Now available to industry clients through Bioengineering Solutions. Bioprocess Engineering (TEA, Scale-Up / Scale Down, Tech Transfer)

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Alexina Glenn
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HO BDO OH Bioengineering Toolbox Now available to industry clients through Bioengineering Solutions Ideas Technology Platform Sector Independent Robust

Engineering Fermentation Engineering Separations Engineering Product Pathway Predictor SimPheny TM Bioinformatics Metagenomics Directed evolution TX-TL

1 HO BDO OH Bioengineering Toolbox Now available to industry clients through Bioengineering Solutions Ideas Technology Platform Sector Independent Robust Commercial Processes Bioprocess Engineering (TEA, Scale-Up / Scale Down, Tech Transfer) Idea (molecule) Gene Pipeline Pathway Prototyping HT Strain Engineering Fermentation Engineering Separations Engineering Product Pathway Predictor SimPheny TM Bioinformatics Metagenomics Directed evolution TX-TL QSS Genomatica s Bioengineering Toolbox 3 C-fluxome Metabolome Proteome Transcriptome Genome HT Cloning Genome editing Precision Fermentation Scalable Kilo Piloting

HO BDO OH Bioengineering Solutions Key Offerings

techno-economic analysis and computational biology

provides a view inside the cell to gain deep

modeling and omics Pathway Enzyme Engineering develop

activity and specificity on nonnatural substrates;

scale-up; improve strain and process robustness for

Quantitative Small Scale develop a predictive and

evaluation Integrated Strain/Process Dev.

fermentation processes Idea (molecule) Pathway

Down, Tech Transfer) Gene Pipeline Bioinformatics

TX-TL QSS Genomatica s Bioengineering Toolbox HT Strain

2 HO BDO OH Bioengineering Solutions Key Offerings Evaluation Studies assess new bioprocess ideas using techno-economic analysis and computational biology tools; Technical Metrics (TRY, OUR, impurities, recovery, wastes, etc.) Process Model Cost Impact $/ton Strain Diagnostics provides a view inside the cell to gain deep understanding of metabolism using systems-based modeling and omics Pathway Enzyme Engineering develop enzymes for improved in vivo performance; such as for activity and specificity on nonnatural substrates; Bioprocess Scale-up/Scale down enable reliable process scale-up; improve strain and process robustness for successful technology transfer Feedstock Evaluation Quantitative Small Scale develop a predictive and quantitative small scale in vivo assay for strain evaluation Integrated Strain/Process Dev. enable new business opportunities or improve existing fermentation processes Idea (molecule) Pathway Predictor Bioprocess Engineering (TEA, Scale-Up / Scale Down, Tech Transfer) Gene Pipeline Bioinformatics Metagenomics Directed evolution Pathway Prototyping TX-TL QSS Genomatica s Bioengineering Toolbox HT Strain Engineering HT Cloning Genome editing 3 C-fluxome Metabolome Proteome Transcriptome Genome Fermentation Engineering Precision Fermentation Separations Engineering Scalable Kilo Piloting Product

3 Bioengineering Toolbox Demonstrated on Numerous Organisms Eukaryotes Prokaryotes Mammalian cells Human cells Modeling Proven in yeast and Gram+ and Gram- bacteria Systems-based omics GENO Technology Platform Eukaryotes Prokaryotes Mammalian cells Molecular/Micro Biology Experience in eukaryotes and prokaryotes; yeast, Streptomyces Fermentation Proven in yeast and Gram+ and Gram- bacteria QSS 3

SimPhenyTM High Performance Strains by Design Modeling and Simulation 4 Deep understanding of metabolic network and impact of genetic changes 4 Provides an understanding of byproducts and the causes

4 SimPhenyTM High Performance Strains by Design Modeling and Simulation 4 Deep understanding of metabolic network and impact of genetic changes 4 Provides an understanding of byproducts and the causes for their production (For example, is ATP or redox limiting?) 4 Prioritization of pathways, strain designs and process engineering strategies 4 Analysis and interpretation of -omics data in global network context 4

System Biology Tools Enable High Performance Strains Provides Key Insights into Metabolism

Transcriptomics RNAseq qpcr Proteomics itraq (global) MRM (targeted) Fluxomics 3 C-label tracing

time [h] Iterative multi-omics provides valuable information about cellular environment 4

5 System Biology Tools Enable High Performance Strains Provides Key Insights into Metabolism Genomics Production and evolved strains SNPs, IS elements, deletions, duplications Transcriptomics RNAseq qpcr Proteomics itraq (global) MRM (targeted) Fluxomics 3 C-label tracing Computational tools Metabolomics Triple quad LC-MS Orbitrap - exact mass NADH/NAD time [h] Iterative multi-omics provides valuable information about cellular environment 4 Regular Omics experiments 4 Network analysis tools 4 Statistical data analysis Data-driven hypothesis generation/decisions 5

