Cytochrome P450 enzymes play a crucial role as catalysts in the biosynthesis of numerous plant natural products (PNPs). Enhancing the catalytic activity of P450s in host microorganisms is essential for the efficient production of PNPs through synthetic biology. In this study, we engineered Saccharomyces cerevisiae to optimize the microenvironment for boosting the activities of P450s, including coexpression with the redox partner genes, enhancing NADPH supply, expanding the endoplasmic reticulum (ER), strengthening heme biosynthesis, and regulating iron uptake. This created a platform for the efficient production 11,20-dihydroxyferruginol, a key intermediate of the bioactive compound tanshinones. The yield was enhanced by 42.1-fold through 24 effective genetic edits. The optimized strain produced up to 67.69 ± 1.33 mg/L 11,20-dihydroxyferruginol in shake flasks. Our work represents a promising advancement toward constructing yeast cell factories containing P450s and paves the way for microbial biosynthesis of tanshinones in the future.

To assess the efficacy/safety of ivarmacitinib, a selective Janus kinase (JAK) 1 inhibitor, in patients with moderate-to-severe active rheumatoid arthritis (RA) who had an inadequate response to conventional synthetic disease-modifying antirheumatic drugs (csDMARDs). Patients were randomised (1:1:1) to receive either placebo (n=188), ivarmacitinib 4 mg (n=189) or ivarmacitinib 8 mg (n=189) once daily, with background csDMARDs allowed. After 24 weeks, patients on placebo switched to ivarmacitinib 4 mg for an additional 28 weeks, while those on ivarmacitinib continued their initial dosage. The primary endpoint was the proportion of patients achieving a 20% improvement in the American College of Rheumatology response criteria (ACR20) at week 24. At week 24, ACR20 response rates were significantly higher in the ivarmacitinib 4 mg (70.4%) and 8 mg (75.1%) groups compared with the placebo group (40.4%; both p<0.0001). Both ivarmacitinib doses achieved numerically higher Disease Activity Score 28-joint count C reactive protein of <2.6/≤3.2 response rates compared with placebo. Improvements in efficacy and patient-reported outcomes were sustained through 52 weeks and were noted in patients who switched from placebo after week 24. During the placebo-controlled period, treatment-emergent adverse events (TEAEs) occurred in 81.5% and 90.5% of patients in the ivarmacitinib 4 mg and 8 mg groups, versus 79.3% in the placebo group. Infection-related TEAEs were slightly higher in the ivarmacitinib groups. Ivarmacitinib may offer a potential therapeutic option for patients with RA who have an inadequate response to csDMARDs, with a safety profile that was generally manageable over 1 year of treatment and similar to other JAK inhibitors. NCT04333771.

Spiro-9,13-epoxy-labdane diterpenoids are commonly found in Leonurus species, particularly in Leonurus japonicus Houtt., which is a medicinal herb of long-standing use in Asia and in which such spiro-heterocycles are present in at least 38 diterpenoids. Here, through generation of a transcriptome and functional characterization of six diterpene synthases (diTPSs) from L. japonicus, including three class II diTPSs (LjTPS1, LjTPS3, and LjTPS4) and three class I diTPSs (LjTPS5, LjTPS6, and LjTPS7), formation of the spiro-9,13-epoxy-labdane backbone was elucidated, along with identification of the relevant diTPSs for production of other labdane-related diterpenes. Similar to what has been found with diTPSs from other plant species, while LjTPS3 specifically produces the carbon-9 (C9) hydroxylated bicycle peregrinol diphosphate (PPP), the subsequently acting LjTPS6 yields a mixture of four products, largely labda-13(16),14-dien-9-ol, but with substantial amounts of viteagnusin D and the C13-S/R epimers of 9,13-epoxy-labda-14-ene. Notably, structure–function analysis identified a critical residue in LjTPS6 (I420) in which single site mutations enable specific production of the 13S epimer. Indeed, extensive mutagenesis demonstrated that LjTPS6:I420G reacts with PPP to both specifically and efficiently produce 9,13S-epoxy-labda-14-ene, providing a specialized synthase for further investigation of derived diterpenoid biosynthesis. The results reported here provide a strong foundation for future studies of the intriguing spiro-9,13-epoxy-labdane diterpenoid metabolism found in L. japonicus.