TABLE 2.

WS, DGAT, and ASAT activities in crude extracts of different recombinant S. cerevisiae and E. coli strains^a

Strain	Enzyme sp act (pmol [mg of protein · min]−1)
	WSb	DGAT	ASAT
			With cholesterol	With ergosterol
S. cerevisiae G175(pESC-URA)	1.12 ± 0.03	4.45 ± 0.08	1.73 ± 0.11	0.67 ± 0.06
S. cerevisiae H1246(pESC-URA)	0.12 ± 0.02	0.12 ± 0.01	0.13 ± 0.03	0.16 ± 0.01
S. cerevisiae H1246(pESC-URA::atfA)	55.40 ± 3.22	44.37 ± 2.11	5.87 ± 0.23	19.62 ± 0.18
E. coli XL1-Blue(pBluescript KS)	0.18 ± 0.03	0.12 ± 0.02	0.15 ± 0.04	0.17 ± 0.02
E. coli XL1-Blue(pKS::atfA)	74.41 ± 3.53	19.00 ± 0.82	48.57 ± 5.12	70.94 ± 2.40

^aYeast cells were cultivated for 24 h at 28°C in synthetic minimal dropout medium without uracil and containing 2% (wt/vol) galactose. Cells of recombinant E. coli strains were cultivated for 6 h at 37 °C in LB medium containing 1 mM IPTG. Enzyme activities were measured as described in Materials and Methods by using 1-hexadecanol, 1,2-dipalmitoylglycerol, and cholesterol or ergosterol for measuring WS, DGAT, and ASAT activities, respectively. Data are mean values of two independent experiments ± SD.

^bWax esters and steryl esters almost comigrated under the applied TLC conditions. Therefore, the production of radiolabeled wax esters and sterol esters was virtually indistinguishable, and WS activities had to be corrected for the formation of radiolabeled sterol esters formed by ASAT activity by utilizing endogenous sterols occurring in yeast, which was measured in control assays with [1-¹⁴C]palmitoyl-CoA, but lacking any external acyl acceptor.