Measuring triketide lactone production
After 8 d, a 200-µL sample was acidified with 20 µL of 6 M HCl,
centrifuged 10 min at 15,000 × g, and extracted with 1 mL ethyl acetate.
Ethyl acetate was removed in vacuo , and 0.5 mL methanol was
added. Mass spectra were obtained on an Agilent 6120 single quadrupole
LC/MS, separating on a C18 column (5% acetonitrile:
95% water with 0.1% formic acid to 100% acetonitrile over 12 m;
Agilent ZORBAX Eclipse Plus, 2.1 x 50 mm, 5 µm particle size) and
scanning m/z 50-2000 in positive ionization mode (Data File 80).
These measurements are not perfect indicators of the relative activities
of P1 -P6 -P7 and its point mutants. One
complicating factor is that the introduced mutations could affect the
expression or stability of the second protein ofP1 -P6 -P7 . However, SDS-PAGE analysis shows
similar levels for this protein and its point mutants (Figure S6).