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Copyright 2015, Amyris Inc. All rights reserved
Dawn of a Golden Age for microbial engineering
Track 3, Session 1, Monday 20th July
Darren Platt,
Vice President Data Science
Copyright 2015, Amyris Inc. All rights reserved
ETHANOL
Amyris engineers living factories that produce chemicals from
sustainable sources
SUGARCANE
phosphoenol-pyruvate
pyruvate
“Baker’s Yeast”Saccharomyces cerevisiae
IsoprenoidSynthesis
acetaldehyde
Acetyl CoA
ISOPRENE
GPP
FPP
FRAGRANCE OILS
ARTEMISINIC ACID
monoterpenes
sesquiterpenes
IPP/DMAPP
FARNESENE
CAROTENOIDS
Copyright 2015, Amyris Inc. All rights reserved
Artemisinin – component of WHO recommended treatment for malaria
• Shorter, reliable production cycle
• Stable long term price
• Currently supplying 30% of world need via Sanofi
Artemisinin
http://www.who.int/malaria/areas/treatment/overview/en/
Copyright 2015, Amyris Inc. All rights reserved
Farnesene – versatile C15 building block
Dimer + H2 (squalane)Myralene ™ (partial H2)
Farnesane (full H2)
Polymers / Co polymers
http://biossance.com
http://muckdaddy.com
Copyright 2015, Amyris Inc. All rights reserved
Diesel MetricAmyrisDiesel
HEFA ASTM-D975 Diesel
Energy Density MJ/kg 44.25 40 to 43 n/a
Cetane Number No. 56 48 40 min.
Thermal StabilityPres. Drop (mmHg)Tube Deposit (No.)
0 (@360°C)0
n/a25 max. (@275°C)
3 max.
Flash Point °C 108 38 52
Density kg/m3 765-780 730-770 n/a
Freeze Point °C <-100 -40 n/a
Sulfur PPM 0 n/a 15 max.
GHG Reduction1 % 70 50 n/a
Amyris diesel delivers top tier fuel performance
1 RGHG reduction by EPA RFS methodology and at 24% fermentation yield of cane sugar
Copyright 2015, Amyris Inc. All rights reserved
Industry Challenges
Industry Overview Amyris Solution• Wide range of emollients – Amyris is
focused on addressing needs at the high to mid range (+$900m mkt)
• Amyris squalane is performance and cost advantaged vs. olive/shark today
Source: Nikko Chemicals
Yen/kg
• Chemically convert farnesene into squalane and hemisqualane
Fermentation
Farnesene Squalane
Downstream Processing Hemisqualane
Sugar• Consumers shifting towards
naturals• Price and supply volatility – by-
products of petrochem or agro sources
• Quality inconsistency
Source: SRI, Squalane data provided by Amyris customers (2kta @ $30/kg today)
JapanSharkOliveAmyris
• Volatile price and quality exemplified by squalane
Impact• Currently reaching >100m consumers• Current usage in ~400 brands
$45
$11
$7
$4
$15
• Squalane $30/kg, 2-4 kta• Lanolin $14.24/kg, 8 kta• Silicones $11.3/kg, 33.5 kta• Natural Wax $8.38/kg, 6 kta• Esters $7.26/kg, 33.5 kta
• Alkoxylates• Glycerides• Fatty Alcohols
• Fatty Acids• Other
Am
ino
Aci
ds
and
Po
ly P
epti
des
List
Pri
ce (
$/kg
)
Shark (High)
Shark (Low)
Olive (High)
Olive (Low)
Greening the emollient industry
Copyright 2015, Amyris Inc. All rights reserved
Lessons from millions of strains inform faster progressEf
fici
ency
(yi
eld
)
FarneseneFirst Generation
FarneseneSecond Generation
Fragrance 1
Fragrance 3
Fragrance 2Ph I
Months
Each product benefits from knowledge gained in previous cycles
Copyright 2015, Amyris Inc. All rights reserved
Design Build Test Learn
Data
center
Data
center
QC DNA
Transform
strain
Design
DNA
Assemble
DNA
QC
strain
Grow
strain
Measure
strain
Analyze
data
We engineer yeast strains that make the products you need to compete and meet the needs of consumers.
