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Background  Diterpenoids are a large class of natural products with complex structures and broad commercial applications as food additives, important medicines, and fragrances. However, their low abundance in plants and high structural complexity limit their applications. Therefore, it is important to create an efficient diterpenoid-producing yeast cell factory of the production of various high-value diterpenoid compounds in a cost-effective manner    Results  In this study, 13 R -manoyl oxide (13 R -MO; 2.31 mg/L) was produced by expressing  CfTPS2  and  CfTPS3  from  Coleus   forskohlii  in  Saccharomyces cerevisiae . The 13 R -MO titer was increased by 142-fold to 328.15 mg/L via the stepwise metabolic engineering of the original strain, including the overexpression of the rate-limiting genes ( tHMG1  and  ERG20 ) of the mevalonate pathway, transcription and protein level regulation of  ERG9 , Bts1p and Erg20 F96C p fusion, and the overexpression of  tCfTPS2  and  tCfTPS3  (excision of the N-terminal plastid transit peptide sequences of  CfTPS2  and  CfTPS3 ). The final titer of 13 R -MO reached up to 3 g/L by fed-batch fermentation in a 5 L bioreactor.    Conclusions  In this study, an efficient 13 R -MO yeast cell factory was constructed, which achieved the de novo production of 3 g/L of 13R-MO from glucose. To the best of our knowledge, this is the highest 13 R -MO titer reported to date. Furthermore, the metabolic engineering strategies presented here could be used to produce other valuable diterpenoid compounds in yeast.    Electronic supplementary material  The online version of this article (10.1186/s12934-019-1123-z) contains supplementary material, which is available to authorized users.

High-titer production of 13R-manoyl oxide in metabolically engineered Saccharomyces cerevisiae