Manufacturing with and

Commercializing Biology

Jay D. Keasling

University of California, Berkeley

Lawrence Berkeley National Laboratory
 What is biomanufacturing?

A type of manufacturing or biotechnology that

utilizes biological systems to produce
commercially important biomaterials and
biomolecules for use in medicines, food and
beverage processing, and industrial
applications.

Wikipedia
 Biomanufacturing
In the US, bio-based products contributed $459B in
2016, up 17% since 2014.

In the European Union, the bioeconomy has been

estimated to be worth more than €2 trillion annually.

Worldwide, current bio-based chemicals and polymer

production is estimated to be 50 million tons annually.

Production of industrial chemicals using synthetic biology is expected

to grow by 15-25% per year for the foreseeable future.
 Today’s talk

• Focus on manufacturing using Simple Sugar

Acetyl-CoA
Amorpha-4,11-diene

engineered microorganisms
ERG10
H
ADS
Acetoacetyl-CoA

ERG13 H

HMG-CoA
AMO/CPR
tHMGR X2 H

• Developments in synthetic biology Mevalonate

ERG12
Mevalonate-P
HO
H

AMO/CPR

that support biomanufacturing ERG8

Mevalonate-PP

ERG19
H

Non-Enzymatic
H

H
H
HO H
IDI1 HO
IPP DMAPP O

ERG20

• Commercialization of GPP
ERG20
H

H
AMO/CPR

Artemisinic acid

biomanufacturing technologies
FPP HO

O
Met erg9::PMET3-ERG9
H

Squalene
H
ERG1,7,11,24,25,6,2,3,5,4 HO

Ergosterol
Low-cost, high quality anti-malarial drug

O
O O
H H
O

O
 Malaria and artemisinin (qinghao)

O
O O
H H
O

• 3.3 billion at risk • Traditional Chinese medicine

• 216 ± 67 million people infected • Extracted from Wormwood plant

• 655,000 ± 252,000 deaths (2/3 • Recommended as frontline malaria

children) treatment by WHO in 2004

Stats: WHO’s 2011 World Malaria Report

Map: Malaria Atlas Project
 Previous process for producing artemisinin therapies relied on
large plantations
Artemisia annua Therapies formulated
into drugs
H
H O
O
O H
O H H
O H O
O O H O
O
O H H
O H
H H O
O
O Chemical Artelinate H
Purification OH O
Conversions OH
O
O H

O Artesunate
Artemisinin O O
H H
O
Grown on farms
OEt

Arteether
Large swings in artemisinin price are caused by an inability
to predict the market, weather, and harvest
1,200 250

Artemisinin produced (tons)

Two-year delay
Artemisinin price ($/Kg)

1,000
200

800
150
600
100
400

50
200

0 0
2002 2003 2004 2005 2006 2007 2008 2009 2010 2011

Source: Boston Consulting Group

Benefits of engineering yeast to produce cannabinoids

Simple Sugar

Acetyl-CoA
Amorpha-4,11-diene
ERG10
H
ADS
Acetoacetyl-CoA

ERG13 H

HMG-CoA
AMO/CPR
tHMGR X2 H

Mevalonate

ERG12 HO
H

Mevalonate-P AMO/CPR
ERG8
H H
Mevalonate-PP

ERG19 Non-Enzymatic H
H
HO H
IDI1 HO
IPP DMAPP O

ERG20
AMO/CPR
GPP H

ERG20
Artemisinic acid
H
FPP HO

O
Met erg9::PMET3-ERG9
H

Squalene
H
ERG1,7,11,24,25,6,2,3,5,4 HO

Ergosterol

Artemisia annua
Artemisinic acid

HOOC

Simple sugars
 Replace the plant with yeast engineered to produce
artemisinic acid from sugar
Therapies formulated
into drugs
Simple Sugar

