bioRxiv (Cold Spring Harbor Laboratory),
Journal Year:
2023,
Volume and Issue:
unknown
Published: Oct. 12, 2023
Abstract
Tryptophan
synthase
catalyzes
the
synthesis
of
a
wide
array
non-canonical
amino
acids
and
is
an
attractive
target
for
directed
evolution.
Droplet
microfluidics
offers
ultrahigh
throughput
approach
to
evolution
(>10
7
experiments
per
day),
enabling
search
biocatalysts
in
wider
regions
sequence
space
with
reagent
consumption
minimized
picoliter
volume
(per
library
member).
While
majority
screening
campaigns
this
format
on
record
relied
optically
active
reaction
product,
new
assay
needed
tryptophan
synthase.
not
fluorogenic
visible
light
spectrum
thus
falls
outside
scope
conventional
droplet
microfluidic
read-outs
which
are
incompatible
UV
detection
at
high
throughput.
Here,
we
engineer
DNA
aptamer
into
biosensor
quantitatively
report
production
droplets.
The
utility
was
validated
by
identifying
5-fold
improved
synthases
from
∼100,000
protein
variants.
More
generally
work
establishes
use
DNA-aptamer
sensors
read-out
widening
RSC Chemical Biology,
Journal Year:
2024,
Volume and Issue:
5(7), P. 595 - 616
Published: Jan. 1, 2024
Carbohydrate-active
enzymes
(CAZymes)
constitute
a
diverse
set
of
that
catalyze
the
assembly,
degradation,
and
modification
carbohydrates.
These
have
been
fashioned
into
potent,
selective
catalysts
by
millennia
evolution,
yet
are
also
highly
adaptable
readily
evolved
in
laboratory.
To
identify
engineer
CAZymes
for
different
purposes,
(ultra)high-throughput
screening
campaigns
frequently
utilized
with
great
success.
This
review
provides
an
overview
approaches
taken
how
mechanistic
understandings
can
enable
new
to
screening.
Within,
we
cover
cutting-edge
techniques
such
as
microfluidics,
advances
computational
synthetic
biology,
well
novel
assay
designs
leading
field
towards
more
informative
effective
approaches.
bioRxiv (Cold Spring Harbor Laboratory),
Journal Year:
2023,
Volume and Issue:
unknown
Published: Nov. 22, 2023
Abstract
Enzyme
engineering
and
discovery
are
crucial
for
a
future
sustainable
bioeconomy,
harvesting
new
biocatalysts
from
large
libraries
through
directed
evolution
or
functional
metagenomics
requires
accessible,
rapid
assays.
Ultra-high
throughput
screening
can
often
require
an
optical
readout,
leading
to
the
use
of
model
substrates
that
may
not
accurately
report
on
activity
target
reaction
bespoke
synthesis.
In
contrast,
coupled
assays
represent
modular
‘plug-and-play’
system,
where
any
pairing
enzyme/substrate
be
investigated,
if
produce
common
intermediate
which
links
catalytic
detection
cascade
readout.
Here
we
establish
cascade,
producing
fluorescent
readout
in
response
NAD(P)H
via
glutathione
reductase
subsequent
thiol-mediated
uncaging
reaction,
with
30
nM
limit.
We
demonstrate
its
utility
glycosidase
AxyAgu
115A
(producing
monosaccharides
natural
biofuel
feedstock)
three
orders
magnitude
improved
sensitivity
compared
absorbance-based
systems,
so
less
than
one
turnover
per
enzyme
molecule
expressed
single
cell
is
detectable.
These
advantages
brought
bear
plate
formats,
but
also
picoliter
emulsion
droplets,
enrichments
950-fold
suggest
interrogated
against
specific
query
substrate.
ACS Catalysis,
Journal Year:
2024,
Volume and Issue:
14(17), P. 12884 - 12894
Published: Aug. 12, 2024
In
search
of
efficient
α-galactosidases
that
can
convert
B
red
blood
cells
(RBCs)
to
universal
type
RBCs,
we
have
developed
a
simple
and
robust
system
for
ultrahigh-throughput
droplet-based
microfluidic
screening.
Here,
multienzyme
coupled
assay
with
fluorogenic
antigen
tetrasaccharide
substrate
is
encapsulated
within
single
emulsion
water-in-oil
droplets
alongside
from
metagenomic
libraries.
The
resulting
fluorescent
containing
candidate
cleaving
enzymes
are
sorted
using
commercially
available,
walk-up
droplet
sorting
instrument
before
validation,
cloning,
characterization
the
hits.
Using
this
approach,
identified
characterized
an
α-1,3-galactosidase
(PvGH110)
human
gut
microbiome
capable
converting
B-to-O-type
RBCs.
simplicity,
efficiency,
accessibility
our
microfluidic-based
make
it
suitable
nonspecialist
laboratories
offer
promising
tool
discover
enable
generation
O
type.
bioRxiv (Cold Spring Harbor Laboratory),
Journal Year:
2023,
Volume and Issue:
unknown
Published: Oct. 12, 2023
Abstract
Tryptophan
synthase
catalyzes
the
synthesis
of
a
wide
array
non-canonical
amino
acids
and
is
an
attractive
target
for
directed
evolution.
Droplet
microfluidics
offers
ultrahigh
throughput
approach
to
evolution
(>10
7
experiments
per
day),
enabling
search
biocatalysts
in
wider
regions
sequence
space
with
reagent
consumption
minimized
picoliter
volume
(per
library
member).
While
majority
screening
campaigns
this
format
on
record
relied
optically
active
reaction
product,
new
assay
needed
tryptophan
synthase.
not
fluorogenic
visible
light
spectrum
thus
falls
outside
scope
conventional
droplet
microfluidic
read-outs
which
are
incompatible
UV
detection
at
high
throughput.
Here,
we
engineer
DNA
aptamer
into
biosensor
quantitatively
report
production
droplets.
The
utility
was
validated
by
identifying
5-fold
improved
synthases
from
∼100,000
protein
variants.
More
generally
work
establishes
use
DNA-aptamer
sensors
read-out
widening
