of 6
German
Edition
:D
OI:10.1002/ange.201708408
Biocatal
ysi
s
International
Edition
:DOI:10.1002/anie.201708408
Directed
Evolution
:Bringing
NewChemistry
to Life
Frances
H. Arnold*
biocatalysis
·enzymes
·heme
proteins
·
protein
engineering
·synthetic
methods
Survival
of the
Fittest
In this competitive
age,when new
industries
sprout
and
decay
in the span
of adecade
,weshould
reflect
on how
acompany
survives
to celebrate
its 350th
anniversary
.A
prerequisite
for survival
in business
is the ability
to adapt
to
changing
environments
and
tastes
,and to sense
,anticipate
,
and meet
needs
faster
and better
than
the competition.
This
requires
constant
innovation
as well
as focused
attention
to
execution.
Acompany
that
continues
to provide
meaningful
and
profitable
solutions
to
human
problems
has
ac
hance
to
survive
,even
thrive
,ina
rapidly
changing
and
highly
competitive
world.
Biology
has abrilliant
algorithm
for solving
the problem
of survival
over
time
:evolution.
Those
who
adapt
and
(re)produce
outcompete
the less agile
and less fertile
.Over
the last 30 years—which
seems
along time
but is less than
one-tenth
the time
Merck
KGaA,
Darmstadt,
Germany
has
been
in business—I
have
tried
to adapt
biology

smechanisms
for innovation
and
optimization
to solving
problems
in
chemistry
and
engineering
.Itturns
out that
evolution
is
apowerful
forward-engineering
process
,whose
widespread
adoption
in enzyme
engineering
and
synthetic
biology
has
been
made
possible
through
advances
in molecular
biology
and high-throughput
screening
.
Expanding
Nature’s
Catalytic
Repertoire
for
aSustainable
Chemical
Industry
Nature
,the best
chemist
of all time,solves
the difficult
problem
of being
alive
and
enduring
for billions
of years
,
under
an astonishing
range
of conditions
.Most of the
marvelous
chemistry
that
makes
life possible
is the work
of
nature

smacromolecular
protein
catalysts
,the enzymes
.By
using
enzymes
,nature
can extract
materials
and energy
from
the environment
and convert
them
into
self-replicating
,self-
repairing
,mobile
,adaptable
,and sometimes
even
thinking
biochemical
systems
.These
systems
are good
models
for
asustainable
chemical
industry
that uses
renewable
resources
and recycles
agood fraction
of its products
.And biology
is not
just amodel
from
which
to draw
inspiration
:living
organisms
or their
components
can be efficient
production
platforms
.In
fact,
Ipredict
that
DNA-programmable
microorganisms
will
be producing
many
of our chemicals
in the not-so-distant
future
.
That most
chemicals
are made
using
synthetic
processes
starting
from
petroleum-based
feedstocks
reflects
the re-
markable
creativity
of synthetic
chemists
in developing
reaction
schemes
and catalysts
that
nature
never
discovered.
Synthetic
chemistry
has given
us an explosion
of products
,
which
feed,
clothe
,house,entertain,
and
cure
us.Synthetic
chemistry
,however,
struggles
to match
the efficienc
yand
selectivity
that biology
achieves
with
enzymes
.Inmany cases
,
synthetic
processes
rely on precious
metals
,toxic reagents
and
solvents
,and extreme
conditions
,and they
generate
substan-
tial amounts
of unwanted
byproducts
.DNA-programmable
chemical
synthesis
using
enzymes
promises
to improve
on
synthetic
chemistry
,particularly
if we are able
to expand
biology

scatalytic
repertoire
to include
some
of the most
synthetically
useful
reactions
,under
physiological
conditions
and with
earth-abundant
resources
.Such clean,
green
chemis-
try might
sound
like pie in the sky,but enzymes
already
show
how
aprotein
can
orient
substrates
for reaction,
exclude
water
from
an active
site,activate
ametal or simple
organic
cofactor,
or suppress
competing
reactions
to draw
out new
and admirable
synthetic
capabilities
.Synthetic
chemists
have
been
drawing
inspiration
from
biology
for decades
,and now
is
the
time
for protein
engineers
to use
inspiration
from
synthetic
chemistry
to generate
new
enzymes
that
will
improve
on and
replace
synthetic
catalysts
and
reaction
pathways
.
