1. Selection and Testing of New Microbes for Green and White Biotechnology Applications Industrial Uses of Bacteria 19 May 2010, IOM3, London, UK
Target high value products that are difficult to make using traditional chemistry
Develop novel and proprietary manufacturing routes
High tech mol biol through fermentation process chemistry and scale up
Robert Speight
Ingenza Ltd
Roslin, UK

2. Introduction Ingenza - Who we are and what we do
Finding catalysts from microbes
Using microbes to make industrial products

3. Background to Ingenza INGENZA = “INdustrial GENetics and ENZymes
Our name gives a clue to where our technology and innovation lies
The team compromises a core scientific team with cutting edge skills in chemistry, genetics and biology.
We are a team of twenty highly skilled and educated staff and our company is doubling in size every 2 years.
We relocated to Roslin Biocentre in Dec 2006
The picture flow on the right shows how we work at Ingenza. We start by generating the right genetic construct to engineer a microbe to do the job we want.
Then we take the microbe and ferment it. Then we using the fermentation output to make the products for our customers using process chemistry. 3

4. Innovation at Ingenza is Product Driven
Innovation at Ingenza is product driven. TO SAY IT ANOTHER WAY, WE ENGINEER BUGS TO PRODUCE ENZYMES THAT MAKE INDUSTRIAL PRODUCTS AND THESE PRODUCTS ARE….
We engineer microbes to make products.
We aim to reduce the costs and efficiency of these products.
We are world leaders in these product areas and have achieve significant successes in each of these areas.
We are highly ambitious and eager to exploit our skills and expertise into new markets.
With our platform skills and capabilities in genetics, microbiology and chemistry we believe we are unique placed to fulfill and realise these opportunities.
Our secret power to enable us to deliver this reality is from our understanding of how we can produce, modify, detect and predict the behaviour of enzyme science.
We have three current product areas where we are adding value.
These products are fine chemicals, biopharmaceuticals and biofuels.
Microbe Engineering
Enzymes
Industrial Products 4

5. Background to Ingenza Business Model:
Bioprocess Improvement in Industrial Biotechnology
Business Model:
Proprietary bioprocess technologies
Chemical manufacturing processes (e.g. amino acids, fuels)
Production organisms, engineering, gene expression
Biopharma manufacturing systems
Custom biotechnology services
Enabling technologies
Enzyme discovery and improvement
Strain construction
Gene expression, fermentation
Bioprocess development

6. Background to Ingenza
Bioprocess Improvement in Industrial Biotechnology
19 scientists (12 Ph.D) with integrated skill set:
Molecular Biology, Biochemistry, Strain Engineering
Fermentation, Formulation
Bioprocess/chemical process development and analytical chemistry
Biotechnology (GMP Compatible), Fermentation and Process Development Laboratories
Ingenza History
Founded in 2002
Spin-out from Edinburgh University
Initial focus on bioprocesses
Customers in pharma, food, agrochem, biofuels
Relocated to Roslin in 2006
Merged with Richmond Chemical Corp. in 2007
Economically Sustainable, Still Growing
Multidisciplinary team, stable, grown together and improved.
GMP compatible lab just opened, relevant for biopharma

7. Background to Ingenza Richmond Chemical Corporation RV Labs Ingenza
Bioprocess Improvement in Industrial Biotechnology
Richmond Chemical Corporation
Oak Brook, Chicago, US
RV Labs
Hyderabad, India
Ingenza
Roslin, UK
RC Fuel
Chicago and Roslin
Fully integrated company with key commercial and scientific expertise
55 People world-wide
Extensive customer and manufacturing alliances
Strong Portfolio of Enabling Technologies

