Mining of Novkitasetaline, a New Sulfur-Containing Antimalarial β-Carboline Alkaloid, from Streptomyces sp. PRh3 by Functional Ribosome Engineering Directed Heterologous Expression

The endophytic <i>Streptomyces</i> sp. PRh3 (PRh3), isolated from Dongxiang wild rice (DXWR), exhibited impaired biosynthetic capacity in the laboratory. To address this defect, rifampicin-based ribosome engineering was first applied to PRh3 to generate PRh3-r55, which acquired a characteristic H473Y rifampicin-resistant mutation in <i>rpoB</i> to activate the production of two β-carboline alkaloids JBIR-133 and JBIR-134. Then the biosynthetic gene cluster (BGC) <i>ksl</i> was introduced into PRh3-r55 for heterologous expression, generating PRh3-r55K. This combined approach achieved a synergistic effect, enabling the strain to produce not only the expected JBIR-133, JBIR-134, and kitasetaline, but also a novel sulfur-containing molecule, novkitasetaline. Structural elucidation identified novkitasetaline as a unique tryptamine-substituted kitasetaline derivative at the C-3 position of its pyridine ring. Notably, this structural modification conferred significant antimalarial activity to novkitasetaline, rendering it active against drug-sensitive <i>Plasmodium falciparum</i> 3D7 (IC₅₀ = 32.65 ± 2.93 μM) and three other drug-resistant <i>P. falciparum</i> strains: K13^C580Y, Dd2, and HB3 (IC₅₀ = 45.98 ± 4.17~59.67 ± 3.15 μM), primarily by disrupting late-stage parasite development. These efforts not only identified a promising antimalarial lead compound but also demonstrated that combining ribosome engineering with heterologous expression is an effective strategy for discovering bioactive natural products from <i>Streptomyces</i>.