Heterologous Biosynthesis of Kauralexin A1 in through Metabolic and Enzyme Engineering.

Kauralexin A1 (KA1) is a key intermediate of the kauralexin A series metabolites of maize phytoalexins. However, their application is severely limited by their low abundance in maize. In this study, an efficient biosynthetic pathway was constructed to produce KA1 in . Also, metabolic and enzyme engineering strategies were applied to construct the high-titer strains, such as chassis modification, screening synthases, the colocalization of enzymes, and multiple genomic integrations. First, the KA1 precursor -kaurene was synthesized using the efficient diterpene synthase GfCPS/KS from , and optimized to reach 244.36 mg/L in shake flasks, which displayed a 200-fold increase compared to the initial strain. Then, the KA1 was produced under the catalysis of ZmCYP71Z18 from and SmCPR1 from , and the titer was further improved by integrating the fusion protein into the genome. Finally, an -kaurene titer of 763.23 mg/L and a KA1 titer of 42.22 mg/L were achieved through a single-stage fed-batch fermentation in a 5 L bioreactor. This is the first report of the heterologous biosynthesis of maize diterpene phytoalexins in , which lays a foundation for further pathway reconstruction and biosynthesis of the kauralexin A series maize phytoalexins.