[Construction of a high-yield platform for phlorizin based on Nicotiana benthamiana]

Phlorizin has antioxidant and anti-aging pharmacological activities, which render it a highly promising candidate for applications in functional foods and the pharmaceutical industry. However, the incomplete elucidation of phlorizin biosynthesis in plants has constrained its large-scale production and application. Lithocarpus litseifolius, which contains a notably higher level of phlorizin than most plant species, serves as an ideal model for identifying key enzymes involved in phlorizin biosynthesis. The genes MdDBR, MdCHS, and MdMYB10 from Malus domestica Borkh. and LlP2'GT from L. litseifolius were introduced exogenously, and the successful production of phlorizin was detected using the Agrobacterium-mediated transient expression system in Nicotiana benthamiana L. as a platform. Further investigation revealed that the transcription factor AtMYB12 from Arabidopsis thaliana significantly enhanced the yield of phlorizin, outperforming MdMYB10 from M. domestica. Furthermore, substituting M. domestica-derived MdCHS with L. litseifolius-derived chalcone synthase LlCHS1 further increased phlorizin accumulation. Subsequently, a double bond reductase gene LlDBR4 was cloned from L. litseifolius, which promoted efficient phlorizin synthesis in N. benthamiana. Co-expression of LlDBR4 with AtMYB12, LlCHS1, and LlP2'GT achieved a phlorizin yield of 2 407.57 μg·g~(-1), setting the current highest record for heterologous production in N. benthamiana. This research used Agrobacterium-mediated transient expression system in N. benthamiana as a platform. By introducing the highly efficient AtMYB12 transcription factor and exploring the double bond reductase gene from L. litseifolius, a plant production platform for phlorizin was successfully constructed, providing critical theoretical and technical frameworks for the efficient heterologous production of phloridzin in plant chassis. These findings hold significant implications for advancing phlorizin industrialization.