Figure Legends
Figure 1 . Screening of CYPs involved with GA biosynthesis based on HLDOA. (a) The plasmid pRS425-iGlCPR-CYP5150L8 containing the iGlCPR and CYP5150L8 genes and the pRS426-CYPx plasmids (expressing different candidate CYP genes, respectively) were co-transformed into S. cerevisiae . (b) HPLC analyses of extracts from fermentation of S. cerevisiae transformants which co-expressed CYP5150L8 (L8) and CYP512U6 or other five CYP genes (CYP5150L6, CYP5150L9, CYP5150K5, CYP5150J5, CYP5150J10) which locate in the same cluster as CYP5150L8 in G. lucidum genome. Yeast transformant harboring plasmids pRS425-CYP5150L8-iGLCPR and void plasmid pRS426 served as control. (c) The transformant co-expressing CYP5150L8 and CYP5139G1 showed a new peak at 17.5 min. The expression of CYP5150L8 or CYP5139G1 alone had no new peak here. (d). High resolution MS spectra of compound Aindicated in (c).
Figure 2 . Purification of compound A via Pre-HPLC. (a) Initial purification of Compound A via Pre-HPLC equipped with Kromasil column from which the fractions at different retention time are intercepted for detecting the compound A in different fractions. (b) HPLC analysis of different fractions (Rt 22.4, Rt 23.1, Rt 24.1, Rt 26.3) collected from first round purification indicated in (a). (c) Second round of purification of compound A via Pre-HPLC equipped with ODS-BP C18 column. (d) HPLC detection of different fractions (Rt 20.2, Rt 21.2, Rt 22.5) separated by second round Pre-HPLC.
Figure 3 . The chemical structure of compound A . (a) NOESY spectra of compound A . Red circle represented the interaction between 29-CH3 and 3-β-OH. (b) Compound A was determined to be 3,28-Dihydroxy-lanosta-8,24-dien-26-oic acid (DHLDOA).
Figure 4 . In vitro microsomal reactions by CYP5139G1. (a) Compared to void plasmid and heated-CYP5139G1 control, microsomes containing CYP5139G1 generated a new peak with the same retention time as purified compound A (black line). (b) MS spectra of purified compound A (red line) and the product generated (red line) by microsome reaction with addition of HLDOA as substrate. For in vitro enzymatic assay, microsomes were prepared from YL-T3 containing void plasmid (control, blue line) or YL-T3 containing CYP5139G1 (red line). Heated-CYP5139G1 (gray line) represented that the CYP5139G1 containing microsomes were inactivated by heating at 80 °C.
Figure 5 . Reconstruction of GA biosynthetic pathway in S. cereviae . Single bold arrows represent one step reaction, while triple bold arrows represent multiple step reactions. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; FPP, farnesyl diphosphate; HLDO, 3-hydroxy-lanosta-8,24-dien-26-ol; HLDA, 3-hydroxy-lanosta-8,24-dien-26-al; HLDOA, 3-hydroxy-lanosta-8,24-dien-26-oic acid; DHLDOA, 3,28-Dihydroxy-lanosta-8,24-dien-26-oic acid; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; IDH, isopentenyl- diphosphate isomerase; ERG20, farnesyl diphosphate synthase; ERG9, squalene synthase; ERG1, squalene epoxidase; ERG7, lanosterol synthase.
Figure 6 . Modulation of CYP5150L8 and CYP5139G1 expression via plasmids with tunable copy numbers under different concentration combination of Hyg and G418. (a) Cell growth under different Hyg and G418 combinations including H0G0, H0G300, H100G0, H100G100, H100G300, H100G500, H200G300. (b) HLDOA and DHLDOA production after 5 days fermentation of strain HYGL8-G418G1 under the combination conditions mentioned above as (a). (c) Fold changes of pRS425-Hyg-CYP5150L8-iGLCPR and pRS426-KanMX-CYP5139G1 plasmid copy number under the conditions mentioned above (day 4 of fermentation). (d) CYP5150L8, CYP5139G1 relative expression in HYGL8-G418G1 strain with different conditions mentioned above in 4-day fermentation. The error bars represent the standard deviation of three biological replicates.