6 Systems Biology Example: Eliminating GBL Biological Solution to an Engineering Challenge HO O OH H Cat SCoA HO O Ald H HO Adh 4-HB 4-HB-CoA 4-HBal BDO O HO OH hydrolase O GBL O GBL formed by cyclization of 4HB-CoA (C-yield loss) Boiling point very close to BDO- expensive separation GBL formation enzyme induced and spontaneous Two approaches to eliminate GBL byproduct:. Delete genes responsible for GBL formation, deduced from transcriptomics. Identify and introduce new hydrolase mm BDO mm GBL BDO KO hydrolase GBL No GBL hydrolase 8

Example: Improving Robustness of Commercial Strain Genetic Instability Identified and

for scale-up performed poorly Approach: Ø WGS (MiSeq) revealed a large duplication event in

genes and proteases Ø Region borders have mobile genetic elements Ø Strain stabilized by

7 Example: Improving Robustness of Commercial Strain Genetic Instability Identified and Resolved in 5 Weeks Problem: Ø A new lot of starter cultures for production strain slated for scale-up performed poorly Approach: Ø WGS (MiSeq) revealed a large duplication event in new lot relative to prior lots Ø 3 kb region spans diverse functions including essential genes and proteases Ø Region borders have mobile genetic elements Ø Strain stabilized by removal of flanking IS elements Ø Performance rescued, no genomic instability observed in subsequent lots 7

Genomatica Enzyme Engineering Optimizing in vivo Performance of Pathway Enzymes Enzyme Structure Smart Library Design HTP in vitro Screening In vivo Screening & Validation Outsource structure

8 Genomatica Enzyme Engineering Optimizing in vivo Performance of Pathway Enzymes Enzyme Structure Smart Library Design HTP in vitro Screening In vivo Screening & Validation Outsource structure determination Rosetta enzyme modeling Substrate docking DNA synthesis to construct only desired mutations Predictive HT assays 96/384-well ~ 30,000 clones/day Quantitative smallscale assay Fermentation Systems/Omics Demonstrated applications: Improve substrate specificity Increase specific rate Lower product inhibition Switch cofactor specificity Improve enzyme stability 0

Example: Improving Downstream Pathway Enzymes Improvements in Activity, Tolerance and Stability Accomplished HO O OH H Cat SCoA HO O Ald H HO Adh 4-HB 4-HB-CoA 4-HBal BDO O HO OH Improve Cat BDO

9 Example: Improving Downstream Pathway Enzymes Improvements in Activity, Tolerance and Stability Accomplished HO O OH H Cat SCoA HO O Ald H HO Adh 4-HB 4-HB-CoA 4-HBal BDO O HO OH Improve Cat BDO tolerance Discovery/evolution applied 0 X activity in M BDO Improve Ald stability Discovery/evolution applied 0 X activity, no degradation Variant Ald FermTime (h): Ald BDO (g/l) Parent enzyme 48hr BDO Titer in Fermentations Evolved Cat Evolved Ald Evolved Cat + Ald Parent Cat* ALD* Cat* + ALD* Evolved Cat and Ald: 4Individually no improvement 4Combined 5% increase in titer Multiple strain changes often required for improved performance

10 QSS: Quantitative Small Scale Technology Smart Design to Maximize Information at Optimal Throughput and Low Cost smart design clean experiment Ü V V genomatica technology strain screening & evaluation to predict largescale performance quantitative metabolic profiling, scale-down diagnostics systems-level metabolic phenotyping (omics) information custom tools & modeling commercial tools

QSS: Proprietary In-house Methods Correlation Between Bioreactor and QSS Scale Predictive 7 6 5 4 3 0 Sp. Product Rate 0 8 6 4 0 Sp. Substrate Rate 0.50 0.40 0.30 0.0 0.0 0.00 Product Yield Rankable.

11 QSS: Proprietary In-house Methods Correlation Between Bioreactor and QSS Scale Predictive Sp. Product Rate Sp. Substrate Rate Product Yield Rankable Relative Product Yield StrainA StrainB StrainC StrainD Relative Byproduct Yield StrainA StrainB StrainC StrainD BR QSS BR QSS Systems Biology Ready H6P/H6P QSS Metabolomics BR Cit/akg QSS BR ug/mg total Proteomics time (h) time (h) 0 0

Scalable Bioprocessing: Robust Commercial Process

Nanofiltration Chromatography Solvent Extraction

36 bioreactors, L, 5 L, 30 L Wide range of unit

LIMS (microbe-to-product) Quant small-scale

large-scale environment 00+ process variables

12 Scalable Bioprocessing: Robust Commercial Process Delivery Smaller, Cheaper, Better, Faster Fermentation 30L scale HTST Flexible, high-precision fermentation Kilo-scale integrated piloting S/L Separations Centrifugation Microfiltration Recovery/Purification Nanofiltration Chromatography Solvent Extraction Distillation Full capability process lab, 6000 ft 36 bioreactors, L, 5 L, 30 L Wide range of unit ops for separations and purification Integrated LIMS (microbe-to-product) Quant small-scale fermentation 98% carbon closure, % CVs Mimics large-scale environment 00+ process variables monitored 30 L piloting suite Kilogram samples 80% of the data in 0% of the time/cost Full technology transfer services

Example: Highly Predictable Scale-up/down Consistent performance across development scales Consistent scale-up to commercial Robust performance at

commercial-scale yield 60% 0% L 30L 3kL 60% 40% 0% Rate, g/l.hr (as % of L) 0% 80% 40% 3,000L 0% 0 0 0 30 40 50 Batch No.

fermentation performance ( ~50 runs at commercial scale vs.

