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www.researchgate.net/project/CHASSY
FUNDING
•The CHASSY project received funding from the Horizon 2020 research and innovation programme (grant agreement 720824)
•The YEASTCELL training network received funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework
Programme FP7/2007-2013/under REA grant agreement n◦606795
•BE-Basic R&D Program, Dutch Ministry of Economic Affairs, Agriculture & innovation (EL&I)
A toolkit to expand the synthetic biology
potential of Kluyveromyces marxianus
Arun S. Rajkumar1, Javier A. Varela1, Hannes Juergens2, Jack T. Pronk2, Jean-Marc G. Daran2and John P. Morrissey1
1School of Microbiology, University College Cork, Cork, Ireland
2Department of Biotechnology, Delft University of Technology, Delft, The Netherlands
…CTCGTCCGAGACCTGCGGAGGTCTCCGTTTTA…
…GAGCAGGCTCTGGACGCCTCCAGAGGCAAAAT…
pUCC001
Kluyveromyces marxianus has the potential to be a next-generation chassis for the production of a wide range of bio-based chemicals, with some
key advantages over other yeasts: it is thermotolerant, fast-growing and can use a wide variety of carbon and nitrogen sources.
However, its broader adoption by biotechnologists is limited by: (i) inefficient genetic manipulation and (ii) a lack of standard native regulatory parts
or synthetic biology tools for the rapid assembly, expression and maintenance of genes.
Constitutive
and inducible
promoters
Antibiotic and
metabolic markers
ARSs/CENs
Homology
arms
Terminators
Bacterial markers
as per the YTK standard
Connector/
Homology arms
for integration
Connector
The parts collection is
built on the Yeast
Toolkit standard1using
Golden Gate assembly
for the efficient
hierarchical assembly of
expression cassettes.
We have optimized a cross-species
CRISPR/Cas9 system2for the faster and
more economical construction of
genome editing vectors by Golden Gate
assembly.
A set of promoters allowing precise gene expression, characterized by
YFP reporter assays.
Inducible promoters further allow on/off gene expression under
conditions specific to K. marxianus’physiology.
Diverse terminators to further
fine-tune gene expression.
BIOLOGICAL PARTS GENOME EDITING TOOLS
Chr.I Chr.IV Chr.V
I1
I2
I3
I4
The parts collection includes:
The parts function well in
different lab strains.
Efficient auxotrophic strain
construction by pUCC001
GENE TARGETING TOOLS
Genes responsible for DNA repair3are
deactivated to find the best
background for gene targeting with
minimal random integration.
We selected integration sites from
genomic data (left)4and evaluate
integration efficiency and expression using
a YFP cassette.
then
Inducing the expression of recombinase from S. cerevisiae
improved gene targeting (YBL002: Glc vs Gal), compared to a
background minimizing random integration alone (YBL001)
his3-1
ura3-1 leu2-1
WT
SD-leucine
SD-histidine SD-uracil
BsaI sites allow direct insertion of
new target gRNAs during assembly.
REFERENCES
1. Lee et al., ACS Synth Biol (2015); 2:975-986
2. Juergens et al., FEMS Yeast Res (2018); 18:foy21
3. Nambu-Nishida et al., Sci Rep (2017); 7:8993
4. Lertwattanasakul et al., Biotech Biofuels (2015); 8:47
Low- and high-copy number centromeres/ARSes for plasmids are also
provided , as well as targeting sequences for integrative vectors.
natr
natrura-
NBRC
1777
YBL001
YBL002
w/Glc
YBL002
w/Gal
1.Express the
recombinase
ScRAD52
using an
inducible
promoter
3. Screen resistant colonies for uracil
auxotrophy
2.Integrate a
marker at
URA3 when
un/induced
We have characterized, developed and collected a number of parts and tools to facilitate the synthetic biology and genome
engineering of K. marxianus and present them here, along with some of their applications.