Pasadena, California

Imag

es c

ourt

esy

of

Shutters

tock

What do enzymes do for us?

Enzymes break down proteins and fats—use in laundry lowers energy costs

Green chemistry

Green chemistry

Green chemistry

Fe

O

S

Cys

+

Fe

C

S

Cys

RR

AATAGCCGTTATTTCCGGATGTGCATAGCTGATTTGACCCATCCGGTACAC

CAATDACAAATCCCGATTTGATCGTGTGCGCGACATGTCTTCCGGCGACA

CATGTGTCTCTCACTCCGAGAGATCGGTTAGAGTCTCGGTTAACCACACG

TCCCGGATATATTTAATTGGCCGGAGAGTCTCCCGCGCGACATAAGGAGT

CCTCGTTTCGAGATACGTACGGCATGGTGACACCAGTTGCCCTCTGATTC

CCGGAGCCTCTTTGAAAACGTCGGGACAAATCCCGATTTGATCGTGTGCG

CGACATGTCTTCCGGCGACACATGTGTCTCTCACTCCGAGAGATCGGTTA

GAGTCTCGGTTAACCACACGTCCAATAGCCGTTATTTCCGGATGTGCATAG

Programmable, green chemistry machines

But the catalysts are not designed to make what we want.

And they are complicated!

AATAGCCGTTATTTCCGGATGTGCAT

AGCTGATTTGACCCATCCGGTACAC

CAATDACAAATCCCGATTTGATCGTG

TGCGCGACATGTCTTCCGGCGACAC

ATGTGTCTCTCACTCCGAGAGATCG

GTTAGAGTCTCGGTTAACCACACGT

CCCGGATATATTTAATTGGCCGGAGA

GTCTCCCGCGCGACATAAGGAGTCC

TCGTTTCGAGATACGTACGGCATGGT

GACACCAGTTGCCCTCTGATTCCCG

GAGCCTCTTTGAAAACGTCGAGTCG

AATCGAAGTTCGAACCCCGGATCGG

GTCCACCAACTTAGAGATGTGTGTG

CGCTGACTCAGTCATTTTGACCCATC

CGGTACACCAATGGGTCGGACAAAT

CCCGATTTGATCGTGTGCGCGACAT

GTCTTCCGGCGACACATGTGTCTCT

CACTCCGAGAGATCGGTTAGAGTCT

CGGTTAACCACACGTCCCGGATATAT

TTAATTGGCCGGAGAGTCTCCAATAG

CCGTTATTTCCGGATGTGCATAGCTG

ATTTGACCCATCCGGTACACCAATGG

GTCGGACAAATCCCGATTTGATCGT

GTGCGCGACATGTCTTCCGGCGACA

CATGTGTCTCTCACTCCGAGAGATC

GGTTAGAGTCTCGGTTAACCACACG

AATAGCCGTTATTTCCGGATGTGCAT

AGCTGATTTGACCCATCCGGTACAC

CAATDACAAATCCCGATTTGATCGTG

TGCGCGACATGTCTTCCGGCGACAC

ATGTGTCTCTCACTCCGAGAGATCG

GTTAGAGTCTCGGTTAACCACACGT

CCCGGATATATTTAATTGGCCGGAGA

GTCTCCCGCGCGACATAAGGAGTCC

TCGTTTCGAGATACGTACGGCATGGT

GACACCAGTTGCCCTCTGATTCCCG

GAGCCTCTTTGAAAACGTCGAGTCG

AATCGAAGTTCGAACCCCGGATCGG

GTCCACCAACTTAGAGATGTGTGTG

CGCTGACTCAGTCATTTTGACCCATC

CGGTACACCAATGGGTCGGACAAAT

CCCGATTTGATCGTGTGCGCGACAT

GTCTTCCGGCGACACATGTGTCTCT

CACTCCGAGAGATCGGTTAGAGTCT

CGGTTAACCACACGTCCCGGATATAT

TTAATTGGCCGGAGAGTCTCCAATAG

CCGTTATTTCCGGATGTGCATAGCTG

ATTTGACCCATCCGGTACACCAATGG

GTCGGACAAATCCCGATTTGATCGT

GTGCGCGACATGTCTTCCGGCGACA

CATGTGTCTCTCACTCCGAGAGATC

GGTTAGAGTCTCGGTTAACCACACG

MTIKEMPQPKTFGELKNL...

