Engineered Production of Hapalindole Alkaloids in the Cyanobacterium Synechococcus sp. UTEX 2973

Cyanobacteria produce numerous valuable bioactive secondary metabolites (natural products) including alkaloids, isoprenoids, nonribosomal peptides, and polyketides. However, the genomic organization of the biosynthetic gene clusters, complex gene expression patterns, and low compound yields synthesized by the native producers currently limits access to the vast majority of these valuable molecules for detailed studies. Molecular cloning and expression of such clusters in heterotrophic hosts is often precarious owing to genetic and biochemical incompatibilities. Production of such biomolecules in photoautotrophic hosts analogous to the native producers is an attractive alternative that has been under-explored. Here, we describe engineering of the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 to produce key compounds of the hapalindole family of indole-isonitrile alkaloids. Engineering of the 42-kbp "fam" hapalindole pathway from the cyanobacterium Fischerella ambigua UTEX 1903 into S2973 was accomplished by rationally reconstructing six to seven core biosynthetic genes into synthetic operons. The resulting Synechococcus strains afforded controllable production of indole-isonitrile biosynthetic intermediates and hapalindoles H and 12-epi-hapalindole U at a titer of 0.75-3 mg/L. Exchanging genes encoding fam cyclase enzymes in the synthetic operons was employed to control the stereochemistry of the resulting product. Establishing a robust expression system provides a facile route to scalable levels of similar natural and new forms of bioactive hapalindole derivatives and its structural relatives (e.g., fischerindoles, welwitindolinones). Moreover, this versatile expression system represents a promising tool for exploring other functional characteristics of orphan gene products that mediate the remarkable biosynthesis of this important family of natural products.