Heterologous production of 3-hydroxy-lanosta-8, 24-dien-26 oic acid (HLDOA) was recently achieved by expressing CYP5150L8 from Ganoderma lucidum in Saccharomyces cerevisiae, but post-modification of HLDOA remains unclear. In this study, another P450 from G. lucidum, CYP5139G1, was identified to be responsible for C-28 oxidation of HLDOA, resulting in the formation of a new ganoderic acid (GA) 3,28-dihydroxy-lanosta-8, 24-dien-26 oic acid (DHLDOA) by the engineered yeast, whose chemical structure was confirmed by LC-MS and NMR. In vitro enzymatic experiments confirmed the oxidation of HLDOA to DHLDOA by CYP5139G1. As the DHLDOA production was low (0.27 mg/L), to improve it, the strategy of adjusting the dosage of hygromycin and geneticin G418 to respectively manipulate the copy number of plasmid pRS425-Hyg-CYP5150L8-iGLCPR (harboring CYP5150L8, iGLCPR and hygromycin resistant gene hygR) and pRS426-KanMx-CYP5139G1 (harboring CYP5139G1 and G418 resistant gene KanMx) was adopted. Finally, 2.2 mg/L of DHLDOA was obtained, which was 8.2 fold of the control (without antibiotics addition). The work not only enriches the library of GAs and GA biosynthetic enzymes, but also helps to construct heterologous cell factories for other GA production as well as to elucidate the authentic GA biosynthetic pathway in G. lucidum.