Biochemical Characterization of β‐Amino Acid Incorporation in Fluvirucin B₂ Biosynthesis

Abstract Naturally occurring lactams, such as the polyketide‐derived macrolactams, provide a diverse class of natural products that could enhance existing chemically produced lactams. Although β‐amino acid loading in the fluvirucin B 2 polyketide pathway was proposed by a previously identified putative biosynthetic gene cluster, biochemical characterization of the complete loading enzymes has not been described. Here we elucidate the complete biosynthetic pathway of the β‐amino acid loading pathway in fluvirucin B 2 biosynthesis. We demonstrate the promiscuity of the loading pathway to utilize a range of amino acids and further illustrate the ability to introduce non‐native acyl transferases to selectively transfer β‐amino acids onto a polyketide synthase (PKS) loading platform. The results presented here provide a detailed biochemical description of β‐amino acid selection and will further aid in future efforts to develop engineered lactam‐producing PKS platforms.