva Confidential Material Slide The Process of Making an In Cell Glycosylation for a Biosynthetic Process

2. Finding a suitable glycosyltransferase that can perform the desired glycosylation

Evolva Confidential Material Slide UGT Screening Assay - Finding a Suitable UGT

Purified enzyme 220 UGT enzymes

170 kDa 130 kDa

95 kDa

72 kDa

56 kDa

43 kDa

34 kDa

26 kDa

R1 OH

R4 R2 N

H2N R3 O Regio-specific glycosylation Substrate detected by HPLC or HPLC-MS

Sugar donor

Evolva Confidential Material Slide The Process of Making an In Cellell GlycosylationG for a Biosynthetic Process

2. Finding a suitable glycosyltransferase that can perform the desired glycosylation

3. Testing the glycosylation in the desired in vivo system

Evolva Confidential Material Slide Testing the glycosylation in the Desired In Vivo System

4500 Unwanted Precursor Glycoside 4000 Unwanted Side Product Glycoside 3500 Product Glycoside 3000

2500 mM μM 2000

1500

1000

500

0 UGT1 UGT2 UGT3 UGT4 UGT5 UGT6 UGT7 UGT8 UGT9 UGT10 UGT11 UGT12

An example showing varying glycosylation of heterologous pathway intermediates and end product in vivo □ Though a number of UGTs are found to do the reaction in vitro they may perform quite differently in vivo

Evolva Confidential Material Slide The Process of Making an In Cell Glycosylation for a Biosynthetic Process

1. Prerequisites: R1 OH OH □ Product needs to have a suitable hydroxy , thio or amino group that can be glycosylated N □ The added sugar moiety is to some extent “wasted”. Product therefore needs to be a R2

H N high or medium priced product 2 R3 O □ Not suitable for biofuel production 2. Finding a suitable glycosyltransferase that can perform the desired glycosylation

4500 Unwanted Precursor Glycoside 4000 Unwanted Side Product Glycoside 3500 Product Glycoside 3000

2500

mM 3. Testing the glycosylation in the desired in vivo system 2000 1500

1000

500

0 UGT1 UGT2 UGT3 UGT4 UGT5 UGT6 UGT7 UGT8 UGT9 UGT10 UGT11 UG 4. Find a suitable method for deglycosylation , to release the final product (if necessary) □ Hydrolysis of glycosidic bonds can both be done chemically or enzymatically using β-glycosidases

Evolva Confidential Material Slide Vanillin Case

Biosynthesis in Yeast De-novo Vanillin Biosynthesis Pathway

Yeast Natural Glucose De-novo vanillin pathway assembled in Saccharomyces cerevisisae from genes of O O OH COOH fungal, mammal and bacterial HO O Shikimate origin 3DSDOH ACAR OH OMT O OH OH OH HO O Main issue with microbial

Pathway production of vanillin is the toxicity Aromatic □ Negatively affects growth of amino acids Saccharomyces cerevisiae even at 0.5 g/L

Fungus Bacterium Mammalian (Podospora anserina) (Nocardia sp.) Synthetic

(Hansen et al., 2009 AEM)

Evolva Confidential Material Slide Glycosylation of Vanillin Product to Solve Toxicity Issue?

Yeast Natural Glucose

O O O OH COOH HO O Shikimate HO 3DSDOH ACAR OH OMT O UGT O OH OH OH O O HO O OH HO

Pathway OH

Aromatic amino acids

Fungus Bacterium Mammalian Plant (Podospora anserina) (Nocardia sp.) Synthetic (Arabidopsis thaliana)

(Hansen et al., 2009 AEM)

Evolva Confidential Material Slide Toxicity of Vanillin vs. Vanillin Glucoside

Glycosylation of vanillin solves toxicity issue □ Vanillin toxic even at 0.5 g/l □ Vanillin glucoside non-toxic even at 25 g/l □ Thus VG is at least 50 times less toxic than vanillin

Vanillin toxicity – Feeding assay Saccharomyces cerevisiae 6 25 g/l VG Control (no addition) 5 0,5 g/l vanillin

3 1 g/l vanillin 2

Culture growthgrowth Culture Culture (optical(opticaldensity) density) 5 g/l vanillin 0 0 5 10 15 20 Time (hours)

Evolva Confidential Material Slide Solubility of Vanillin vs. Vanillin Glucoside

Water solubility of vanillin and VG depends on ionization, pH and temperature and different values have been O reported

HO O O O Predicted solubility of vanillin glucoside in water 25°C is 351 OH HO mM (110.5 g/L) OH Vanillin glucoside □ Our experiments showed 114 mM O Predicted solubility of vanillin is 45 mM (6.875 g/L) O OH □ Our experiments showed 41mM Vanillin

Gain in solubility thus in the 2-8 fold range

Evolva Confidential Material Slide Creating a Metabolic Sink in the Vanillin Pathway

Vanillin glucoside is readily excreted from the cell □ The glycosylation step removes the

O O O OH COOH vanillin and thereby helps to create HO O HO 3DSDOH ACAROH OMT O UGT O O sink conditions that provides extra OH OH OH O HO O OH HO OH “pull” in the pathway Transporter Transporter ?