We have state of the art:
• Computational DNA design tools
• DNA construction AND QC tools
• Microscale assays that are highly predictive of fermentor performance
• Lab scale fermentor processes that have near perfect prediction of manufacturing scale
• World’s largest Yeast Engineering Data Repository
Our framework for driving faster product development
Copyright 2015, Amyris Inc. All rights reserved
Amyris has industrialized the science of engineering living factories
350 strains per person20 strains per person
2015: state of the art = 24-48 parts at a time at 6 genomic locations, for 1000 different strains every 3 weeks at ~ $0.02 / basepair
Test platformAutomation
Automation covers most of the DBTL cycle
Copyright 2015, Amyris Inc. All rights reserved
LIMS systems and CAD Platform
Software and Algorithms supporting every stage of DBTL
Copyright 2015, Amyris Inc. All rights reserved
3 CO2
GLUCOSE
Fatty acids
Ergosterol
Ubiquinone
Dolichol
Membrane
localization
Amino acids
Fatty acids
Cell wall
Histone acetylation
Growth regulation
Amino acids
NAD+
Ubiquinone
Glycolysis
PPP
Solved fundamental challenges in metabolic engineering
FARNESENE
Second generation yeast metabolism for acetyl coA production
Copyright 2015, Amyris Inc. All rights reserved
Time (h)
No switch
Withswitch
When grown in the OFF state, growth rate (µ) is improved dramatically
Ce
ll G
row
th
v
Solved fundamental synthetic biology challenges
Robust, Industrial Scale Switch
200h
Copyright 2015, Amyris Inc. All rights reserved
Open strain engineering platform: partnership with Genome Compiler
Drive quality through serving a larger community
Copyright 2015, Amyris Inc. All rights reserved
What are the resource drivers for microbial engineering?
Copyright 2015, Amyris Inc. All rights reserved
Challenges of optimizing 100 molecules concurrently
• Sheer scale of molecule pipeline forces automation– designing strains by hand could easily consume every strain engineer we have
– analyzing those data could easily consume every computing person we have
– (and we still wouldn’t be done)
• Computers are better at crunching complex data– phenotype data are multivariate, multi-dimensional
– new data of gigabyte volumes flowing in constantly
– decisions must be made quickly to inform next cycle of strain development or testing
• Computers excel at combinatorial design & learning without bias– better random number generators
– it’s boring to do by hand => error-prone
– avail of extensive literature on machine learning
– intentionally generate poor designs, fully crossed/nested DOEs for uncovering genotype-phenotype relationships
Copyright 2015, Amyris Inc. All rights reserved
execution
prefrontal
cortex
memoryperception,
learning
KNOWLEDGE
STORE
facts ~ inferences
targets ~ routes
methods ~ models
genotypes ~ lineages
priorities
hitpick,
retest
prob.
assertions
INITIAL
PATHWAY
DESIGN
“know-
ledge”
pass/fail
data
PROCESS
ANALYZER
probabilistic decision making
design of experiments
control engineering
bandwidth allocation
tactics ~ strategy
DESIGNER
BUILD
ENZYME
IMPRVMT
MUTA-
GENESIS
pipeline selection
DECON-
VOLUTION
RATIONAL
ENGG
DNA
design
routes to
molecule
raw
data
PHENOTYPE
ANALYZER
LIMS
strains
TEST
high
level
designGENOTYPE
GENERATOR
Building an Automated Scientist for closed-loop strain engineering
Copyright 2015, Amyris Inc. All rights reserved
Where will the next 100-500 compounds come from ?
