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8 October 2020 · 16:00–17:00 CET

Spotlight on ...Unlocking the potential of industrial yeasts for production of high value consumer products

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SPEAKERS

John Morrissey – CHASSY Project Coordinator, Senior Lecturer in Microbiology, University College Cork, Ireland

Else-Jasmijn Hassing – PhD candidate, Delft University of Technology, Netherlands

Florian David – CSO & Co-Founder, Biopetrolia AB, Gothenburg, Sweden

Tristan Rossignol – Senior Researcher, MICALIS Institute at INRAE, France

Verena Siewers – Senior Researcher, Chalmers University of Technology, Gothenburg, Sweden

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www.pulp2value.eu www.chassy.euEFIB 2020: Unlocking the potential of industrial yeasts for production of high value consumer products

1. Introduction to CHASSY (John Morrissey, UCC)……………............................4

2. Yeast platforms for aromatics (Jasmijn Hassing, TUD)……........................14

3. Yeast platforms for fatty acids and derived products (Florian David, Biopetrolia)………………………………………………………………………………………….…24

4. Applications of Kluyveromyces marxianus for food and industrial biotechnology (John Morrissey, UCC) ……………………………………………………33

5. Yarrowia lipolytica as a platform for sustainable lipids production (Tristan Rossignol, INRAE)………………………………………………………………………45

6. Computational models of industrial yeast metabolism and their role in changing the landscape of industrial biotechnology (Verena Siewers, Chalmers)……………………………………………………………………………………………….53

Table of contents


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Unlocking the potential of industrial yeasts for production of high value consumer products

European Forum for Industrial Biotechnology & the Bioeconomy (EFIB)

8th October 2020

Dr John MorrisseyUniversity College Cork [email protected]

Session Moderator Speakers and Panel

Florian David, SwedenTristan Rossignol, FranceJasmijn Hassing, NetherlandsJohn Morrissey, IrelandVerena Siewers, SwedenJean-Marc Nicaud, France

mailto:[email protected]


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Funded by the European Union in H2020 under the Leadership in Enabling and Industrial Technologies (LEIT) Pillar (2017 – 2021)

Model-Based Construction And OptimisationOf Versatile Chassis Yeast Strains For ProductionOf Valuable Lipid And Aromatic Compounds

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 720824

Microbial chassis platforms with optimised metabolic pathways for industrial innovations through systems biology


Development of efficient cell factories for the industrial production of novel high-value products


Dr John MorrisseyUniversity College Cork CHASSY Project [email protected]



Development of efficient cell factories for the industrial production of novel high-value products


Dr John MorrisseyUniversity College Cork CHASSY Project [email protected]

Lead ScientistsJohn MorrisseyJean-Marc DaranJack PronkVerena SiewersJens NielsenTristan RossignolJean-Marc NicaudCeline HenryMislav OrebEckhard BolesFlorian DavidAnastasia KrivoruchkoHenning Markmann




Traditional Products

Fermented BeveragesEthanolCitric AcidEnzymesFragrancesFlavours

Biopharma Products

InsulinGrowth factorsVaccinesEnzymes

Artemsinin

Bio-economy products

2G ethanolLactic AcidSuccinic acid

Flavours

Wide range of yeastsTorulaspora delbrueckii

Kluyveromyces lactisKluyveromyces marxianusSaccharomyces cerevisiaeDebaryomyces hansenii

Yarrowia lipolytica

Saccharomyces cerevisiae




Traditional Products

Fermented BeveragesEthanolCitric AcidEnzymesFragrancesFlavours

Bio-economy products

2G ethanolLactic AcidSuccinic acid

Flavours

Wide range of yeastsTorulaspora delbrueckii

Kluyveromyces lactisKluyveromyces marxianusSaccharomyces cerevisiaeDebaryomyces hansenii

Yarrowia lipolytica


Other Industrial hosts?


