Metabolic Engineering of Escherichia\ncoli for <i>De Novo</i> Biosynthesis of\nthe Platform Chemical Pelletierine

Pelletierine is a versatile plant\nalkaloid having a C5N–C3\nstructure from which numerous chemicals can be derived. One notable\nderivative is huperzine A (HupA) which may alleviate the symptoms\nof Alzheimer’s disease. Currently, industrial production of\npelletierine relies primarily on chemical synthesis and plant extraction.\nHowever, chemical synthesis leads to analogues that complicate product\nseparation, and plant extraction is constrained by limited resources.\nHerein, we report that pelletierine can be produced by recombinant Escherichia coli in which the engineered pelletierine\nbiosynthesis pathway comprises four modules involving seven key genes\nnative to E. coli, three genes from\nother bacteria, and three genes from plants. To overproduce pelletierine,\nthe intrinsic l-lysine biosynthesis pathway in E. coli was simplified, and a clustered regularly\ninterspaced short palindromic repeats (CRISPR) interference (CRISPRi)\nsystem was engineered to minimize the byproducts. Moreover, the transporter\nMatC was overexpressed to enhance the intracellular concentration\nof 3-oxoglutaryl ketide, which is another precursor of pelletierine.\nBased on the aforementioned manipulations, the resulting recombinant E. coli harboring the pelletierine biosynthesis pathway\nand CRISPRi system produced 3.40 and 8.23 mg/L pelletierine in a shake-flask\nand a 5 L bioreactor, respectively. This is the first report of microbial\nproduction of pelletierine, which represents a sustainable route to\nproduce the precursor of HupA and beyond.