8. Enabling Technologies
Cost-effective
processes
Adapting enzymes
to new targets
Bioprocess Development
Biocatalyst Formulation
High-throughput
Screening
Enzyme immobilization
Lyophilization
Enabling Technologies
Fermentation
Directed Evolution
High cell density fermentation
4 x 5 L in house, Scaled to 40,000 L
Enzyme improvement
by mutation/screening
Biochemistry and Genomics
Strain Engineering
Bio-production of
natural products
Enzyme
Characterization

9. Enantiopure amino acids and amines by deracemisation
Enantioselective
Oxidase
biocatalyst
Chemical Reductant
Platform technology
Cheap starting materials to high value products
D/L-amino acid oxidases, R/S-amine oxidases
Genes from microbes - enzymes made in other microbes
Needs wide variety of enzymes with wide substrate specificity and/or adaptability
Need to be able to access a wide range of substrates as platform technology takes a long time and cost to develop

10. Speeding Up Enzyme Discovery
New oxidases from diverse sources
Bioinformatics - Genome Sequencing Projects
1138 microbial sequenced genomes completed and submitted to NCBI
Genome sequencing becoming much cheaper
Data mining, BLAST searching
Gene cloning, enzyme expression, assay
Gene synthesis and custom cloning
In-house expression systems
E. coli - diverse plasmid collections
Yeast - IP free integration and expression systems
Graph reproduced from: Nature 458, (2009).

11. SEASCREEN: Organism Screening
Direct identification of oxidase activity
TSB, EPSRC and BBSRC Funding Project
Ingenza Limited Heriot-Watt University
Aquapharm Biodiscovery Limited Plymouth Marine Laboratory
Novel Oxidase
Amine/Amino Acid + H2O + O2
Ketone/Keto Acid + H2O2 + NH3
Or look directly for activities
Detection of by-products
Cell survival if substrate is the only source of nitrogen that it needs to survive
Colorimetric assay
Cell Survival

12. SEASCREEN: Organism Screening
Cell Survival - Direct selection of oxidase activity
Very Low Nitrogen
High Nitrogen
Very Low Nitrogen
+ amine substrate
Selective growth on amines
Screening of >600 strains yielded new broad spectrum L-amino acid and amine oxidases
Methods developed to control induction, lysis, heterologous expression and assay/screening

13. Changing Existing Enzymes
Mutation and Selection
Generation of large libraries of variants
Random mutagenesis
e.g. error prone PCR
Targeted mutagenesis
from structure and mechanism information
hot-spots selected from random mutagenesis
High efficiency plasmid library construction
Ligation-free approaches
Libraries containing up to 2 million independent variants built
Take the enzymes you have and mutate them to change their properties (such as which substrates they accept)

14. Directed Evolution Strategy
Random Mutagenesis
Parent Gene
Targeted Mutagenesis
High Throughput Screening
Hit confirmation
Activity Quantification
Validation
Oxidation Reaction
Key aspect is how to select out of 2 million random variants which ones have the characteristics we desire - needle in the haystack
Benefits from interdisciplinary team
Analysis
Fermentation

15. Mutation and Screening
Variety of oxidases (e.g. ScDAO, TvDAO)
Commercial substrate
Variable conditions (temperature, pH, inhibitors)
Stability screening used due to our previous experience with ScDAO
H141Y selected in first round of ScDAO screening
Watch for diagram flipping over.
1st screen
Re-assay

16. ScDAO Mutation and Selection
Streptomyces coelicolor enzyme previously uncharacterised.
Initial poor activity towards target compound, low stability and low expression improved through directed evolution
X 294
Activity at 20 mM racemate.Corrected for total protein concentration. Good/fast way of assaying Vmax AND KM
141 position in all other proteins is Tyr. Is this just a correction? Is 218 therefore the most important for specificity?
Round 1
Round 2
Round 3
Round 4

17. Biocatalyst development Biocatalyst stability and process robustness
Not stable under process conditions
Susceptible to chemical denaturation
Susceptible to physical denaturation
High risk for scale-up
Not only more thermally stable but
More resistant to chemical denaturation
More resistant to physical denaturation
Applied in process scale-up

