Search

Abstract Phloretin (PT) belongs to the dihydrochalcones (DHCs) family and is found in apple and rooibos tea. Its glycosides, including phlorizin (PT 2′-O-glucoside), trilobatin (PT 4′-O-glucoside), and nothofagin (NF, PT 3′-C-glucoside), are present in various plants. Phloretin and its related glycosides possess health benefits, including antioxidant, anti-inflammatory, and antibacterial activities. To biosynthesize PT and its glycosides, the relevant pathways in plants were studied and introduced into Escherichia coli. We reconstructed the biosynthetic pathways pertaining to PT and three PT C-glycosides (NF, PT 3′, 5′-di-C-glucoside [PDG], and PT 3′-C-arabinoside [PARA]) in E. coli. To prevent the undesirable synthesis of flavonoids instead of PT, we strategically divided the entire pathway into two parts: the first involved the synthesis of tyrosine to phloretic acid (PA), while the second involved the synthesis of PA to PT and its glycosides. The gene set pertaining to each part was incorporated into a different engineered microbe. We optimized phloretin microbial biosynthesis by improving enzyme affinity, identifying the gene that increased the output, refining the production design to a stepwise culture approach, and analyzing the culture conditions (substrate and yeast extract concentrations and pH) conducive to maximum output and the prevention of product degradation. Using the stepwise culture approach, 12.8 mg/L of PT, 26.1 mg/L of NF, 30.0 mg/L of PDG, and 18.1 mg/L of PARA were synthesized. This study provides valuable information for future approaches in the microbe-based synthesis of PT derivatives.

Objective: In humans, aging is associated with increased susceptibility to most age-related diseases. Phloretic acid (PA), a naturally occurring compound found in Ginkgo biloba and Asparagus, exhibits has potential as an anti-aging agent and possesses antioxidant, anti-inflammatory, and immunomodulatory properties. This study aimed to investigate the effects of PA on longevity and stress resistance in Caenorhabditis elegans (C.elegans) and the mechanisms that underlie its effects.Methods: First, we examined the effects of PA on lifespan and healthspan assay, stress resistance and oxidative analysis, lipofuscin levels. Second, we examined the insulin/insulin-like pathway, mitochondria, autophagy-related proteins, and gene expression to explain the possible mechanism of PA prolonging lifespan.Results: Our findings demonstrated that PA dose-dependently extended the C.elegans lifespan, with 200 μM PA showing the greatest effect and increased the C.elegans lifespan by approximately 16.7%. PA enhanced motility and the pharyngeal pumping rate in senescent C.elegans while reducing the accumulation of aging pigments. Further investigations revealed that daf-16, skn-1, and hsf-1 were required for mediating the lifespan extension effect of PA in C.elegans since its impact was suppressed in mutant strains lacking these genes. This suggests that PA activates these genes, leading to the upregulation of downstream genes involved in stress response and senescence regulation pathways. Furthermore, PA did not extend the lifespan of the RNAi atg-18 and RNAi bec-1 but it attenuated SQST-1 accumulation, augmented autophagosome expression, upregulated autophagy-related gene expression, and downregulated S6K protein levels. These findings suggest that the potential life-extending effect of PA also involves the modulation of the autophagy pathway.Conclusion: These findings results highlight the promising anti-aging effects of PA and warrant further investigation into its pharmacological mechanism and medicinal development prospects.