Acetyl-CoA H
Amorpha-4,11-diene
ERG10

O
H
ADS
Acetoacetyl-CoA

ERG13
O
H

HMG-CoA
AMO/CPR
O H
tHMGR X2 H

H H
H O
Mevalonate

ERG12 H
O
O
HO

Mevalonate-P AMO/CPR
ERG8
H O
H
H H

O
Mevalonate-PP
H H
ERG19

IDI1
HO
H Non-Enzymatic
H
H
O O
O
HO
IPP DMAPP O

Chemical Artelinate
ERG20
H
Fermentation
AMO/CPR

OH
GPP
O
H

ERG20

Conversions OH
Artemisinic acid
H
FPP
HOOC
HO

O
Met erg9::PMET3-ERG9
H

O
Squalene

ERG1,7,11,24,25,6,2,3,5,4 HO
H H
Artesunate
O

O
Ergosterol

Artemisinic acid O O
H H
Microbial synthesis O

OEt

Arteether
 Low-cost, high quality anti-malarial drug

O
O O

H H
O

Simple Sugar

Acetyl-CoA
Amorpha-4,11-diene
ERG10
H
ADS
Acetoacetyl-CoA

ERG13 H

HMG-CoA
AMO/CPR
tHMGR X2 H

Mevalonate

ERG12 HO
H

Mevalonate-P AMO/CPR
ERG8
H H
Mevalonate-PP

ERG19 Non-Enzymatic H
H
HO H
IDI1 HO
IPP DMAPP O

ERG20
AMO/CPR
GPP H

ERG20
Artemisinic acid
H
FPP HO

O
Met erg9::PMET3-ERG9
H

Squalene
H
ERG1,7,11,24,25,6,2,3,5,4 HO

Ergosterol
 Low-cost, high quality anti-malarial drug

O O
O
We need an industrial microorganism
to host the biosynthetic pathway.
H H
O

E. coli Yeast
 How do you engineer a yeast to
Yeast
Intracellular
produce a new chemical?
metabolites
Step 1: Find an enzymatic
pathway to transform intracellular
metabolites into your desired
product.
Genes

atoB HmgR PMK IspA FPPS ADS

HmgS MK MPD IDI ADS

Intracellular
Enzymes Artemisinic
metabolites acid
 How do you engineer a yeast to
Yeast
Intracellular
produce a new chemical?
metabolites
atoB
Step 2: Put the genes that encode the
enzymatic pathway inside the yeast.
HmgS

HmgR

MK

PMK

MPD

IspA

FPPS
IDI
Genes
ADS

ADS

atoB HmgR PMK IspA FPPS ADS

HmgS MK MPD IDI ADS

Intracellular
Enzymes Artemisinic
metabolites acid
 How do you engineer a yeast to
Yeast
Intracellular
produce a new chemical?
metabolites
atoB
Step 3: Express the genes that encode the
enzymatic pathway
HmgS

HmgR

MK

PMK

MPD

IspA

FPPS
IDI
Genes
ADS

ADS

Artemisinic atoB HmgR PMK IspA FPPS ADS

acid
HmgS MK MPD IDI ADS

Intracellular
Enzymes Artemisinic
metabolites acid
 Yeast
Production of the new chemical
Intracellular
metabolites Step 4: Grow the engineered yeast
on sugar, and it produces the
atoB

HmgS

HmgR

MK
desired chemical.
PMK

MPD

IspA

IDI

FPPS

ADS

ADS

Artemisinic
acid
 Known genes can be made synthetically

Databases Sequences DNA synthesis Synthetic

HMGR
ATCGDTA… MK
ATCGDTA…
companies gene
HMGS
ATCGDTA… PMK
ATCGDTA…
ML
ATCGDTA… MPD
ATCGDTA…
IspA
ATCGDTA…

Genes
Known genes and
enzymes that are atoB HmgR PMK IspA FPPS ADS
common to many
organisms HmgS MK MPD IDI ADS

Intracellular
Enzymes Artemisinic
metabolites acid
 For unknown genes/enzymes, we must
hunt for them from natural producer

Unknown genes and

enzymes that are unique
to Artemisia annua

Genes

atoB HmgR PMK IspA FPPS ADS

HmgS MK MPD IDI ADS

Intracellular
Enzymes Artemisinic
metabolites acid
Artemisinin is produced in oil sacs on Artemisia annua
leaves