[1]
Unfortunately
,our understanding
of the link
between
sequence
and
function
lags
well
behind
our desire
for new
[*] Prof.
F. H. Arnold
Division
of Chemist
ry and Chemical
Engineeri
ng
California
Institute
of Technology
210-41
1200
E. California
Blvd.
,Pasadena,
CA 91125
(USA)
E-mail
:frances@chem
e.caltech.edu
Homepag
e: http://fhalab.c
altech.edu
The ORCID
identifica
tion number
for the author
of this article
can be
found
under
https
://doi.org/
10.1002/ani
e.2017084
08.

2017
The Authors.
Published
by Wiley-V
CH Verlag GmbH
&
Co.
KGaA.
This
is an open
access
article
under
the terms
of the Creative
Commons
Attribution
Non-C
ommercial
License,
which
permits
use,
distributi
on and reprod
uction
in any medium,
provided
the original
work
is properly
cited,
and is not used
for commercial
purposes.
This
article
is part of the Special
Issue
to commemora
te the 350th
anniversar
yofMerck KGaA,
Darmstadt,
Germany
.More articles
can
be found
at http://doi.wiley
.com/10.
1002/anie
.v57.16.
A
ngewan
dte
Che
mi
e
Essays
4143
Angew
.Chem.
Int.Ed.
2018
,
57
,4143 –4148

2018
The
Autho
rs. Publish
ed by Wiley-VCH
VerlagG
mbH
&C
o. KGaA
,Weinhei
m
enzymes
.Given
that
our ability
to predict
protein
sequences
,
or even
just changes
to asequence
,which
reliably
give
rise to
whole
new,finely
tuned
catalytic
activities
is rudimentary
at
best,
creating
new
enzymes
capable
of improving
on current
synthetic
processes
is apretty
tall order
.Wealso dream
of
going
beyond
known
chemistry
to create
enzymes
that
catalyze
reactions
or make
products
that
are simply
not
possible
with
any
known
method,
synthetic
or otherwise
.
Requiring
that
these
new
enzymes
assemble
and function
in
cells,where
they
can be made
at low cost
and incorporated
into synthetic
metabolic
pathways
to generate
abroader
array
of products
,represents
an even
greater
set of engineering
constraints
and challenges
.
Nature

senzymes
are the products
of evolution,
not
design.
By using
generations
of mutation
and
selection
for
fitness
advantages
,evolution
allows
organisms
to continu-
ously
update
and
optimize
their
enzyme
repertoires
.New
enzymes
even
appear
in real
time
in response
to challenges
(e.g. the need
to resist
antibiotics
or pesticides)
or oppor
-
tunities
(e.g. the chance
to occupy
anew food
niche
by
degrading
recently
introduced,
manmade
substances).
Iargue
that the process
that gave
rise to all the remarkable
biological
catalysts
in nature
should
be able
to produce
yet more
.Inthe
laboratory
.Quickly
.Advances
in molecular
biology
over
the
past
few decades—the
ability
to write
,cut, and paste
DNA
and to have
that
DNAread and translated
into
proteins
in
recombinant
organisms—have
given
us the ability
to breed
enzymes
much
like
we breed
sheep
or sake
yeast.
We can
direct
the evolution
of enzymes
in the laboratory
by requiring
them
to perform
in ways
that may
not be useful
to abacterium
but
are
useful
to us.Directed
evolution
achieves
these
desirable
functional
outcomes
while
circumventing
our deep
ignorance
of how
sequence
encodes
them.
Directed
evolution
mimics
evolution
by artificial
selec-
tion,
and is accelerated
in the laboratory
setting
by focusing
on individual
genes
expressed
in fast-growing
microorgan-
isms.Westart with
existing
proteins
(sourced
from
nature
or
engineered),
introduce
mutations
,and then
screen
for the
progeny
proteins
with
enhanced
activity
(or another
desirable
trait).