Fermentation performance across lab, pilot, and demo scales is highly consistent, enabling rapid lab-to-commercial scale development path Average

13 Example: Highly Predictable Scale-up/down Consistent performance across development scales Consistent scale-up to commercial Robust performance at commercial-scale 0% Fermentation Run Top 5 Fermentation Runs BDO, g/l (as % of L) 90% 60% 30% ~ 50x scale-up 0% 00% 80% Percent average commercial-scale yield 60% 0% L 30L 3kL 60% 40% 0% Rate, g/l.hr (as % of L) 0% 80% 40% 3,000L 0% Batch No. Percent average commercial-scale IRR' 0% L 30L 3kL 0% 00% Yield, g/g (as % of L) 0% 90% 60% 30% 0% L 30L 3kL Demonstration Scale Average fermentation performance ( ~50 runs at commercial scale vs. average demo scale) Commercial Scale Campaign Strain Titer 98% Rate 04% Yield 00% 80% 60% 40% 0% 0% Batch No. Fermentation performance across lab, pilot, and demo scales is highly consistent, enabling rapid lab-to-commercial scale development path Average commercial-scale performance over ~50 campaign fermentations equivalent to demonstration-scale performance for same strain (+/-%) Low variability in performance across ~50 campaign fermentations, indicates process robustness and predictability 03 Kirkpatrick Chemical Engineering Achievement Award 3

14 Integrated Solutions Example: Correlation Between O Supply and Yield Integrated Approach to Improving Fermentation Performance BDO titer (g/l) Normal O Lower O BDO BDO productivity (g/l/hr) Rate Biomass (g) Growth % of Glc to Excess CO CO BDO Yield Commercialization Yield Target Yield 4Lowering O decreases excess CO and increases yield - good 4Lowering O decreases growth, rate, and BDO titer - bad Ü Diagnostics, omics, strain engineering required to increase BOTH rate and yield under low O conditions 4

Reducing Flux Through CO Forming Pathways Systems Biology Based Diagnostics Results in Key Discovery Deleted three CO PP pathway forming pathways: zwf Glucose CO malate OAA G6P PEP CO

!. 3C isotope tracing revealed a rogue flux, converting succinyl-coa to succinate & forming excess CO.

15 Reducing Flux Through CO Forming Pathways Systems Biology Based Diagnostics Results in Key Discovery Deleted three CO PP pathway forming pathways: zwf Glucose CO malate OAA G6P PEP CO acetyl-coa acebak CO isocitrate akg CO. pentose phosphate pathway. complete oxidative TCA cycle 3. glyoxylate shunt BDO decreased Growth rate decreased Excess CO % increased!!. 3C isotope tracing revealed a rogue flux, converting succinyl-coa to succinate & forming excess CO.. Excess CO pathways may be required to supply cofactors (ATP, NADPH, NADH) for growth & maintenance. Ü The cell is telling us it needs more of these cofactors succinate succd CO NADH NADH ATP NAD(P)H NADPH,4-BDO SSA 4HB Rogue Flux - complements DsucCD and completes TCA cycle

16 Identifying and Reducing the Rogue Flux (RF) Critical solution identified based on systems-based understanding of metabolism malate OAA citrate isocitrate CO 4 Potential succinyl-coa hydrolase candidates prioritized for deletion using bioinformatics & omics data Insufficient ATP? NAD(P)H? => Both needed fumarate DsucCD X akg CO Rogue flux TCA-impaired strains perform poorly cyd+ Dcyd, pntab OE BDO Low BDO (0 g/l) Slow growth Growth 4TCA-impaired strains poor BDO production and growth 6

17 Genomatica s Capabilities: Geno BDO Commercialization Proven in Practice: 5 years from Concept to Commercialization 40 g/l GENO BDO TM Process 80 g/l 00 g/l 50 g/l 5 mg/l 0 g/l 0 (3 m 3 ) Integrated demo plant 00 (3 m 3 ) Piloting 009 (30 L) Purified Bio-BDO 008 ( L) Bio-BDO producing organisms 0 Shipping tons at a time 0/3 (650 m 3 ) Commercial scale 7

Industrial biotechnology enables the replacement of traditional

Industrial biotechnology enables the replacement of traditional SBE SUPPLEMENT: Synthetic Biology A Bioengineering Platform to Industrialize Biotechnology Stephanie Culler Genomatica, Inc. An integrated bioengineering platform that harnesses the synthetic biology toolbox