Unveiling a pyrroloindoline diketopiperazine biosynthetic pathway featuring a phytoene-synthase-like family prenyltransferase with distinct regioselectivity.

Pyrroloindoline-containing natural products are a group of molecules with diverse biological activities. Herein, we identified a cryptic cyclodipeptide synthase (CDPS) gene cluster (lan) from a deepsea-derived Streptomyces strain using the phytoene-synthase-like (PSL) family prenyltransferase (PT) as a probe. Heterologous expression of the lan gene cluster in Streptomyces albus J1074 led to the production of two reversely C5'-prenylated pyrroloindoline-containing diketopiperazines (DKPs), lansais A (4) and B (5). Gene inactivation and biochemical assays established its biosynthetic pathway as follows: LanA, a fused CDPS-methyltransferase, sequentially generates mono- and bis-pyrroloindoline intermediates (2 and 3) viacyclo (l-Trp-l-Trp) formation and C3/C3'-methylation; subsequently, N-methyltransferase LanC installs the methyl group at N1/N1' followed by installation of the prenyl moiety at C5' by the PSL family PT LanB. Notably, compounds 4 and 5 demonstrated potent anti-vesicular stomatitis virus (VSV) activity, with a 50 % virus inhibitory concentration (IC50) values of 1.98-2.22 μM, significantly stronger than that of ribavirin (IC50 = 14.27 μM), revealing the critical role of the prenyl moiety in anti-VSV activity. Taking advantage of the catalytic promiscuity of LanB, two new lansai derivatives with C5'-reverse or C7'-regular dimethylallyl moiety (9 and 10) were obtained, achieving IC50 value as low as 0.31 μM (10). Of note, the regioselectivity of LanB is distinct from all the other reported PSL family PTs. Our study enriches the biosynthetic machineries of pyrroloindoline-containing DKPs, and lays the foundation for further increasing the structural diversity of this compound family.