18. Fine Chemical Manufacture
For example: 2-Aminobutyric Acid, Norvaline
Methodology, scalability and economics all validated by Ingenza process and RC Corp commercial groups
Engineered microbes and enzymes
High yield fermentations
Defined media, fed batch
High volumetric productivity
High enantiomeric purity
Rapid adaptation
Platform process
Adapted readily between ABA, norvaline and also an L-amino acid oxidase based process but relies on high enzyme activity
IF YOU FEEL LIKE IT AND TIME IS OK YOU CAN SAY THAT MANY BIO BASED METHODS EXIST FOR AMINO ACID MANUFACTURE
(FERMENTATION, PATHWAY ENG, BIOTRANS, RESOLUTION ETC...BUT THIS FACT ILLUSTRATES NO SINGLE METHOD HAS DOMINATED)
OUR VIEW IS THAT TO MAXIMISE ADAPTABILITY OF OUR ROUTE WILL GIVE GREATEST PAYBACK AND MITIGATE RISKS OF EXPENSIVE
BIOPROCESS DEVELOPMENT

19. Biopharmaceuticals Biopharma production
E.coli expression
HCD fermentation
Protease knockouts
Protein refolding
Active products
H.T. Screens
Biopharma
production
Yeast expression
HCD fermentation
Enabling technology in gene expression and strain improvement

20. Screening for Improved Biopharma Production
Test Protein is Soluble
Linker
R is functional
Test Protein
Reporter Protein R
Colour
Growth
Test Protein is Insoluble X
R is non-functional
No Colour
No Growth
Waldo, G. S. (2003), Current Opinion in Chemical Biology 7, 33-38

21. The Ingenza System Test gene mutated
Reporter
Test gene mutated
The oxidase reporter protein is constant
Assay response proportional to fusion protein concentration

22. Key Advantages The screening system is highly tunable
Choice of promoter
Inducer strength
Choice of DAO substrate
Concentration of substrate
Growth and assay time
Growth temperature
Total assay response is dependent on each test protein
Subtle improvements can be seen by finding the right assay ‘window’
Sensitive, cheap and rapid
Need to find right window to see subtle improvements

23. Biofuels (RC Fuel)
Project founded December 2008 to improve biofuel production process efficiencies through biotechnology
Commercially Driven
Strain Engineering
Molecular Biology, Directed Evolution
Biochemistry
High throughput screening for improved strains
Strain and process characterization
Analytical method development for process characterization
Fermentation
Process modeling and validation

24. Acknowledgements Heriot-Watt: Prof. Mark Keane and Alec Foster
Plymouth Marine Laboratory: Sohail Ali, Mike Allen
Aquapharm Biodiscovery: Kim McKendrick, Andrew Mearns-Spragg
Università degli Studi dell'Insubria: Prof. Loredano Pollegioni and Group
ACIB, Graz: Prof. Toni Glieder and Group
CoE Bio3, Manchester: Prof. Nick Turner, Paul Goddard
Everyone at Ingenza and RC Corp.
Our customers
TSB, EPSRC, BBSRC, Scottish Enterprise, Scottish Executive
At Ingenza we strongly believe that educating and sharing knowledge is key to our future success.
Last November we hired 2 modern apprentices on the Scottish government backed 2 for 1 scheme for modern apprentices in life sciences.
Ross and Kirsty are study one day a week at Forth Valley College for a HNC in life sciences.
We also engage with the local community the world-leading science we are exploiting. Last month we opened our doors to a local high school to enthuse our next generation of scientific innovators to become excited about the opportunities they have in science. 24

25. To summarise Ingenza is an exciting world leading bioprocessing company which is gaining a international reputation to deliver high quality products in various markets. Our unique selling point is our ability to use our multi skills set in synthetic chemistry, fermentation, and molecular biology to the customer to exploit the power of enzyme technology. 25