Co-cultivation Serratia marcescens with Trichoderma harzianum for improving production of Huperzine A

Plant-derived (-)-Huperzine A (HupA), the bioactive enantiomer of the acetylcholinesterase inhibitor used for Alzheimer's disease (AD) therapy, is limited by the scarcity of Huperzia serrata (HS), while chemically synthesized (+)-HupA is clinically unviable due to high toxicity and low activity (1/30 that of (-)-HupA), creating a critical bottleneck for HupA pharmaceutical development that necessitates microbial biosynthesis solutions. In this study, we isolated the endophytic bacterium Serratia marcescens HL-1 from HS (identified via morphological characterization and 16S rRNA sequencing) and developed a novel co-cultivation strategy with Trichoderma harzianum NSW-V in modified PDA medium (26 °C, 2 days): This co-cultivation system sustained the stable HupA biosynthetic capacity of both Serratia marcescens HL-1 and Trichoderma harzianum NSW-V, synergistically enhancing the (-)-HupA yield of the endophytic bacterium to 32.976 ± 0.21 mg/L (biosynthetic HupA, BHA) and concurrently boosting the HupA production of the fungal strain; this revealed a positive upward trend in HupA yield with co-cultivation intervention, but no statistically significant differences were observed between groups (p > 0.05), and notably co-cultivation restored the strain's HupA-synthesizing capacity when its native production potential declined. BHA exhibited physicochemical properties and crystal structure identical to plant-derived (-)-HupA (PHA), as validated by NMR spectroscopy and molecular docking analyses. Furthermore, we identified a novel pharmacological role for HupA: BHA protected pancreatic islet β-cells in a palmitic acid-induced injury model, where cell viability increased from 58.2% to 71.2% (one-way ANOVA followed by Dunnett's test, p < 0.01, n = 3 independent experiments), revealing an unprecedented role of HupA in pancreatic β-cell protection beyond its well-established anti-AD activity. KEY POINTS: • Co-cultivation of endophytic fungi and bacteria could highly express prior HupA.