From: Application of synthetic biology for production of chemicals in yeast Saccharomyces cerevisiaeFEMS Yeast Res. 2015;15(1):1-12. doi:10.1111/1567-1364.12213

Yeast offer huge potential for production of bio-molecules, but ……..



Yeast offer huge potential for production of bio-molecules, but ……..

• Strain construction is increasingly complex

• Time for strain development is too slow

• Cost of development is prohibitive



CHASSY focus: overcoming bottlenecks to accelerate innovation

“We offer an innovative platform and toolbox

for accelerated delivery of high-value yeast-

derived products for the bio-economy”


Outputs from CHASSY that can accelerate industrial innovation

Systems strategies for

optimisng yeast for

biololecule production

Florian DavidBiopetroliaSweden

Jasmijn HassingTU DelftNetherlands

John MorrisseyUCCIreland

Tristan RossignolINRAEFrance

Verena SiewersChalmersSweden

Product Classes New Platforms New Thinking


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Yeast platforms for aromatics



Jasmijn Hassing, Delft University of Technology, Netherlands

October 2020


Edit titleModel-Based Construction And OptimisationOf Versatile Chassis Yeast Strains For ProductionOf Valuable Lipid And Aromatic Compounds

One

(aromatic)

Ring to make

them all…


One Ring to make them all


Exploiting the market

2-phenyl

ethyl acetate

500 tonnes

~4.5 USD/kg

Flavonoids

410 M USD

7-90 USD/kg

Vanillin

18000 tonnes

60-3300 €/kg

Lignans

70 M USD

~375 USD/kg

2-phenylethanol

5000 tonnes

350 USD/kg


- Unstable/limited supply- Mixed compounds- Difficult extraction+ Natural

+ Stable/Unlimited supply

- Limited products- Harsh conditions- Not natural

+ Stable/Unlimited supply+/- Pure compounds+ Natural

Production methods


Microbial production

Phenylalanine(Tyrosine)

Malonyl-Coa


Yeast chassis


Our yeast chassis

Kluyveromycesmarxianus

Thermotolerant

Wide substraterange

> 0.8 g L-1

precursor

Yarrowialipolytica

Oleaginous

Good secretion

> 0.6 g L-1

precursor

Saccharomycescerevisiae

Robust

Well characterized

> 1.5 g L-1

precursor


We offer

Chassis strains Know-how on strain engineering

Collaborations


Contact us

Jean-Marc Daran [email protected] (PI – Saccharomyces)

Else-Jasmijn Hassing [email protected] (Phd)

John Morrissey [email protected] (PI – Kluyveromyces)

Jean-Marc Nicaud [email protected] (PI – Yarrowia)





www.pulp2value.eu www.chassy.euEFIB, 2020: Unlocking the potential of industrial yeasts for production of high value consumer products


EFIB, October 2020

Assist. Prof. Florian David CSO, Biopetrolia [email protected]


Yeast platforms for fatty acids and derived products



Why an alternative for fatty acid production?


Our Solution Fatty acids through Yeast Biotechnology


Key advantages of yeast-based production of fatty acid-derived products

Sustainable sourcingProduction from wide-variety of feedstocks (incl. waste/side-streams), resulting in reduced land use and greenhouse gas emissions

EconomicCost-efficient alternative to production of many fatty acid-derived products.

€

Tailor made structuresSpecific design parameters (chain length, saturation, assembly into complex products, etc.)à specialized properties for specific applications


Our technology platformRapid engineering of yeast cell factories

Fatty acids

Cell mass

Yeast strains producing g/L of

fatty acids

We can rapidly optimize production and create industrially-relevantstrains for production of many different fatty acid-based products.