We use the improved
enzymes
as parents
for the next
round
of mutation
and
screening,
recombining
beneficial
mutations
as needed,
and continuing
until
we reach
the target
level
of performance
.
Engineering
enzymes
in the 1980s
and 1990s
,Ilearned
the
hard
way
that
there
was
no reliable
method
to predict
performance-enhancing
mutations
.Turning
instead
to ran-
dom
mutagenesis
and screening
,Iquickly
realized
that
such
mutations
were
easy
to find
and
accumulate
with
the right
evolutionary
optimization
strategy
.Mystudents
and
Iob-
served
that proteins
,the products
of evolution,
are themselves
readily
evolvable
.Properties
we and
others
targeted
in the
early
days
of directed
evolution
(the
mid-1990s)
included
recovering
activity
in unusual
environments
(e.g.organic
solvents),
improving
activity
on non-native
substrates
,en-
hancing
thermostability
,and changing
enantioselectivity
.We
learned
the then-surprising
fact
that
beneficial
mutations
could
be far from
an active
site,and often
appeared
on the
protein
surface
(which
in those
days
was generally
deemed
insensitive
to mutation
and functionally
neutral).
To this day,
no one
can explain
satisfactorily
how
such
mutations
exert
their
effects
,much less predict
them.
Evolution
of Novelty
:Enzymes
that
Catalyze
Reactions
Invented
by Synthetic
Chemists
Although
we could
enhance
activity
(and
many
other
properties)
by accumulating
beneficial
mutations
over
gen-
erations
of random
mutagenesis
and
screening,
evolving
awhole
new
catalytic
activity
seemed
amuch more
difficult
problem.
After
all, evolution
is not good
for problems
that
require
multiple
,simultaneous
,low-probability
events
,
[2]
and
the active
sites
of enzymes
are so beautifully
and
precisely
configured
that
it was
hard
to imagine
how
the stepwise
accumulation
of beneficial
mutations
could
create
anew one.
Evolution

sinnovation
mechanisms
,however,
are more
simple
than
they
might
appear
:evolution
works
best
when
it
does
not need
to generate
awhole
new
active
site
from
scratch.
Instead,
evolution
can generate
anew enzyme
from
one that
is “close”,
that
is,shares
elements
of mechanism
or
machinery
from
which
the new
activity
can be built.
Nature
co-opts
old machinery
to do new
jobs.And sometimes
the
ability
to do the new job is already
there
,atleast at alow level.
Thebiological
world
is replete
with
proteins
whose
capabil-
ities
extend
well
beyond
what
may
be used
at any given
time.
Thus new
enzymes
are
built
from
promiscuous
or side
activities
that
become
advantageous
in anew biological
context,
such
as when
anew food
source
becomes
available
.
[3]
Thus aconservative
process
of accumulating
beneficial
mutations
can
innovate
because
the innovation
is already
there
!The magnificent
diversity
of the biological
world
provides
the fuel for further
innovations
.
Fordirected
evolution
to be areliable
approach
to
creating
new
enzymes
,wethe breeders
of proteins
must
first
identify
potential
catalytic
novelty
in the form
of starting
proteins
which
have
at least
low levels
of anew activity
.We
therefore
look
for activities
that
are known
to synthetic
chemistry
,but perhaps
not explored
in nature
.Cytochrome
P450s
,whose
native
functions
include
avariety
of extremely
challenging
transformations
such
as hydroxylation,
epoxida-
tion,
heteroatom
oxidations
,nitration
and more
,looked
to me
like
apromising
place
to start
hunting
for new
activities
.
Nature
had
already
exploited
this
evolvable
heme-protein
assembly
and
the
various
reactive
intermediates
in the
catalytic
cycle
to create
all the natural
P450
functions
.We
Frances
Arnold
is the Linus
Pauling
Profes-
sor of Chemical
Engineerin
g, Bioenginee
ring,
and Biochem
istry at the California
Institute
of Technology
,where
her research
focuses
on enzyme
engineering
by directed
evolu-
tion,
with
applications
in sustainable
fuels
and chemicals.