□ Concentration ratio Intracellular:extracellular is approx. O

HO 1:5, which indicates that active O O O OH HO excretion is involved OH

Evolva Confidential Material Slide Purification of Vanillin via Deglucosylation

Vanillin glucoside produced in yeast gets excreted efficiently Differential solubility of vanillin and vanillin glucoside means vanillin can be purified after glucosidase treatment

Evolva Confidential Material Slide Towards Getting Fermentation Derived Vanillin on Market

The use of glycosylation technology has allowed for a high vanillin glucoside titer □ Production cost is now at a level where fermentation derived vanillin is competitive in certain markets

2011 targets for vanilla programme was achieved □ Scale -up scheduled to be initiated early 2013

Evolva Confidential Material Slide Saffron Case

Biosynthesis in yeast Saffron

The characteristic saffron flavour, colour and odour come from several components □ Most important are picrocrocin, crocin and safranal – all present in the crocus stigma

The colour of saffron is primarily the result of α-crocin (trans -crocetin di-(β-D-gentiobiosyl) ester) Crocus sativus

Principal aroma constituent is safranal , which is released from picrocrocin

Purpose of the project is to produce the key compounds picrocrocin and crocin in yeast via fermentation □ Crocin is a tetra glucosylated crocetin molecule □ Picrocrocin is a mono glucoside

Crocetin (The aglycon of crocin)

Evolva Confidential Material Slide Saffron

The end products are glycosides themselves

Advantages of producing a glycoside still applies □ Solubility: Crocin is water soluble, while crocetin is almost insoluble

Crocetin Crocin Sparingly soluble in water Freely soluble in hot water (Merck index) (Merck index)

Main advantages of producing key saffron components by fermentation: □ Allows “saffron” to be available at a much decreased price, which will both expand existing markets and open new ones □ Eliminate the many complexities involved in the current supply chain □ By making each of the key components separately it will enable the production of customised forms that are for examxample particularly rich in aroma, taste or colour and that can be adapted to specific food formulations and regional preferences

Evolva Confidential Material Slide Saffron Pathways

UGT: Aglycon; O-glycosyl β-carotene BCH β-cryptoxanthin UGT3 Enzymatic BCH Physical conversion Zeaxanthin HTCC ZCO Picrocrocin Safranal Crocetin dialdehyde ALD

Crocetin UGT1: Aglycon; O-glycosyl UGT1 UGT2: β1-6 linkage; Glu -Glu

Crocin Crocetin monoglucosyl ester

UGT2 Crocetin diglucosyl ester UGT1 UGT1 Crocetin digentiobiosyl glucosyl ester Crocetin monogentiobiosyl ester UGT2

Evolva Confidential Material Slide Stevia

Biosynthesis in yeast Stevia – Production of Rebaudiosides in Yeast

AcetylCoA/ Steviol glycosides originating from the plant Stevia rebaudiana are pyruvate widely used as natural high intensity sweeteners

Functional pathway established in bakers yeast (Sacchromyces cerevisiae )

Taste pattern of the stevia glycosides is PPPs modulated by the glycosylation pattern □ Difference in sweetness, bitterness and liqurice aftertaste

Steviol

Rebs Stevia rebaudiana

Evolva Confidential Material Slide The Rebaudioside A Biosynthetic Pathway

Naturally occuring glycosylation pathway in Stevia rebaudiana Isopentenyl -PPB Farnesyl-PP

6 1 GGPPS Sr UGT85C2 3 Glycosylation pathway OP2O6 functional in yeast Geranylgeranyl-PP Steviol-13-O-monoside

2 CDPS (cyclase) 7 UGT 3 From glucose to OP2O6 rebaudiosides H Ent-Copalyl-PP Steviol-1,2-bioside H □ Precursor pathways 3 KS (cyclase) 8 Sr UGT74G1 □ 5-7 steps to prenyl phosphates H Ent-Kaurene Stevioside H □ Steviol pathway 4 KO 9 Sr UGT76G1

□ 5 steps to steviol H Kaurenoic acid Rebaudioside A

HO H □ Glycosylation pathway OH 5 O KAH □ 4 steps to RebA, 5 to RebD H Steviol HO H O

Evolva Confidential Material Slide Overview of the Main Steviol Glycosylations

1 sugar 2 sugars 3 sugars 4 sugars 5 sugars

Glucose Glucose O Glucose O Glucose Glucose

UGT74G1

COOH COOH Steviol-1,3- Reb B bioside

Glucose Glucose Glucose O Glucose O Glucose G O Glucose O Glucose Glucose O Glucose Glucose

UGT UGT COO Glucose COO Glucose Glucose COOH COOH COO Glucose Steviol-13- Steviol- 1,3-Stevioside Reb A Reb D O-glucoside 1,2-bioside OH

COOH OH O Glucose O Glucose Glucose O Glucose Glucose Steviol UGT85C2 UGT UGT COO Glucose COO Glucose COO Glucose COO Glucose Glucose Steviol-19- Rubusoside Stevioside Reb E O-Glucoside Glucose

O Glucose Rhamnose O Glucose Rhamnose

UGT76G1

COO Glucose COO Glucose Dulcoside A Reb C

Evolva Confidential Material Slide Stevia by Fermentation

Production by fermentation allows pure Stevia sweetener components to be produced □ Several of the most attractive Stevia components are present at very low concentrations in the plant and thus very hard to extract □ Glycosylation platform allows for production of specific steviosides /rebaudiosides by selection and use of regio - specific UGTs

Regio-specific glycosylation detected by HPLC or HPLC-MS

Evolva Confidential Material Slide Conclusions

Glycosylation is a very valuable tool for improving productivity of heterologous biosynthesis pathways in micro-organisms □ Main benefits are:

□ Improved solubility

□ Decreased toxicity

□ Improved “sink” conditions in pathway

Several high value products are glycosides □ Exact glycosylation patterns crucial for product profile

Evolva Confidential Material Slide Thank You

Esben Halkjær Hansen Research Scientist , Evolva

[email protected] Direct: +45 35 200 235 Mobile: +45 26 87 24 05