O
O O
H H
O

Artemisinin... ...is produced by trichomes... ...found on

Artemisia annua leaves...
 Artemisia annua trichomes produce and store
artemisinin
Cells at base of trichomes are enriched in enzymes that produce artemisinin and the
mRNA that encode them

O
O O
H H
O

O
O O
H H
O
CH 2OH OHC HOOC HOOC
O
For unknown genes/enzymes, we must hunt for
Simple Sugar

Acetyl-CoA
Amorpha-4,11-diene
ERG10
H
ADS
Acetoacetyl-CoA

ERG13 H

HMG-CoA
AMO/CPR
tHMGR X2 H

Mevalonate

them from natural producer

ERG12 HO
H

Mevalonate-P AMO/CPR
ERG8
H H
Mevalonate-PP

ERG19 Non-Enzymatic H
H
HO H
IDI1 HO
IPP DMAPP O

ERG20
AMO/CPR
GPP H

ERG20
Artemisinic acid
H
FPP HO

O
Met erg9::PMET3-ERG9
H

Squalene
H
ERG1,7,11,24,25,6,2,3,5,4 HO

Ergosterol

Previously
unknown genes

Genome Genome
FPPS ADS
sequencing annotation
ADS

Artemisin
acid
Previously
unknown enzymes
Choosing the appropriate genetic controls results
in a yeast that produces high levels of artemisinin Artemisinin
Yeast chromosomes pathway genes

Yeast
ADS TT
HMGS TT

MPD TT
HMGR TT
ispA TT
idi TT
AMO TT
PMK TT
MK TT
Artemisinic acid is transported out of the cell

Simple Sugar H

Acetyl-CoA H
Amorpha-4,11-diene
ERG10
H H
ADS HO
Acetoacetyl-CoA

ERG13 H
O

HMG-CoA
AMO/CPR
tHMGR X2 H H
Mevalonate

ERG12 HO
H

Mevalonate-P H
AMO/CPR HO
ERG8
H H O
Mevalonate-PP
ERG19 Non-Enzymatic H H
H
HO H
IDI1 HO
IPP DMAPP O

ERG20
AMO/CPR H
GPP HO
H

ERG20 O
Artemisinic acid
H H
FPP HO

Met erg9::PMET3-ERG9 O H
H

Squalene H
HO
H
ERG1,7,11,24,25,6,2,3,5,4 HO
O
O

Ergosterol
Ro. 2006. Nature 440:940-943
 Artemisinic acid precipitation

From Chris Paddon, Amyris

 O
H Light-based conversion of
ArtemisininO O
O
AA to artemisinin
H H
O

rom Pilot to Industrial Scale O

evelopment and industrial

Artemisinic Acid Artemisinin

ompleted.
H
dOequipment are in place.
• Process development and industrial
scale-up completed.
sfer
O to •manufacturing site
Facility and equipment are in place.
• Tech transfer to manufacturing site
d.O H completed.
• Validation completed.
completed.
• Routine Production Started
O
Production Started
Artemisinin
Production facility in Italy
Glucose Artemisinic Acid Artemisinin
Public-Private Partnership to deliver artemisinin

Patents Licenses Licenses

Patents Licenses

• Royalty-free, exclusive licenses for production of

artemisinin for the Developed World
• Must be produced at cost
 Current Status
• 51 million treatments have been delivered to Africa
• Heuvepharma has the capacity to produce 100-150 million
treatments annually, roughly one-half of the world’s need
 Repurposing yeast to produce other products
Artemisinin
Yeast chromosomes pathway genes

Yeast

Intracellular
metabolites
HMGS TT
XADS TT

a toB

Hm gS
MPD TT
Hm gR

MK
HMGR TT
PMK

Is pA
MPD
ispA TT
idi TT

X
IDI

FPPS

X
ADS

ADS AMO TT

Products
acid X
Artemisinic PMK TT
MK TT
What else can you do with
Flavors & Fragrances
the engineered yeast? Menthol

OH

Yeast
Intracellular
metabolites Fuels Bisabolene
atoB
Nutraceuticals
HmgS Lycopene
HmgR