She uses evolution’s
innova-
tion mechanisms
to bring
new chemical
reactions
to biology
.Her honors
include
the
Millenniu
mTechnology
Prize
(2016).
She
has been
elected
to the US National
Academies
of Science,
Medic
ine, and Engi-
neering.
A
ngewandte
Chem
i
e
Essays
4144
www
.angewandte.org

2018
The
Autho
rs. Published
by Wiley-
VCH
Verlag
GmbH
&C
o. KGaA,
Weinhei
m
Angew
.Chem.
Int.
Ed.
2018
,
57
,4143 –4148
quickly
discovered
that
many
more
new,non-natural
func-
tions
were
possible
.Inthe last few years
we have
engineered
P450s
and
other
heme
proteins
to carry
out aplethora
of
reactions
known
to synthetic
chemists
,but not
found
in
biology
.
[4]
Forexample
,olefin
cyclopropa
nation
by carbene
transfer
is areaction
well
known
in the area
of transition-metal
catalysis
,but not known
to be catalyzed
by an enzyme
.In
2012,
inspired
by much
older
reports
of heme
mimics
performing
such
reactions
in organic
solvents
,wediscovered
that heme
proteins
catalyze
cyclopropanation
when
provided
with
diazo
carbene
precursors
and asuitable
olefin,
in water.
[5]
This promiscuous
activity
is manifested
when
the protein
encounters
the diazo
reagent,
forms
the reactive
carbene
,and
then
transfers
it to the olefin.
Our
lab took
advantage
of this
inherent
ability
of abacterial
cytochrome
P450
to evolve
ahighly
efficient
enzyme
for production
of the chiral
cis
-
cyclopropane
precursor
to the
antidepressant
medication
levomilnacipran.
[6]
Our
group
and
that
of Rudi
Fasan have
since
pushed
avariety
of heme
proteins
to synthesize
other
chiral
cyclopropane
pharmaceutical
precursors
,including
one
used
in the synthesis
of ticagrelor
,amedication
used
to
prevent
the reoccurrence
of heart
attacks
.
[7]
In our case,we
identified
atruncated
globin
from
Bacillus
subtilis
,which
catalyzes
the reaction
at low levels
and
also
showed
some
selectivity
for producing
the single
,desired
diastereomer
of
the ticagrelor
cyclopropane
precursor
from
ethyl
diazoacetate
and 3,4-difluorostyrene
(Figure
1). Just afew generations
of
directed
evolution
improved
the activity
and selectivity
of the
enzyme
so that,
of the four
possible
stereoisomers
,itproduces
the ticagrelor
cyclopropane
almost
exclusively
.Because
the
reaction
proceeds
in whole
Esch
erichia
coli
cells
which
express
the evolved
enzyme
,producing
the catalyst
is as
simple
as growing
bacteria.
While we were
investigating
carbene-transfer
reactions
catalyzed
by heme
proteins
,wealso looked
into the possibility
of evolving
enzymes
for nitrene-transfer
reactions
.Inspired
by ahint in the
literature
from
the
1980
stoattempt
intramolecular
C
@
Hamination,
we were
delighted
to find
that
acytochrome
“P411”
exhibited
alow level
of promiscu-
ous activity
with
an aryl sulfonyl
azide
nitrene
precursor
,and
that
activity
could
be improved
by directed
evolution.
[8]
We
purposefully
engineered
the P411
by replacing
the completely
conserved
cytochrome
P450
cysteine
ligand,
which
is bound
to
the iron
center
,with serine
,aligand
not found
in any known
natural
heme
protein.
This change
shifts
the characteristic
peak
in the CO difference
spectrum
from
l
=
450 to 411 nm
and
abolishes
the native
monooxygenase
activity
.Italso
greatly
promotes
carbene-transfer
and nitrene-transfer
activ-
ities.
After
demonstrating
that
the P411
derived
from
Bacillus
megaterium
cytochrome
P450
could
be engineered
for intra-
molecular
C
@
Hamination,
and intermolecular
aziridination
and
sulfimidation
activities
unknown
in biological
systems
,
our efforts
culminated
in cytochrome
P411
CHA
,which
cata-
lyzes
intermolecular
benzylic
C
@
Hamination.
