Chloroplast Cell-Free Systems from Different Plant Species as a Rapid Prototyping Platform
ACS Synthetic Biology,
Journal Year:
2024,
Volume and Issue:
13(8), P. 2412 - 2424
Published: July 19, 2024
Climate
change
poses
a
significant
threat
to
global
agriculture,
necessitating
innovative
solutions.
Plant
synthetic
biology,
particularly
chloroplast
engineering,
holds
promise
as
viable
approach
this
challenge.
Chloroplasts
present
variety
of
advantageous
traits
for
genetic
but
the
development
tools
and
part
characterization
in
these
organelles
is
hindered
by
lengthy
time
scales
required
generate
transplastomic
organisms.
To
address
challenges,
we
have
established
versatile
protocol
generating
highly
active
chloroplast-based
cell-free
gene
expression
(CFE)
systems
derived
from
diverse
range
plant
species,
including
wheat
(monocot),
spinach,
poplar
trees
(dicots).
We
show
that
work
with
conventionally
used
T7
RNA
polymerase
well
endogenous
polymerases,
allowing
detailed
prototyping
regulatory
sequences
at
both
transcription
translation
levels.
demonstrate
platform
promoters
5'
3'
untranslated
regions
(UTRs)
higher
expression,
analyze
collection
23
5'UTRs,
10
3'UTRs,
6
promoters,
assessed
their
spinach
extracts,
found
consistency
patterns,
suggesting
cross-species
compatibility.
Looking
forward,
our
CFE
open
new
avenues
offering
understanding
developing
engineered
plants,
which
could
help
meet
demands
changing
climate.
Language: Английский
MoCloro: an extension of the Chlamydomonas reinhardtii modular cloning toolkit for microalgal chloroplast engineering
Physiologia Plantarum,
Journal Year:
2025,
Volume and Issue:
177(1)
Published: Jan. 1, 2025
Abstract
Photosynthetic
microalgae
are
promising
green
cell
factories
for
the
sustainable
production
of
high‐value
chemicals
and
biopharmaceuticals.
The
chloroplast
organelle
is
being
developed
as
a
chassis
synthetic
biology
it
contains
its
own
genome
(the
plastome)
some
interesting
advantages,
such
high
recombinant
protein
titers
diverse
dynamic
metabolism.
However,
engineering
currently
hampered
by
lack
standardized
cloning
tools
Design‐Build‐Test‐Learn
workflows
to
ease
genomic
metabolic
engineering.
MoClo
(Modular
Cloning)
toolkit
based
on
Golden
Gate
assembly
was
recently
in
model
eukaryotic
Chlamydomonas
reinhardtii
facilitate
nuclear
transformation
Here,
we
present
MoCloro
an
extension
that
allows
Briefly,
Gate‐compatible
vector
(pWF.K.4)
constructed,
which
homologous
arms
integration
at
petA
site
plastome.
A
collection
parts
(promoters,
terminators,
reporter
selection
marker
genes)
created
following
syntax
enable
easy
combinatorial
multi‐cassettes
destination
pWF.K.4
vector.
functionality
biobricks
assayed
constructing
assessing
expression
several
multigenic
constructs.
Finally,
generic
pK4
constructed
5′
3′
arms,
allowing
targeting
alternative
plastome
sites.
This
work
represents
significant
advancement
technology
aimed
facilitating
more
efficient
rapid
microalgae.
Language: Английский
Advancing chloroplast synthetic biology through high-throughput plastome engineering ofChlamydomonas reinhardtii
bioRxiv (Cold Spring Harbor Laboratory),
Journal Year:
2024,
Volume and Issue:
unknown
Published: May 10, 2024
Abstract
Chloroplast
synthetic
biology
holds
promise
for
developing
improved
crops
through
improving
the
function
of
plastids.
However,
chloroplast
engineering
efforts
face
limitations
due
to
scarcity
genetic
tools
and
low
throughput
plant-based
systems.
To
address
these
challenges,
we
here
established
Chlamydomonas
reinhardtii
as
a
prototyping
chassis
biology.
We
developed
an
automation
workflow
that
enables
generation,
handling,
analysis
thousands
transplastomic
strains
in
parallel,
expanded
repertoire
selection
markers
transformation,
new
reporter
genes,
characterized
over
140
regulatory
parts,
including
native
promoters,
UTRs,
intercistronic
expression
elements.
integrated
system
within
Phytobrick
cloning
standard
demonstrate
several
applications,
library-based
approach
develop
promoter
designs
Finally,
provide
proof-of-concept
novel
traits
plastids
by
introducing
chloroplast-based
photorespiration
pathway
demonstrating
twofold
increase
biomass
production.
Overall,
our
study
advances
engineering,
provides
promising
platform
rapidly
prototype
manipulations
before
their
transfer
into
higher
plants
crops.
Language: Английский