Engineering Monobactam Biosynthesis towards the Generation of Novel β-Lactam Antibiotics
Organic Molecular Chemistry - Synthesis and Characterisation
Final Report Abstract
The wide-spread use of β-lactam antibiotics subjected their bacterial targets to an immense selective pressure to develop ever more powerful mechanisms of resistance, including the production of β-lactamases that render an increasing number of prescribed β-lactam antibiotics ineffective and urgently call for the discovery of new compounds to treat these pathogens. Sulfazecin, produced by Pseudomonas acidophila, belongs to the sub-class of monobactams that, due to their characteristic N-sulfonation, are intrinsically resistant towards metallo-βlactamases. Through the supplementation of suitable precursor molecules, the principal aim of this project was to utilize the native sulfazecin biosynthetic pathway to generate functionalized derivatives of sulfazecin by mutasynthesis that are potentially showing an altered spectrum of bioactivity. Therefore, a divergent synthesis was developed to prepare various functionalized congeners of the non-canonical amino acid L-2,3-diaminopropionate (L-Dap) as alternative biochemical building blocks. In vitro assays revealed that the gatekeeping adenylation domain (A3), that controls their incorporation into the biosynthetic pathway, shows a high diastereoselectivity and activates analogs of L-Dap bearing an additional methyl (MeDap)- or fluoromethyl substituent to a notable degree. When these were supplemented to a knockout strain of P. acidophila that is deficient in production of the native precursor L-Dap, high resolution mass spectrometry confirmed that these analogs were in fact carried through the entire biosynthetic pathway to yield the corresponding C4-substituted sulfazecin derivatives through fermentation. Bioassays revealed an altered antibacterial activity compared to the native sulfazecin and highlights the intrinsic capability of this platform to produce functionalized monobactams in detectable quantities. Initial efforts to genetically engineer A3 towards improved activity on functionalized L-Daps yielded an increase of 65 % in relative activity for MeDap by a single mutation, further emphasizing the potential of this biosynthetic system. During the screening of mutant A3 it was realized that most established adenylation domain assays are either insensitive or require additional co-enzymes or expensive chemicals and equipment. To provide a sensitive and cost-efficient alternative, a novel colorimetric adenylation assay was developed that visualizes enzymatic activity by a color change from blue to orange through detection of enzymatically released pyrophosphate at micromolar levels. Finally, it was shown that the terminal biosynthetic enzymes that complete sulfazecin biosynthesis through hydroxylation and O-methylation, respectively, have appreciable activity on synthetic monobactams bearing non-native side chains. These studies highlight the potential of a chemoenzymatic approach to develop new monobactam antibiotics in order to overcome bacterial resistance enzymes.
Publications
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Colorimetric Determination of Adenylation Domain Activity in Nonribosomal Peptide Synthetases by Using Chrome Azurol S. ChemBioChem, 24(5).
Kahlert, Lukas; Lichstrahl, Michael S. & Townsend, Craig A.
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Synthesis of functionalized 2,3-diaminopropionates and their potential for directed monobactam biosynthesis. Chemical Science, 14(14), 3923-3931.
Lichstrahl, Michael S.; Kahlert, Lukas; Li, Rongfeng; Zandi, Trevor A.; Yang, Jerry & Townsend, Craig. A.
