| Abstrakt: |
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. [ABSTRACT FROM AUTHOR] |
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