Expanding Polycyclic Tetramate Macrolactam (PoTeM) Core Structure Diversity by Chemo-Enzymatic Synthesis and Bioengineering

Abstract

Polycyclic tetramate macrolactams (PoTeMs) represent a growing class of bioactive natural products that are derived from a common tetramate polyene precursor, lysobacterene A, produced by an unusual bacterial iterative polyketide synthase (PKS)/non-ribosomal peptide synthetase (NRPS). The structural and functional diversity of PoTeMs is biosynthetically elaborated from lysobacterene A by pathway-specific cyclizing and modifying enzymes. This results in diverse core structure decoration and cyclization patterns. However, approaches to directly edit the PoTeM carbon skeleton do currently not exist. We thus set out to modify the PoTeM core structure by exchanging the natural l-ornithine-derived building block by l-lysine, hence extending macrocycle size by an additional CH₂ group. We developed streamlined synthetic access to lysobacterene A and the corresponding extended analog and achieved cyclization of both precursors by the cognate PoTeM cyclases IkaBC in vitro. This chemo-enzymatic approach corroborated the catalytic competence of IkaBC to produce a larger macrolactam yielding homo-ikarugamycin. We thus engineered the adenylation domain active site of IkaA to directly accept l-lysine, which upon co-expression with IkaBC delivered a recombinant bacterial homo-ikarugamycin producer. Our work establishes an entirely new PoTeM structural framework and sets the stage for the biotechnological diversification of the PoTeM natural product class in general.

Keywords: PoTeM; chemo-enzymatic; natural products; protein engineering; total synthesis.

Scheme 1

Biosynthesis and structural diversity of PoTeMs. All PoTeMs derive from a common precursor lysobacterene A (2 a), which is produced by an iPKS/NRPS (e.g., IkaA). Oxidoreductases (light blue) catalyze the formation of different carbocyclic patterns, leading to a broad variety of PoTeM core structures. Tailoring enzymes further diversify the PoTeM structural spectrum by introducing functional groups. PoTeM hydroxylases (PH, purple) can act on a broad variety of PoTeMs and introduce a hydroxy group at C‐3 (3–6). Monooxygenases like CftA and IkaD (green) add epoxides, ketones, and hydroxy groups, resulting in, e.g., 8 and 9. Additionally, the tetramic acid can be methylated by a methyl transferase (red) towards 10. Recently, also halogenated derivatives have been discovered, e.g., 12 (orange).

Scheme 2

Retrosynthetic analysis of IkaA intermediate 2 with key reactions. Individual building blocks are depicted in green (tetramic acid), red (C₂ moiety for attachment of southern side chain), blue (polyene aldehyde), and yellow (C₁₂ polyene carboxylic acid). Both polyene chains (blue and yellow) can be accessed by Iridium‐catalyzed stepwise chain elongation.[ ¹⁸ , ¹⁹ ]

Scheme 3

Preparation of the central building blocks. A. Synthesis of ylides 16 and B. stepwise chain elongation towards conjugated olefin side chains (acid 25 and aldehyde 26).

Scheme 4

Fusing the individual compounds towards 2. A. Wittig‐olefination of ylides 16 and B. completion of the synthesis of IkaA intermediates 2.

Figure 1

NRPS expression plasmid and substrate‐conferring codes of IkaA and 15 representative l‐lysine activating A domains. The residues were determined according to PheA and contained two to eight differences (grey) compared to IkaA. Mutations of lysine‐activating construct mut6 are labeled in green.

Figure 2

HRMS analysis of the cell extracts from Streptomyces expressing mutated ika. A. EIC for mass of ikarugamycin (1). B. EIC for the mass of homo‐ikarugamycin (13) with extended carbon skeleton (green). I) Wild‐type, II) I308V, III) double mutant, and IV) E281D. The intensities of chromatograms in B were scaled up by a factor of five.

Figure 3

Active site of the IkaA structure predicted by AlphaFold and modeling of substrate binding. A. In the wild‐type IkaA, ionic interactions between Glu281 (2.2 Å) and Asp342 (3.2 Å) to the amino group of l‐ornithine are possible. B. In the double mutant mut6 (E281D, I308V), Asp342 can still interact with l‐ornithine (pink). C. Through mutation of Glu281 to Asp the active site pocket is extended, making space and allowing binding for the longer l‐Lys (purple) substrate. In addition, the interaction between the carboxylic acid group of Asp281 and the ϵ‐amino group of l‐Lys provides specificity.