resulted in the production of MCFOHs (C10–C12) in Saccharo-
Table S1). However, the C16 fatty alcohol was still the major
product (>60%) in this study, for which the substrate preference
of TaFAR might be one of the reasons. Our study, on the other
hand, provides new insight into MCFOH production. Considering
the difference in cultivation conditions, we achieved a significant
the template for the first round of evolution and the mutant CAR with
mutations Q182R; Q371R was used for the second round. The sizes of the
4
two libraries were both around 5 × 10 . The same workflow for the library
enrichment was used as described above, but different concentrations of C8
FA were used, which were 290 mg/L, 300 mg/L, and 310 mg/L for the first
round and 310 mg/L, 330 mg/L, and 350 mg/L for the second round. Around
6
0 single colonies from first round and second round were cultivated in
minimal medium with histidine containing 330 mg/L and 370 mg/L C8 FAs,
respectively. The growth curves were monitored by a Bioscreen C MBR in-
strument (Growth Profiler 960 was used for the second round).
MmCAR was also investigated both in vitro and in vivo. The ef-
ficient reduction of MCFAs by the engineered CAR enzymes will
enable the synthesis of versatile aldehyde intermediates with
broad applications for further production of, for example, the
corresponding alka(e)nes and FA acyl esters. Although CAR
enzymes were previously modified by different strategies (39, 42,
Site-Directed Saturation Mutagenesis Library. The primers used for estab-
used as described above with concentrations of 290 mg/L, 310 mg/L, 330 mg/
L, and 350 mg/L C8 FAs. After isolating the variants on an SD-URA plate, 80
of the colonies were picked to grow in medium with 380 mg/L of C8 FA.
Based on the growth curves generated with the help of a Growth Profiler
4
5), this work is unique in comprehensively engineering the CAR
enzyme for more selective biosynthesis of MCFOHs. In addition,
our engineering strategies may inspire and promote the engi-
neering of other complex multidomain enzymes.
9
60, the clones with the highest growth rates were selected.
Enzymatic Assays. The yeast cells (50 OD) were harvested after 24 h by
centrifuging at 4,000 × g at 4 °C for 5 min, then washed twice with PBS
buffer. After discarding the supernatant, the cells were resuspended in
Methods
Directed Evolution of Full-Length MmCAR. The mutagenesis library of MmCAR
was constructed by error-prone PCR (GeneMorph II Random Mutagenesis
Kit, Agilent Technologies) with a low mutation frequency (0 to 4.5 muta-
tions/kb) using the primers MmCARwm-F/MmCARwm-R. The product was
then cotransformed with two backbone fragments of pZW01 into yeast
0
.5 mL extraction buffer (50 mM Tris·Cl, 1 mM EDTA, 1 mM KCl, pH 7.5)
containing 10 mM DTT and 1% (vol/vol) protease inhibitor. The suspensions
were vortexed for 20 s ×5 (5-min intervals on ice) with 300 mg 0.2- to 0.4-mm
glass beads. The supernatants of the samples were collected by centrifuging
at 20,000 × g at 0 °C for 20 min. Proteins were quantified using a Pierce BCA
Protein Assay Kit (ThermoFisher Scientific). The MmCAR activity assay was
performed in 96-well microplates; 90-μL reaction mix containing 50 mM
7
The library sizes for the two rounds of evolution were around 6 × 10 and
7
4
.5 × 10 , respectively. All of the colonies from the SD-URA (synthetic com-
2
Tris·Cl (pH 7.5), 10 mM MgCl , 1 mM NADPH, 1 mM ATP, and FAs (0.5 mM
plete medium without uracil) plate were all scratched into 20 mL minimal
medium with 290 mg/L C8 FA (330 mg/L for the second round). After 24 h,
the cells (1:100 diluted) were transferred into 20 mL minimal medium with
C10, C12, C14, C16:1, C18:1 FA, and 5 mM C6, C8 FA) and 10 μL of extracts (5
μg total protein) were mixed to initiate the reaction. Then the reactions
were monitored at 340 nm for 30 min at room temperature with a FLUOstar
Omega micropate reader (BMG Labtech). The MmCAR activity was defined
as NADPH oxidation rate (1 μmol/min) of total protein.
3
10 mg/L C8 FAs (350 mg/L for the second round) for 48 h. Then the cells
were diluted 1:50, and transferred into 20 mL minimal medium with 330 mg/
L C8 FA (370 mg/L for the second round) for 48 h. The enriched cells were
plated on SD-URA solid medium, and around 60 single colonies were picked
randomly into 96-well plate with minimal medium containing 330 mg/L C8
FAs (370 mg/L for the second round). The growth curves were monitored by
a Bioscreen C MBR instrument (Growth Profiler 960 was used for the second
round). The plasmids from fast-growing colonies were extracted and used to
transform strain ZWE243 to determine MCFOH production. The mutant CAR
with mutations D241E, H454R, L567M was used as the template for the
second round of evolution. The same process was conducted as described
above.
profiler, and enzymatic assays, can be found in Dataset S1.
ACKNOWLEDGMENTS. We thank Quanli Liu, Yi Liu, Martin Engqvist, and
Tao R. Yu for critical discussion; and the Chalmers Mass Spectrometry
Infrastructure for assistance with metabolite analysis. This work was funded
by the Novo Nordisk Foundation (Grant NNF10CC1016517), the Swedish
Foundation for Strategic Research, and the Knut and Alice Wallenberg
Foundation. We also thank the Energimyndigheten for support. M.W. thanks
the Austrian science fund (Elise-Richter Fellowship V415-B21) for financial
support. Z.Z. acknowledges support from the Fundamental Research Funds for
the Central Universities [DUT20RC(3)044].
Directed Evolution of MmCAR a Domain. The random mutagenesis library
based on the A domain (residues 88 to 541) of MmCAR was established using
error-prone PCR (GeneMorph II Random Mutagenesis Kit) with the low
mutation frequency (0 to 4.5 mutations/kb) by primer pair CAR-A-F/CAR-A-R.
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