MK

PMK

MPD

IspA

IDI

FPPS

Sweateners
ADS

Therapeutics
ADS

Artemisinic
acid Taxol

Rebaudioside
 There are 18 new molecules in
development

5 8 10 8 8-10

2010-2015
2016-2020
2021-2023
Clean Beauty
Health & Wellness
Flavor & Fragrance
Various End-markets 2024-2025
Discovery

13 ingredients in market 18 ingredients in development

 Amyris has repurposed the yeast to
produce 13 different molecules

Yeast Amyris Partners Amyris brands

Intracellular
metabolites
atoB

HmgS

HmgR

MK

PMK

MPD

IspA

IDI

FPPS

ADS

ADS

Artemisinic
acid
 Amyris has reduced the time to develop new yeast-
synthesized products via its industry-leading biofoundry
From first strain to pilot run
45
From first strain to first pilot run
40

35

30

25
Months

20

15

10

Product 1 Product 2 Product 3 Product 4 Product 5

2012 2020
 Cannabinoids are produced by Cannabis sativa

OH
∆9-THC
O

OH
CBD
HO

• Widely used for treating pain and relieving side-effects of cancer

treatment
• Extraction from Cannabis is difficult
• It is expensive to produce CBD and THC using synthetic
chemistry (>$40,000 - 70,000/Kg)
 Some natural cannabinoids produced by Cannabis
Tetrahydrocannabinol (THC) Cannabidiol (CBD) Cannabinol (CBN)
OH OH OH

O HO O

Tetrahydrocannabiviran (THCV) Cannabiviran (CDBV) Cannabigerol (CBG)

OH OH OH
COOH

O HO HO

O OH

HO O
Cannabichromene Cannabicyclol
 Production of cannabinoids in Cannabis
Synthesis of cannabinoids is compartmentalized in trichromes

THC

CA

GPP

OA

Kim and Mahlberg, 2003

 FDA-approved cannabinoid therapeutics
Dronabinol Epidiolex Nabilone

Synthetic THC CBD Synthetic THC

analogue
Prescribed for Prescribed for
§ Weight loss (HIV) § Epilepsy in Prescribed for
§ Nausea/vomiting children § Nausea/vomiting
(cancer) (cancer)
§ PTSD
OH
(experimental)
CBD
HO
 Cannabinoids are projected to grow
to be a very large market in the US

• By 2025, $60 billion • By 2030, >$100 billion

Plants versus microbes for production of cannabinoids

VS

Not scalable Scalable

Benefits of engineering yeast to produce cannabinoids

Intracellular
metabolites

Benefits
atoB

HmgS

HmgR

• Produce rare or unnatural

MK

PMK

MPD

cannabinoids
IspA

IDI

FPPS

ADS

ADS

Cannabinoids

• Produce low-cost
cannabinoids Cannabinoids

• Lower inputs of water ∆9-THC

OH

and energy
O

OH CBD

Simple sugars HO
 The cannabinoid biosynthetic pathway is more
complicated than that for artemisinin
Sugar

O
Genes
G3P Hex1
SCoA
OH
Hexanoyl-CoA COOH
PEP
HO Hex2
O O
Olivetolic acid
PYR HO SCoA Hex3
Malonyl-CoA
AcCoA MEV IPP DMAPP GPP OAC

OH CBGA
COOH TKS
OAA CIT atoB HmgR PMK HO

CBGAS
MAL HmgS MK GPPS
CBDS
OH
THC OH
CBD
MPD IspA IDI
Mitochondrion O HO
 THC and CBD are produced …
but so are THCV and CBDV

OH
THC OH
CBD
Intracellular
metabolites

O
atoB
HO
HmgS

HmgR

MK

PMK

MPD

IspA

IDI

OH FPPS
OH
THCV ADS
ADS

CBDV
Cannabinoids
O HO
 Sugar
Butyryl-CoA, an intermediate in the hexanoyl-
CoA pathway, is incorporated into cannabinoids
O
G3P
SCoA
Butyryl-CoA
OH
COOH
PEP
HO
O O
Olivetolic acid
PYR HO SCoA
Malonyl-CoA
AcCoA MEV IPP DMAPP GPP
OH CBGVA
COOH