KETSPIPQPKTFGPLGNL...

KQASAIPQPKTYGPLKNL...

WRRRGIPGPLGYPLVGSF...

WIRKGVKGPRGLPFLGVI...

FIRKGIKGPRGFPGIGML...

WIRKGVKGPRGFPFFGVI...

WIRKGVKGPRGFPFFGVI...

WMRKGIKGPRGLPFFGII...

WMRKGVKGPRGRPFVGVL...

WRRRGVVGPMGFPVLGVF...

REKIGLSGPEPHWFLGNL...

REKIGLTGPEPHWFMGNL...

RSSIGIPGPPVHWLWGNL...

KVSKYPKGPLPLPFIGNI...

...

Enzyme families are the products of millions of years of

mutation and natural selection.

mutation and selection

Ancestral enzyme

Evolution makes new enzymes extremely well.

Each one is well adapted to its biological role.

Humans have altered the biological world using evolution by artificial selection for

thousands of years.

Natural mechanisms for generating sequence diversity are limited.

Not so in the test tube.

Protein space:

VASTLY (Very much more than astronomically) large

Mostly empty

sequences20 450

20 natural amino acids

EVOLUTION IS HARD!

~ 450 amino acids long

Directed evolution is a molecular optimization on a fitness landscape, where fitness is performance, defined by the user.

Fitn

ess

Evolution works because…

?

…the regions that life

has discovered and

explored are rich in

function.

And, at least in some of its

many dimensions, the

fitness landscape is smooth.

This allows adaptation, one

step (one mutation) at a

time.

select/screen for

improvements

(cells)

~10

(a few)

random

mutations

4

~10 ~10715

NO

Parent gene

(= parent protein)

Directed evolution exploits smooth paths in the fitness landscape.

select/screen for

improvements

YES

repeat

(cells)

~10

(a few)

random

mutations

4

~10 ~10715

NO

Parent gene

(= parent protein)

Evolved gene

(= evolved protein)

Directed evolution exploits smooth paths in the fitness landscape.

Performance

Generation

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6 7

0 0 Gen1 Gen2 Gen3 Gen4 Gen5

Directed evolution by iterative mutagenesis and screening can re-optimize the enzyme

Evolved enzyme doing a new job

Native enzyme doing its native job

Native enzyme doing a new job

Diabetes affects 422 million people(5.9%) of the world’s population in 2014.5

Sit

Sitagliptin synthesis

Solvent waste

Multi-step chemical process

is expensive and generates

a lot of waste.

Toxic heavy metals

Sit

Rela

tive p

roductivity

Directed enzyme evolution

Savile et al. Science, 2010, 329, 5989

Replace all that with a single enzyme step!

Round of Evolution

1

10

100

1000

10000

100000

1 2 3 4 5 6 7 8 9 10 11 12

100000

10000

1000

100

10

1

Improved

70,000 fold

Started here

Sit

Rela

tive p

roductivity

Solvent waste

reduced 60%

Increased yield

from starting

materials

Directed enzyme evolution

Replace all that with a single enzyme step!

Round of Evolution

1

10

100

1000

10000

100000

1 2 3 4 5 6 7 8 9 10 11 12

100000

10000

1000

100

10

1

Improved

70,000 fold Toxic heavy metals

eliminated

Savile et al. Science, 2010, 329, 5989

Sit

Replace all that with a single enzyme step!

FDA Approval in 2012

AWARDThomas Edison

Patent Award

Toxic heavy metals

eliminated

Solvent waste

reduced 60%

Increased yield

from starting

materials

Savile et al. Science, 2010, 329, 5989

PRESIDENTIAL

We need better cellulases to break

down biomass and generate sugars

for microbial chemicals and fuel production.

MTIKEMPQPKTFGELKNL...

KETSPIPQPKTFGPLGNL...

KQASAIPQPKTYGPLKNL...

WRRRGIPGPLGYPLVGSF...

WIRKGVKGPRGLPFLGVI...

FIRKGIKGPRGFPGIGML...

WIRKGVKGPRGFPFFGVI...

WIRKGVKGPRGFPFFGVI...

WMRKGIKGPRGLPFFGII...

WMRKGVKGPRGRPFVGVL...

WRRRGVVGPMGFPVLGVF...

