Summary Cytochromes P450 (CYPs) play key role in generating the structural diversity of terpenoids, the largest group of plant natural products. However, functional characterization of CYPs has been challenging because of the expansive families found in plant genomes, diverse reactivity and inaccessibility of their substrates and products.Here we present the characterization of two CYPs, CYP76AH3 and CYP76AK1, that act sequentially to form a bifurcating pathway for the biosynthesis of tanshinones, the oxygenated diterpenoids from the Chinese medicinal plant Danshen.These CYPs had similar transcription profiles to that of the known gene responsible for tanshinone production in elicited Danshen hairy roots. Biochemical and RNA interference studies demonstrated that both CYPs are promiscuous. CYP76AH3 oxidizes ferruginol at two different carbon centers, and CYP76AK1 hydroxylates C-20 of two of the resulting intermediates. Together, these convert ferruginol into 11,20-dihydroxy ferruginol and 11,20-dihydroxy sugiol en route to tanshinones. Moreover, we demonstrate the utility of these CYPs by engineering yeast for heterologous production of six oxygenated diterpenoids, which in turn enabled structural characterization of three novel compounds produced by CYP-mediated oxidation.Our results highlight the incorporation of multiple CYPs in diterpenoids metabolic engineering, and a continuing trend of CYPs promiscuity generating complex networks in terpenoid biosynthesis.

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Co-fermentation of lignocellulose-based carbohydrates is a potential solution to improve the economics of microbial lipid production. In the present paper, experiments were performed to optimize the media composition for lipid production by the oleaginous yeast Lipomyces starkeyi AS 2.1560 through co-fermentation of glucose and xylose (2 : 1 wt/wt). Statistical screening of nine media variables was performed by a Plackett-Burman design. Three factors, namely mixed sugar, yeast extract and FeSO 4 , were found as significant components influencing cellular lipid accumulation. Further optimization was carried out using a Box-Behnken factorial design to study the effects of these three variables on lipid production. A mathematical model with the R 2 value at 96.66% was developed to show the effect of each medium composition and their interactions on the lipid production. The model estimated that a maximal lipid content of 61.0 wt-% could be obtained when the concentrations of mixed sugar, yeast extract and FeSO 4 were at 73.3 g/L (glucose 48.9 g/L, xylose 24.4 g/L), 7.9 g/L and 4.0 mg/L, respectively. The predicted value was in good accordance with the experimental data of 61.5%. Compared with the initial media, the optimized media gave 1.59-fold and 2.03-fold increases for lipid content and lipid productivity, respectively.

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