LEARN

Different organism can provide different benefits:Saccharomyces cerevisiae: robust, well characterized, easy to engineerYarrowia lipolytica: naturally high fat production


Fatty AcidsStarter molecules for a lot of products

Fuels

Cosmetics

Fragrances

Nutraceuticals

AgrochemicalsAldehydes, acetates, alcohols

Alkanes, Alkenes, FAEE

Waxes, long chain alcohols

Food ingredients Triacylglycerols SC alcohols, aldehydes, lactones

VLC fatty acids, PUFA


Hydrocarbons$600B/yr

Adipic acid$6B/yr

Dodecanedioic acid

$400M/yrSemiochemicals

>$540M/yr

Musks$180M/yr

Fatty alcohols

$5B/yr

PUFAs$4B/yr

MCT oil$1.6B/yr

VLC wax esters$900M/yr

Phospholipids$4B/yr

Vegetable oil(Palm & others)

$180B/yr

Vegetable oil(Palm & others)

$180B/yr

Wide spectrum of market opportunitiesFatty acid-based products are used across a wide spectrum of market segments for a

wide array of applications worth $billions/yr


Chassy Platform Strains

Developed platform strains Applications for platforms

Example products

S. cerevisiae strains producing high level of fatty acids and derived products (C16/C18)

Food fats/oils, biofuels, building blocks, personal care.

Palmitic acid, oleic acid etc.

S. cerevisiae strains producing high level of very long chain fatty acids and derived products (>C18)

Nutraceuticals, personal care, pharmaceuticals.

Docosanol

S. cerevisiae strains producing high level of short chain fatty acids and derived products (<C16)

Personal care, food ingredients, pharmaceuticals.

Octanoic acid, Hydroxyoctanoic acid

These platform strains are ideal starting points to develop a variety of fatty acid and fatty acid-derived producing strains.


Contact usBiopetrolia

[email protected]@biopetrolia.com

Info on short-chain fatty acids:

Goethe University FrankfurtDr Eckhard Boles

[email protected]





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Applications of Kluyveromyces marxianus for food and industrial biotechnology



John Morrissey, University College Cork

October 2020


Kluyveromyces marxianus• QPS and GRAS status• “Milk”, “dairy” or “lactic” yeasts• Involved in traditional dairy fermentations• Putative health benefits

QPS status (qualified

presumption of safety)

Kluyveromyces yeasts have a long history in traditional food and beverages


Examples of industrial use of K. marxianus1 – Bioethanol from whey permeates

yeast

Ethanol


Examples of industrial use of K. marxianus2 – Bioconversion of amino acids to flavours

2-phenylalanine

2-phenylethanol

OH

O

O

O

NH2

OH

O

OH

L-phenylalanine

O

O-

O

O

OH

LeucineNH2

OH

OO

O-

O

O

O-

OH

OH

2-Phenylethylacetate Isoamyl acetate

Market data

5,000 tonnes

350 USD/kg (natural 2-PE)


• Thermotolerant (up to 52oC)

• Short generation time (as low as 45 min)

• Assimilates lactose, cellobiose and xylose

• GRAS / QPS

Kluyveromyces marxianus – a versatile yeast for industrial biotechnology

CHALLENGES AND LIMITATIONS?


Knowledge of strain physiology

Molecular tools for strain engineering

Host strains

Exemplar strains to demonstrate utility

Scale-up experience and expertise

Challenges to developing K. marxianus for IB applications


Knowledge of strain physiology



Molecular tools for strain engineering

Host strains



CRISPR – based genome engineering

Development and application of tools for engineering K. marxianus

• Optimised host strains

• Gene engineering pipelines


Synthetic Biology infrastructure – pathway construction

Development and application of tools for engineering K. marxianus


A case study: de novo synthesis of 2- Phenylethanol (2-PE) in K. marxianus

Shikimate pathway

2- Phenylethanol~ 750mg/L 2-PE

Flask culture

AAA pathway


New industrial host

Wide substrate range

Capacity for aerobic production of aromatics

Amenable to genetic manipulation and synthetic biology

The offer from CHASSY

Know-how to construct cell factory strains

Knowledge of genetics, physiology and metabolism

Optimised strains and methods for aromatics

Collaborative partnerships to develop strains and processes

What is this of interest to industry?