OAA CIT HO

MAL

OH
THCV OH
CBDV
Mitochondrion O HO
 By feeding the yeast fatty acids OH
COOH OH
COOH

it can make unnatural cannabinoids HO

O
OH
COOH
OH
COOH
HO

Yeast O

OH
Intracellular
Sugar
COOH
OH
metabolites COOH
HO
atoB

O
HmgS

HmgR OH
OH

Fatty acid PMK

MK
COOH
COOH

HO
O
MPD O
O HO IspA

OH
HO
IDI
COOH OH
O FPPS

COOH
HO ADS
HO
O
ADS

O
HO
O
Cannabinoids OH

HO COOH OH
O COOH
HO
HO
O
 The engineered yeast is capable of producing many
natural and unnatural cannabinoids
Natural cannabinoids Unnatural cannabinoids
Yeast OH OH
OH
COOH
OH
COOH

HO O

Intracellular O O
OH
metabolites OH

Tetrahydrocann Tetrahydrocann COOH COOH

abinol (THC) abiviran (THCV)

atoB

HO O

HmgS

OH
OH
HmgR

COOH COOH
MK

PMK
OH OH HO O

MPD OH OH
IspA HO HO COOH COOH

FPPS
IDI
Cannabidiol (CBD) Cannabiviran HO O

ADS
(CDBV) OH
OH
OH COOH COOH
ADS
COOH
O
Cannabinoids HO

HO

OH OH

Luo, Reiter et al. 2019. Cannabigerol COOH COOH

(CBG) HO O
Nature 567(7746):123
 Unlocking the potential
of a >$30B industry
Supplements

Personal Care

Food & Beverage

Pharmaceuticals

Unlocking the potential for a >$30B industry promoting

health and wellness for people around the world.
[Forbes 2020]
 Hoppy beers are popular!

Denby, Li, et al. 2018. Nat. Commun. 9:965

 a The brewing process
Brewing process

Brewer’s Yeast Hops

Wort Beer Hoppy

Beer

Engineered Brewer’s yeast

Denby, Li, et al. 2018. Nat. Commun. 9:965
 Why replace hops?
• Hops are expensive and contracts can be difficult to secure
• Hops require a large amount of natural resources
• Flavor content is highly variable

Denby, Li, et al. 2018. Nat. Commun. 9:965

a
a
Brewing process
Goal: engineer yeast to produce hops flavors
Brewing process

Brewer’s Yeast Hops

Brewer’s Yeast Hops

Wort Beer Hoppy

Wort Beer Hoppy
Beer
Beer
Engineered Brewer’s yeast
Engineered Brewer’s yeast

Denby, Li, et al. 2018. Nat. Commun. 9:965

 Flavor determinants of Cascade hops

Denby, Li, et al. 2018. Nat. Commun. 9:965

 We will need various linalool and geraniol levels in order
to produce hoppy beer

Denby, Li, et al. 2018. Nat. Commun. 9:965

 Sugar Four key enzymes to produce different
hoppy flavors
G3P hmgR fppS* t67-McLIS ObGES

PEP DXP HO

PYR
Linalool
HMGR t67-McLIS
AcCoA MEV IPP DMAPP

FPPS*
GPP
OAA CIT
ObGES
OH
MAL FPP
Geraniol

Denby, Li, et al. 2018.

Mitochondrion Ergosterol Nat. Commun. 9:965
 Most promising strains were scaled up in fermentations
performed at the UC Davis brewery

Denby, Li, et al. 2018. Nat. Commun. 9:965

 Tasting was performed by Lagunitas

Denby, Li, et al. 2018. Nat. Commun. 9:965

 Little Feller Sniff Test (Waimea) Sniff Test (ARFQ) Smart Blonde
21st Amendment Brewery Fieldwork Fieldwork The Bruery

Y-20 Dos Doses Strange Love

21st Amendment Brewery Modern Times & Creature Comforts Almanac
Artemisinic Acid Artemisinin
Acknowledgements

Funding: Bill & Melinda Gates Foundation

Department of Energy
National Science Foundation

#Jay Keasling has a financial interest in Amyris, Lygos, Demetrix, Maple Bio, Napigen, Ansa
Biotechnologies, Berkeley Brewing Sciences, Apertor Pharma, and Zero Acre Farms.