10.1002/cbic.201800606
ChemBioChem
FULL PAPER
gene, which are tetracycline-, rhamnose- and IPTG-inducible,
performed chromatographically on silica gel (pore size 60, 220-240 mesh
size, particle size 35-75 µm), which was equilibrated with 100% PET. The
compounds were eluted with a mix of PET and ether (5-40%), and each
elution fraction was analysed by thin-layer chromatography (TLC) using a
mobile phase composed of a PET/ether in 70:30 ratio. The TLC plate
was stained with phosphomolybdic acid stain (PMA; 12 g in 250 mL
ethanol) and exposed to UV-light. The fractions containing the desirable
metabolites were pooled, and the solvent was removed as before.
respectively. PCR linearisation of L6H -SmCPR-CDH was performed
M
between the 3’ end of CDH and the terminator region, while amplification
of the PETNR-promoter-CHMO
inserts included rbs2 upstream of
3M
PETNR. Following each PCR reaction, template removal and DNA clean
up was performed as above. The oligonucleotide sequences encoding
the PCR primers can be found in Supporting Information Table S8. The
correct assembly of each construct was confirmed by DNA sequencing
(L6HCCP-tet-C
3M
,
L6HCCP-rha-C
3M
and L6HCCP-trc-C ). Each
3M
1H and 13C NMR spectra of the up-scaled purified product(s) (10 mg/mL)
in deuterated chloroform were recorded on a Bruker Avance 400 MHz
NMR spectrometer at 298 K without the addition of an internal standard.
Chemical shifts are reported as δ in parts per million (ppm) and are
calibrated against the residual solvent signal. 1H AND 13NMR spectra
were analysed using MestreNova.
construct was co-transformed with plasmid pJBEI6410 into competent
cells of E. coli strain NEB10β for functional overexpression according to
the manufacturers protocols. A summary of all the gene constructs is
found in Supporting Information Table S9.
In vivo biotransformations: A single colony of E. coli NEB10β co-
transformed with pJBEI6410 and pBbB8k-containing biosynthetic
constructs was used to inoculate 5 mL of Terrific broth, containing
phosphate salts (9.4 g/L KH2PO4 and 2.2 g/L K2HPO4), 0.7 % (w/v)
glucose 60 µg/mL kanamycin and 100 µg/mL ampicillin. The culture was
incubated at 37°C, and 200 rpm until reaching OD600 = 0.4, followed by
Acknowledgements
This work was funded by grants provided by the BBSRC and
EPSRC (BB/M000354/1 and BB/M017702/1). UK Catalysis Hub
is kindly thanked for resources and support provided via our
membership of the UK Catalysis Hub Consortium and funded by
EPSRC (grants EP/K014706/2, EP/K014668/1, EP/K014854/1,
EP/K014714/1 and EP/M013219/1)’
IPTG, arabinose, δ-aminolevulinic acid
+ tetracycline addition +
rhamnose (25 µM, 25 mM, 500 µM, 100 nM and 0.05%, respectively).
The cultures were incubated at 30 °C for 72 hours unless otherwise
stated. Each culture aliquot (3 mL) was cooled for 10 minutes on ice,
followed by extraction with ethyl acetate (2 x 375 µL) containing 0.0[8]1 %
sec-butyl benzene. Product yields and identification were determined by
GCMS analysis.
Analytical techniques: Monoterpenoid content quantification was
performed using an Agilent Technologies 7890A GC system with an FID
detector. Biotransformation extracts (1 mL) were analysed on a DB-WAX
column (30 m; 0.32 mm; 0.25 µm film thickness; JW Scientific). In this
method the injector temperature was at 220 °C with a split ratio of 20:1.
The carrier gas was helium with a flow rate of 1 mL/min and a pressure
of 5.1 psi. The program began at 40°C with a hold for 2 minutes, followed
by an increase of temperature to 210°C at a rate of 15°C/minute, with a
final hold at 210 °C for 3 min. The FID detector was maintained at a
temperature of 250°C with a flow of hydrogen at 30 mL/min. Product
quantitation was performed by comparing the peak areas to those of
authenticated standards of known concentration. Where authentic
standards were not commercially available (by-products only), the
concentrations were estimated using an average concentration per peak
area value based on 11 related monoterpenoid standards.
Keywords: (+)-Dihydrocarvide monomer • Baeyer-Villiger
monooxygenases • synthetic biology • bioplastics • engineering
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