Screening of -copalyl Diphosphate Synthase and Metabolic Engineering to Achieve Biosynthesis of -copalol in

The diterpene -copalol is an important precursor to the synthesis of andrographolide and is found only in green chiretta . biosynthesis of -copalol has not been reported, because the catalytic activity of -copalyl diphosphate synthase (CPS) is very low in microorganisms. In order to achieve the biosynthesis of -copalol, was selected as the chassis strain, because its endogenous mevalonate pathway and dephosphorylases could provide natural promotion for the synthesis of -copalol. The strain capable of synthesizing diterpene geranylgeranyl pyrophosphate was constructed by strengthening the mevalonate pathway genes and weakening the competing pathway. Five full-length CPSs were screened by transcriptome sequencing of and CPS2 had the best activity and produced -CPP exclusively. The peak area of -copalol was increased after the CPS2 saturation mutation and its configuration was determined by NMR and ESI-MS detection. By appropriately optimizing acetyl-CoA supply and fusion-expressing key enzymes, 35.6 mg/L -copalol was generated. In this study, biosynthesis and identification of -copalol were achieved and the highest titer ever reported. It provides a platform strain for the further pathway analysis of andrographolide and derivatives and provides a reference for the synthesis of other pharmaceutical intermediates.