Table 1 Semi-quantitative analysis of CYP716 substrates and products in transformed yeasts.

Compounds produced by *C. asiatica* CYP716s	% β-Amyrin [218]+	% Oleanolic acid [203]+	% 6β-OH oleanolic acid [203]+	% Maslinic acid [203]+
Yeast strain	Compared with strain 2	Compared with strain 4	Compared with strain 10	Compared with strain 11
2/ BAS	100±12
3/ BAS+CYP716A86	104±9
4/ BAS+CYP716A83	28±3	100±2
10/ BAS+CYP716A83+CYP716E41	36±2	25±4	100±56
11/ BAS+CYP716A83+CYP716C11	35±10	10±1		100±11
14/ BAS+CYP716A83+CYP716E41+CYP716C11	25±2	6±1	0±0	5±1

Compounds produced by *P. grandiflorus* CYP716s	% β-Amyrin [218]+	% Oleanolic acid [203]+	% 16β-OH β-amyrin [216]+	% 12,13α-epoxy-β-amyrin [514]+	% 16β-OH oleanolic acid [318]+
Yeast strain	Compared with strain 2	Compared with strain 30	Compared with strain 32	Compared with strain 33	Compared with strain 36
2/ BAS	100±12
30/ BAS+CYP716A140	11±1	100±4
32/ BAS+CYP716A141	26±2		100±4		29±7
33/ BAS+CYP716AS5	114±12			100±11
36/ BAS+CYP716A140+CYP716A141	7±1	24±3	10±0		100±25
37/ BAS+CYP716A140+CYP716S5	18±1	117±7		0±0
39/ BAS+CYP716A140+CYP716A141+CYP716S5	7±1	30±3	10±0	0±0	103±26

Compounds produced by *A. coerulea* CYP716s	% β-Amyrin [218]+
Yeast strain	Compared with strain 2
2/ BAS	100±12
46/ BAS+CYP716A110	67±8
47/ BAS+CYP716A111	115±8

Known triterpenoids were analysed and quantified from spent medium of MβCD-treated yeast cultures. The values indicate % of substrate left in comparison with the strain indicated±s.e. (n=4). These percentages are calculated from the means of peak areas of extracted ion intensities of representative ions of known triterpenoids, which are provided in Supplementary Table 3. Production rates of all products were consistent between the four replicates, except for the strains with C. asiatica CYP716E41, which showed larger quantitative variability although the same products were present in all four replicates. The comparison of substrate accumulation between producer yeast strains with and without additional CYP716s from C. asiatica, P. grandiflorus and A. coerulea showed diminished accumulation of the substrate compounds with active CYP716s.

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