Image courtesy of James Kennedy (http://tinyurl.com/qhjmen8)
60% of known natural products are Terpenoid
Copyright 2015, Amyris Inc. All rights reserved
Terpenes have the richest chemical repertoire on the planet
Phytochemisty 70 (2009) 1621-1637
Copyright 2015, Amyris Inc. All rights reserved
Homologous
recombination
+ PPS
TS
P450
PPS TS P450
PPS TS P450
PPS TS P450
PPS TS P450
PPS TS P450
PPS TS P450
PPS TS P450
PPS TS P450
PPS TS P450
PPS TS P450
PPS TS P450
PPS TS P450
PPS TS P450
PPS TS P450
PPS TS P450
PPS TS P450
PPS TS P450
PPS TS P450
PPS TS P450
PPS TS P450
b-farnesene
patchoulol
amorphadiene artemisinic acid
santalene
limonene g-terpinene terpinolene
3-carene b-pinene a-pinene
myrcene C10 C15
C30
geraniol
valencene
(+)-d-cadinene 5-Epi-aristolochene
vetispiradiene
Epi-cedrol
taxadien-5a-ol taxadiene
casbene
C20
lanosterol
> 3
0,0
00
ter
pen
oid
sin
nat
ure
µPharmsm
: Applying our platform to drug discovery
Amyris’ multi-loci integration technology allows rapid combinatorial library construction
Copyright 2015, Amyris Inc. All rights reserved
glucose
TCACycle
Phenylpropenes Stilbenes
Flavonoids
Isoflavonoids
Trp Tyr Phe
Shikimate pathway
Flavonoid pathway
Arg
His
Gln
ProGluGlutamate pathway
Ergosterol
Isoprenoid pathway
C5's
C10's
C15's
Metabolic node
IPP
GPP
FPP
Replicate process for other key metabolic nodes
Copyright 2015, Amyris Inc. All rights reserved
Architecture for Genome Compiler joint development
customer
Genome Compiler Portal
Order + Design input
AmyrisBioFab
Design specification,Cost feedback
DNA Delivery
T_CYC1 3’ Gene2
P_GAL1
5’ Gene1 Gene2 URA3
Copyright 2015, Amyris Inc. All rights reserved
BUILD STRAINS
A predictable path from lab to Industrial Manufacturing
TEST STRAINS & PROCESSES
96-wellScreening
Design and Learn: Automation, Computing
Mutagenesis,Meiosis &
AutomatedStrain
Engineering
0.5L & 2LFermentation
300 LFermentation
&Recovery Ops
120,000 strains/mo 60 strains/mo 2 strains/mo 2-4 strains/yr
MANUFACTURE
Millions strains/mo
200,000 LFermentation
&Recovery Ops
Copyright 2015, Amyris Inc. All rights reserved
Warehouse to integrate data sources
LIMS system
Data drivendecision making
LIMS system LIMS system Large data streams
Primary warehouse
Larg
e sc
ale
par
alle
l co
mp
uti
ng
Process QCETL
Secondary warehouse
Mining, design tools
Quality tagging
Quality tagging
Quality tagging
FermentationHTSGenotypes
Quality Tagging is
critical: we want to
distinguish operational
outliers from scientific
ones.
Tremendousamount of analytic work to summarizea tremendousamount of experimentalwork
Copyright 2015, Amyris Inc. All rights reserved
Quality: high resolution / throughput DNA QC
SNP
indel
Copyright 2015, Amyris Inc. All rights reserved
Multivariate regression predicts tank performance better than any
single assay
Tan
kp
erfo
rman
ce
Plate performance (various assays)
0.92
Copyright 2015, Amyris Inc. All rights reserved
Genome browsing / alternative host support
Courtesy of Amyris, Inc.
H. polymorpha K. marxianus K. lactis A. adeninivorans
© Kable © Scipio © Getty Images
E. coli
© LBL
Y. lipolytica
© Bioline Int.
CAT-1; PE-2(Industrial Yeast)
© GRC© Eye Of Science
Copyright 2015, Amyris Inc. All rights reserved
The rabit hutch: 120 million base pairs . . .
-
20
40
60
80
100
120
140
0
5,000
10,000
15,000
20,000
25,000
30,000
35,000
40,000
45,000
50,000
Jan
-10
Ap
r-1
0
Jul-
10
Oct
-10
Jan
-11
Ap
r-1
1
Jul-
11
Oct
-11
Jan
-12
Ap
r-1
2
Jul-
12
Oct
-12
Jan
-13
Ap
r-1
3
Jul-
13
Oct
-13
Bas
e p
airs
(m
illio
ns)
Ban
k si
ze (
bas
e p
airs
)
Date
Bank size(parts)
Human Genomecoding sequences
Yeast Genome
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