[9]
Efficient
and
highly
enantioselective
intermolecular
amination
of C(sp
3
)
@
Hbonds
has long
been
achallenge
in chemical
catalysis
.
Despite
screening
many
different
heme
proteins
and protein
variants
,however,
we never
found
one
with
the desired
activity
until
postdoctoral
fellow
Chris
Prier
discovered
that
the P411
variant
“P4”,
evolved
for an intermolecular
sulfimi-
dation
and rearrangement
reaction,
had acquired
promiscu-
ous activity
for benzylic
C
@
Hamination.
Chris
Prier
and
doctoral
student
Kelly
Zhang
then
directed
the evolution
of
P4 to create
P411
CHA
,which
exhibits
hundreds
of turnovers
for the amination
of benzylic
C
@
Hbonds
with
excellent
enantioselectivities
(
>
99%
ee
).
[9]
Free heme
does
not catalyze
any of these
nitrene-transfer
reactions
,and small-molecule
catalysts
for
direct
C
@
H
amination
rely
heavily
on precious
metals
which
are not
sustainable
.The protein,
however,
can
impart
this
new
reactivity
to earth-abundant
iron
in its porphyrin
cofactor
,
and
it is evolvable
.Evolution
enabled
P411
CHA
to promote
nitrenoid
formation
and
transfer
to
as
econd
substrate
over
the competing
nitrene
reduction
heavily
favored
in the parent
enzyme
,
[9]
aproperty
that
would
be extremely
challenging,
if
not impossible
,todesign.
In fact,
we think
of these
proteins
as
chiral,
self-assembling
,DNA-encoded
macromolecular
tran-
sition-metal
complexes
whose
steric
and electronic
properties
are readily
tuned
by directed
evolution
to achieve
desired
activities
and selectivities
.
Recently
we have
been
exploring
enzymes
that
open
yet
more
chemical
space
for biocatalysis
,including
enzymes
that
form
chemical
bonds
unknown
in biology
.Inthe last year
we
described
heme
enzymes
that
catalyze
carbene
insertion
into
Si
@
Hand B
@
Hbonds,thus giving
living
systems
their
first
carbon–silicon
[10]
and
carbon–boron
[11]
bond-forming
activi-
ties.C
@
Si bonds
are useful
in medicinal
chemistry
,imaging
agents
,elastomers
,and awide variety
of consumer
products
,
but they
have
never
been
found
in biological
systems
.Until
now,the only
methods
to create
these
bonds
enantioselec-
tively
involved
multistep
syntheses
just
to prepare
chiral
reagents
or chiral
transition-metal
complexes
.The resulting
catalysts
are often
only
poorly
active
,and an iron-based
catalyst
had
never
been
reported
for this carbene-insertion
reaction.
Upon
screening
acollection
of heme
proteins
,
postdoctoral
fellow
Jennifer
Kan
and
her team
discovered
that
asmall
(124
aa),
highly
stable
cytochrome
c
from
Rhodothermus
marinus
(
Rma
cyt
c
)could
catalyze
the
reaction
between
ethyl
2-diazopropanoate
and phenyldime-
thylsilane
to form
the chiral
organosilicon
product
with
high
enantioselectivity
(Figure
2A). Directed
evolution
discov-
ered
three
mutations
that
enable
the enzyme
to form
C
@
Si
bonds
with
up to 8200
total
turnovers
(based
on
Rma
cyt
c
concentration)
and
enantioselectivities
with
greater
than
99%
ee
for awide range
of silicon-containing
substrates
.
Figure
1.
A
B. subtilis
globin
variant,
engineered
by directed
evolution
,
catalyzes
the cyclopropan
ation
of 3,4-difluo
rostyrene
to make
the
desired
stereois
omer
of aticagrelor
precursor
with
high
selectiv
ity and
yield.
[7a]
A
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dte
Che
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Essays
4145
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Int.Ed.
2018
,
57
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2018
The
Authors.
Publish
ed by Wiley-VCH
Verlag
GmbH
&C
o. KGaA,
Weinheim
www
.angewandte.org