One‐Pot Hetero‐Di‐C‐Glycosylation of the Natural Polyphenol Phloretin by a Single C‐Glycosyltransferase With Broad Sugar Substrate Specificity
Biotechnology and Bioengineering,
Journal Year:
2025,
Volume and Issue:
unknown
Published: Feb. 7, 2025
The
structural
motif
of
hetero-di-C-glycosyl
compound
is
prominent
in
plant
polyphenol
natural
products
and
involves
two
different
glycosyl
residues
(e.g.,
β-d-glucosyl,
β-d-xylosyl)
attached
to
carbons
the
same
phenolic
ring.
Polyphenol
hetero-di-C-glycosides
attract
attention
as
specialized
ingredients
herbal
medicines
their
tailored
synthesis
by
enzymatic
C-glycosylation
promising
overcome
limitations
low
availability
expand
molecular
diversity
new-to-nature
glycoside
structures.
However,
installing
these
di-C-glycoside
structures
with
synthetic
precision
efficiency
challenging.
Here
we
have
characterized
syntheses
C-β-galactosyl-C-β-glucosyl
C-β-glucosyl-C-β-xylosyl
on
phloroglucinol
ring
phloretin,
using
kumquat
(Fortunella
crassifolia)
C-glycosyltransferase
(FcCGT).
FcCGT
uses
uridine
5'-diphosphate
(UDP)-galactose
(5
mU/mg)
UDP-xylose
(0.3
U/mg)
at
lower
activity
than
UDP-glucose
(3
U/mg).
3'-C-β-glucoside
(nothofagin)
~10-fold
less
reactive
non-glycosylated
phloretin
all
UDP-sugars,
suggesting
practical
order
hetero-di-C-glycosylation
C-galactosylation
or
C-xylosylation
followed
C-glucosylation
resulting
mono-C-glycoside.
Each
performed
presence
twofold
excess
UDP-sugar
proceeds
completion
appears
be
effectively
irreversible,
evidenced
absence
residue
exchange
extended
reaction
times.
Synthesis
shown
10
mM
concentration
quantitative
conversion
cascade
synthase,
allowing
for
situ
formation
from
more
expedient
donor
substrate
UDP-glucuronic
acid.
desired
Xyl
Gal
was
obtained
a
single
product
its
structure
confirmed
NMR.
Language: Английский
Discovery, characterization, and comparative analysis of new UGT72 and UGT84 family glycosyltransferases
Communications Chemistry,
Journal Year:
2024,
Volume and Issue:
7(1)
Published: June 28, 2024
Abstract
Glycosylated
derivatives
of
natural
product
polyphenols
display
a
spectrum
biological
activities,
rendering
them
critical
for
both
nutritional
and
pharmacological
applications.
Their
enzymatic
synthesis
by
glycosyltransferases
is
frequently
constrained
the
limited
repertoire
characterized
enzyme-catalyzed
transformations.
Here,
we
explore
glycosylation
capabilities
substrate
preferences
newly
identified
plant
uridine
diphosphate
(UDP)-dependent
(UGTs)
within
UGT72
UGT84
families,
with
particular
focus
on
polyphenol
from
UDP-glucose.
Four
UGTs
are
classified
according
to
their
phylogenetic
relationships
reaction
products,
identifying
as
biocatalysts
either
glucoside
(UGT72
enzymes)
or
glucose
ester
(UGT84
members)
formation
selected
phenylpropanoid
compounds.
Detailed
kinetic
evaluations
expose
unique
attributes
these
enzymes,
including
specific
activities
regio-selectivities
towards
diverse
polyphenolic
substrates,
characterizations
validating
capacity
family
members
perform
di-
O
-glycosylation
flavones.
Sequence
analysis
coupled
structural
predictions
through
AlphaFold
reveal
an
unexpected
absence
conserved
threonine
residue
across
all
four
trait
previously
linked
pentosyltransferases.
This
comparative
broadens
understood
specificity
range
enhancing
our
understanding
utility
in
production
phenolic
glycosides.
The
findings
this
in-depth
characterization
provide
valuable
insights
into
functional
versatility
UGT-mediated
reactions.
Language: Английский
Solvent Engineering for Nonpolar Substrate Glycosylation Catalyzed by the UDP-Glucose-Dependent Glycosyltransferase UGT71E5: Intensification of the Synthesis of 15-Hydroxy Cinmethylin β-d-Glucoside
Journal of Agricultural and Food Chemistry,
Journal Year:
2023,
Volume and Issue:
71(36), P. 13419 - 13429
Published: Sept. 1, 2023
Sugar
nucleotide-dependent
glycosyltransferases
are
powerful
catalysts
of
the
glycosylation
natural
products
and
xenobiotics.
The
low
solubility
aglycone
substrate
often
limits
synthetic
efficiency
transformation
catalyzed.
Here,
we
explored
different
approaches
solvent
engineering
for
reaction
intensification
β-glycosylation
15HCM
(a
C15-hydroxylated,
plant
detoxification
metabolite
herbicide
cinmethylin)
catalyzed
by
safflower
UGT71E5
using
UDP-glucose
as
donor
substrate.
Use
a
cosolvent
(DMSO,
ethanol,
acetonitrile;
≤50
vol
%)
or
water-immiscible
(n-dodecane,
n-heptane,
n-hexane,
1-hexene)
was
ineffective
due
to
enzyme
activity
stability,
both
impaired
≥10-fold
compared
pure
aqueous
solvent.
Complexation
in
2-hydroxypropyl-β-cyclodextrin
enabled
dissolution
50
mM
while
retaining
(∼0.32
U/mg)
stability.
Using
recycling,
converted
completely,
β-d-glucoside
isolated
90%
yield
(∼150
mg).
Collectively,
this
study
highlights
requirement
mild,
enzyme-compatible
strategy
enhancement
glycosyltransferase
catalysis
applied
glycoside
synthesis.
Language: Английский
Construction and Optimization of Engineered Saccharomyces cerevisiae for De Novo Synthesis of Phloretin and Its Derivatives
Journal of Agricultural and Food Chemistry,
Journal Year:
2024,
Volume and Issue:
unknown
Published: Dec. 26, 2024
Phloretin
and
its
derivatives
are
dihydrochalcone
compounds
with
diverse
pharmacological
properties
biological
activities,
offering
significant
potential
for
applications
in
the
food
pharmaceutical
industries.
Due
to
their
structural
similarity
flavonoids,
extraction
isolation
were
highly
challenging.
Although
biosynthesis
of
phloretin
via
three
distinct
pathways
has
been
reported,
a
systematic
comparison
within
same
host
yet
be
conducted.
In
this
study,
we
employed
rational
design
synthetic
biology
approaches
engineer
Saccharomyces
cerevisiae
de
novo
synthesis
derivatives.
We
constructed
evaluated
biosynthetic
S.
cerevisiae,
demonstrating
that
effective
is
achievable
only
p-coumaryl-CoA
pathway.
Additionally,
by
optimizing
enzyme
screening,
strain
engineering,
coordinating
heterologous
endogenous
metabolism,
achieved
highest
reported
titer
287.2
mg/L
phloretin,
184.6
phlorizin,
103.1
trilobatin,
164.5
nothofagin
first-time
4-methylphloretin
hesperetin
dihydrochalcone.
This
study
was
committed
addressing
growing
demand
dihydrochalcones
while
laying
foundation
more
complex
Language: Английский