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Utilizing site-directed mutagenesis and heterologous expression, we discovered that unantimycin productions are correlated because of the task of a chorismatase homolog, the nat-hyg5 gene, from a type-I PKS gene cluster. Biochemical analysis verified that the catalytic activity of Nat-hyg5 creates 3-HBA from chorismate. Eventually, we accomplished discerning production of unantimycins B and C by engineering a chassis number. On the basis of these results, we suggest that unantimycin biosynth