{Reference Type}: Journal Article
{Title}: Screening of ent-copalyl diphosphate synthase and metabolic engineering to achieve de novo biosynthesis of ent-copalol in Saccharomyces cerevisiae.
{Author}: Li S;Luo S;Yin X;Zhao X;Wang X;Gao S;Xu S;Lu J;Zhou J;
{Journal}: Synth Syst Biotechnol
{Volume}: 9
{Issue}: 4
{Year}: 2024 Dec
{Factor}: 4.692
{DOI}: 10.1016/j.synbio.2024.06.005
{Abstract}: The diterpene ent-copalol is an important precursor to the synthesis of andrographolide and is found only in green chiretta (Andrographis paniculata). De novo biosynthesis of ent-copalol has not been reported, because the catalytic activity of ent-copalyl diphosphate synthase (CPS) is very low in microorganisms. In order to achieve the biosynthesis of ent-copalol, Saccharomyces cerevisiae was selected as the chassis strain, because its endogenous mevalonate pathway and dephosphorylases could provide natural promotion for the synthesis of ent-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 ApCPSs were screened by transcriptome sequencing of A. paniculata and ApCPS2 had the best activity and produced ent-CPP exclusively. The peak area of ent-copalol was increased after the ApCPS2 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 ent-copalol was generated. In this study, de novo biosynthesis and identification of ent-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.