Recombinant Macrocyclic Lanthipeptides Incorporating Non-Canonical Amino Acids

Abstract

Nisin is a complex lanthipeptide that has broad spectrum antibacterial activity. In efforts to broaden the structural diversity of this ribosomally synthesized lantibiotic, we now report the recombinant expression of Nisin variants that incorporate non-canonical amino acids (ncAAs) at discrete positions. This is achieved by expressing the nisA structural gene, cyclase (nisC) and dehydratase (nisB), together with an orthogonal nonsense suppressor tRNA/aminoacyl-tRNA synthetase pair in E.coli. A number of ncAAs with novel chemical reactivity were genetically incorporated into NisA, including an α-chloroacetamide-containing ncAA which allowed for the expression of Nisin variants with novel macrocyclic topologies. This methodology should allow for the exploration of lanthipeptide variants with new or enhanced activities.

Playing with the Molecules of Life

“…71, 270 Finally, E. coli has been engineered for the recombinant production of ribosomally synthesized posttranslationally modified peptides (RIPPs) that contain ncAAs, and analogues of the lanthipeptide nisin with altered ring structures and sizes have been produced. 271–275 …”

Preparative Asymmetric Synthesis of Canonical and Non‐canonical α‐amino Acids Through Formal Enantioselective Biocatalytic Amination of Carboxylic Acids

“…[7] a-HAs can be further oxidized into a-keto-acids [8] to serve as precursors for a-amino acid (a-AAs). [11] While fermentation processes for most proteinogenic l-a-AAs are well established, [9] ncAAs are often produced via chemical synthesis (Scheme 1). [9][10] In particular, non-canonical l-a-AAs (ncAAs) are of increasing interest and demand, driving innovations in the field of synthetic biology, pharma, bio-imaging, biosensors, medicinal applications or hybrid-/organocatalysts design.…”

“…[9][10] In particular, non-canonical l-a-AAs (ncAAs) are of increasing interest and demand, driving innovations in the field of synthetic biology, pharma, bio-imaging, biosensors, medicinal applications or hybrid-/organocatalysts design. [11] While fermentation processes for most proteinogenic l-a-AAs are well established, [9] ncAAs are often produced via chemical synthesis (Scheme 1). [10,12] Despite the high efficiency and broad substrate scope, toxic cyanides (KCN), strong acids and petroleum-based aldehydes (derived from multistep high energy processes involving rare-metals) [1b,13] are typically required for chemical manufacturing (Scheme 1).…”

Therapeutic applications of genetic code expansion

“…More recently, co-translational and site-specific incorporation of ncAAs has been applied to the biosynthesis of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products to expand their chemistry and structure repertoire. For example, incorporation of ncAAs at discrete positions on Nisin expanded building blocks that can be biosynthetically incorporated into lanthipeptide and diversified macrocyclic topologies of this antibacterial peptide [ 100 ]. Likewise ncAAs have been successfully introduced into other lanthipeptides [ 101 , 102 ], lasso peptides [ 103 , 104 ], macrocyclic peptides [ 105 ], thiopeptides [ 106 ] as well as in the natural product cinnamycin, recently [ 107 ].…”