Cocultivation of pigeon pea hairy root cultures and Aspergillus for the enhanced production of cajaninstilbene acid.

Pigeon pea hairy root cultures (PPHRCs) have been proven to be a promising alternative for the production of health-beneficial phenolic compounds, such as the most important health-promoting compound, i.e., cajaninstilbene acid (CSA). In this study, PPHRCs were cocultured with live Aspergillus fungi for further improving phenolic productivity via biological elicitation. Aspergillus oryzae CGMCC 3.951 (AO 3.951) was found to be the optimal fungus that could achieve the maximum increment of CSA (10.73-fold increase) in 42-day-old PPHRCs under the inoculum size of mycelia 0.50% and cocultivation time 36 h. More precisely, the contents of CSA in hairy roots and culture media after fungal elicitation increased by 9.87- and 62.18-fold over control, respectively. Meanwhile, the contents of flavonoid glycosides decreased, while aglycone yields increased upon AO 3.951 elicitation. Moreover, AO 3.951 could trigger the oxidative stress and pathogen defense response thus activating the expression of biosynthesis- and ABC transporter-related genes, which contributed to the intracellular accumulation and extracellular secretion of phenolic compounds (especially CSA) in PPHRCs. And PAL2, 4CL2, STS1, and I3'H were likely to be the potential key enzyme genes regulating the biosynthesis of CSA, and ABCB11X1-1, ABCB11, and ABCG24X2 were closely related to the transmembrane transport of CSA. Overall, the cocultivation approach could make PPHRCs more commercially attractive for the production of high-value phenolic compounds such as CSA and flavonoid aglycones in nutraceutical/medicinal fields. And the elucidation of crucial biosynthesis and transport genes was important for systematic metabolic engineering aimed at increasing CSA productivity. KEY POINTS: • Cocultivation of PPHRCs and live fungi was to enhance CSA production and secretion. • PPHRCs augmented CSA productivity 10.73-fold when cocultured with AO 3.951 mycelia. • Several biosynthesis and transport genes related to CSA production were clarified.