Contact

Dr John Morrissey

University College Cork

Email: [email protected]



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Yarrowia lipolytica as a platform for sustainable lipids production



Tristan Rossignol, INRAE France

EFIB 2020


Yarrowia lipolytica

Oleaginous yeast

Accumulates high amount of lipids

Grows on hydrophobic substrates

Isolated from lipid-rich or protein-rich environments

GRAS status (generally regarded as safe)

QPS status (qualified presumption of safety)


Yarrowia lipolytica, an industrial chassis

Eicosapentaenoic acid (omega 3)

Feeding of farm animalsSingle cell protein1970’s

Long history in industry


image from www.pngfuel.com

Sustainable & environmentally friendly production

Host for biolipids production

Lipid metabolism well known

Naturally a good chassis for biolipids production

http://www.pngfuel.com/


Metabolic Engineering

Carbon sources

Biolipids

Development of chassis optimized for acetyl-CoA precursors

Modification of metabolic pathways

Heterologous pathway “plug-in”

Increase quantityControl composition


Expanding genetic engineering tools

New versatile and efficient tools in the frame of CHASSY dedicated to synthetic biology

Gene targeting Metabolic pathway insertion


Metabolic Engineering

Carbon sources

Biolipids

Acetyl-CoA

(6.5 g/L) Larroude et al 2018

Glucose

Glycerol

Lipidic wastes

Hemicellulose

Starch

Molasses

Carotenoid pathway

Biofuels

Odd-chain fatty acids Ricinoleic acids

Cyclopropane fatty acids Poly unsaturated fatty acids

Acetylated triglyceride….

Unusual lipids


Yarrowia lipolytica, an industrial chassis for biolipids production

GRAS - QPS

Robust industrial organism (pH etc)

High potential for biolipids and acetyl-CoA derivatives production

Contact

Dr Jean-Marc Nicaud

INRAE – Micalis, Jouy-en-Josas


Dr Tristan Rossignol

INRAE – Micalis, Jouy-en-Josas





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Verena Siewers, Chalmers University of Technology, Gothenburg, Sweden

8 October 2020

Computational models of industrial yeast metabolism and their role in changing

the landscape of industrial biotechnology


Design-build-test-learn cycle

Genome-scale metabolic models(GEMs) assist in strain design


Genome-scale metabolic models

Each gene and reaction is represented in a stoichiometric matrix


Genome-scale metabolic modelsH

igh

-val

ue

pro

du

ct

Glucose Xylose

Ove

rflo

w m

eta

bo

lite

• Determine maximum biochemical yield

• Evaluate alternative pathways

• Estimate internal flux distributions

• Predict beneficial genetic manipulations


Use of GEMs for cell factory design

Use of GEM to select target genes for CRISPR-based regulation

Increase of 3-hydroxypropionic acid production

ACS Synth Biol (2019) 8, 2457–2463



Problem 1: Spatial constraint

Limited amount of total protein per cell (enzyme mass not accounted for in model)


Problem 2: Varying enzyme efficiency


Pathway YZ requires less proteinPathway AB is faster

Hig

h-v

alu

e p

rod

uct

Glucose

A

B

Z

Y Enzyme Size Reaction rate

A 33 kDa +++

B 312 kDa +++

Y 27 kDa +

Z 33 kDa +



S. cerevisiaeK. marxianusY. lipolytica

Solution: GECKOEnzyme amounts and catalytic enzyme efficiencies included in the model

ecModels for 3 CHASSY yeasts:



Model prediction Exp. Measurement Decision

(high product yield) (low product yield)

• Deletion of non-essential enzymes

• Fine tuning levels of key enzymes



Modelling pipeline:Used for prediction of genetargets for 110 industriallyrelevant chemicals

• Alcohols• Alkaloids• Amino acids• Aromatics• Amines• Fatty acids• Flavonoids• Isoprenoids/steroids• Organic acids• Proteins


Contact us

[email protected]@[email protected]

Systems and Synthetic BiologyChalmers University of Technology

Gothenburg, Sweden




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