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 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 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.

Identification, Characterization, and Application of a Highly Sensitive Lactam Biosensor from Pseudomonas putida

“…For example, a better understanding of pestalactam A-C biosynthesis may provide new and more efficient chemoenzymatic routes to seven-ring cyclization . Recent work biochemically characterizing the fluvirucin β-amino acid loading pathway may also help open the door to effective PKS-based routes to non-natural lactam synthesis …”

“…27 Recent work biochemically characterizing the fluvirucin β-amino acid loading pathway may also help open the door to effective PKS-based routes to non-natural lactam synthesis. 28 In addition to the value of OplR as a biosensor for metabolic engineering purposes, it may also be useful as an inducible system. Valerolactam is inexpensive (∼$2/gram), and highly water-soluble (291 mg/mL).…”

Technical Advances to Accelerate Modular Type I Polyketide Synthase Engineering towards a Retro-biosynthetic Platform

“…Although to our knowledge there are no reported cases of a biosensor being coupled with type I PKS production in vivo, some biosensors are reported to detect chemicals that are made or can be possibly made by engineered PKSs, such as diacids [74,81], lactams [82][83][84], and pyrones [85,86]. These compounds are promising targets for high throughput PKS engineering (Fig.…”

Polyketide synthases as a platform for chemical product design

“…In contrast, a more recent effort swapped the promiscuous loading module of avermectin with the loading module of phoslactomycin that more selectively loads cyclohexanecarboxylic acid to improve the yield of doramectin . While swapping among and between Type A and Type B loading modules has been successful, Type C loading modules provide the entryway to even more diverse loading substrates, including β‐alanine, benzoyl CoA, among many others. While less thoroughly investigated, Type C modules have been successfully swapped into 6‐deoxyerythronolide synthase (DEBS), generating exotic triketide lactones …”