REKIGLSGPEPHWFLGNL...

REKIGLTGPEPHWFMGNL...

RSSIGIPGPPVHWLWGNL...

KVSKYPKGPLPLPFIGNI...

...

We can also use products of natural evolution to breed new enzymes by recombination (molecular sex)

Sexual recombination populates fitness peaks (conservative of structure and function while making large jumps in sequence)

Parent proteins, found in nature

Chimeric progeny can be better than their parents.

Sexual recombination populates fitness peaks (conservative of structure and function while making large jumps in sequence)

We used recombination and random mutations to make thermostable cellulases that hydrolyze more biomass at

elevated temperatures

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

35 45 55 65 75 85 95

Cel

lob

iose

(mM

)

Temperature (ºC)

3C6P

HJPlus

HICel6

HJCel6

Enzymes (and other proteins) are highly ‘evolvable’

Proteins can adapt via simple uphill walks

New functions by changing tiny fractions of

(<2 %) of the sequence

Scary fact: Many beneficial

mutations are far from the

active site.

Directed evolution can optimize enzyme function

in real time, circumventing our profound ignorance

of how sequence encodes function.

How can we create whole new enzymes to expand the chemistry of the biological world?

(catalyze reactions not known in nature)

How can an inherently conservative process like this create novelty?

At any given time, nature takes the most probable paths

(the fewest mutational ‘steps’).

(Molecular)

Evolution does it: the internet of living things

Crowd-sourcing problem solving for 3+ billion years: trillions of organisms at work 24/7

Atrazine

• 1950–1993 non-biodegradable; accumulated in soil

• From 1993 onwards, fast degradation observed

“Novelty” is often already there.

Access to new features relies on some (even very small) functional

overlap that can be drawn out and optimized during evolution.

Native reaction New reaction

Natural ‘oxene’

transfer

Non-natural

Fe-carbene

Non-natural

Fe-nitrene

We evolved heme proteins that catalyze reactions not known in nature

Ser

Ser

O

O

Heme enzyme

Cyclopropane-containing serotonin-norepinephrine

reuptake inhibitor (SNRI) used to treat clinical depression

Jane Wang, Hans Renata et al., ACIE (2014)

Second most

abundant element

in the Earth’s crust

Si14

28.0855

Silicon

...the literature is void of examples of biologically synthesized, either in vivo or in vitro,

silicon-carbon bonds.

Silicon 2009, 1, 147.

NH

Si

OH

RR

catalysts / ligands

SiR3

reagents for cross-coupling

Si

18F-acceptors for

PET imaging

Si(i-Pr)3

Si(i-Pr)3

materials for organic LED

biological probes

and drug leads

O

NH

H2N

Me3Si O

OH

18F

RR

3

Rhodothermus marinus cytochrome c

SiH

MeMe

N2

Me

O

OEtMe

O

OEtSi

MeMeRma cyt c+

Physically possible molecules…

Relevant to biology

Freed from the constraints of biological

function, we can explore the physically

possible proteins to find new capabilities.

Bacterial enzymes that mimic human drug metabolism so that we can test the toxicity of new drug candidates

New ‘human’ enzymes to treat cancer

Proteins to monitor brain chemistry or enable brain

stimulation with light

A palette of fluorescent sensor proteins for imaging

Better agricultural practices: combat crop pests, effects of climate change…

Fe

O

S

Cys

+

Fe

C

S

Cys

RR

AATAGCCGTTATTTCCGGATGTGCATAGCTGATTTGACCCATCCGGTACAC

CAATDACAAATCCCGATTTGATCGTGTGCGCGACATGTCTTCCGGCGACA

CATGTGTCTCTCACTCCGAGAGATCGGTTAGAGTCTCGGTTAACCACACG

TCCCGGATATATTTAATTGGCCGGAGAGTCTCCCGCGCGACATAAGGAGT

CCTCGTTTCGAGATACGTACGGCATGGTGACACCAGTTGCCCTCTGATTC

CCGGAGCCTCTTTGAAAACGTCGGGACAAATCCCGATTTGATCGTGTGCG

CGACATGTCTTCCGGCGACACATGTGTCTCTCACTCCGAGAGATCGGTTA

GAGTCTCGGTTAACCACACGTCCAATAGCCGTTATTTCCGGATGTGCATAG

The future of production (the bioeconomy)