The biosynthesis of the three """"""""non-classical"""""""" clans of the beta-lactam antibiotics will be investigated. Represented by the """"""""classical"""""""" penicillins and cephalosporins, collectively these drugs constitute 65% of the world antibiotic market and account for >$25B/yr in economic value. They remain an important mainstay of human health and longevity. But with wide spread use has come the inevitable rise of antibiotic-resistant infections. Structural modifications have slowed these effects, but increased reliance on the newer, non-classical classes, for example, the beta-lactamase inhibitor clavulanic acid and the potent, broad-spectrum carbapenems like thienamycin, will benefit from fundamental understanding of their biosynthesis for improved, lower cost manufacture by fermentation, semi-synthesis and, as we propose, chemoenzymatic synthesis using engineered biosynthetic pathways to produce clinically-used drugs and new structures with improved characteristics. The monocyclic beta-lactams are the simplest structurally but ironically the mechanism of their formation is unknown. A series of breakthroughs in the current grant period have set the stage to finally solve this problem. This understanding will guide experiments with the biosynthetically related monobactams, distinct for their N-sulfonated beta-lactam rings that confer the increasingly important property of comparative resistance to the Class B beta-lactamases (Zn++ metalloproteases). While in the current grant period we discovered and examined in detail the ATP-dependent beta-lactam forming reactions in both clavulanic acid and carbapenem biosynthesis, the essential oxidative bicyclic ring inversion processes to the antibiotic-active stereochemistry will be determined. The four known classes of beta-lactam antibiotics arise by convergent evolution and are emblematic of remarkably varied enzyme mechanisms of impressive synthetic efficiency. Research to understand them holds great training potential for students and affords the opportunity to make valuable contributions academically and practically to their manufacture and structural alteration to overcome resistance mechanisms.

Public Health Relevance

The non-classical beta-lactam antibiotics, notably the beta-lactamase inhibitor clavulanic acid and the potent, broad-spectrum carbapenems, have become increasingly important in healthcare to overcome resistant bacterial infections. Study of their biosynthesis will improve and lower the costs of manufacture, and combined chemical and enzymatic syntheses using engineered biosynthetic proteins can enable these advances and provide routes to agents with improved properties.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
High Priority, Short Term Project Award (R56)
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Synthetic and Biological Chemistry B Study Section (SBCB)
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Xu, Zuoyu
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Johns Hopkins University
Schools of Arts and Sciences
United States
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Long, Darcie H; Townsend, Craig A (2018) Mechanism of Integrated ?-Lactam Formation by a Nonribosomal Peptide Synthetase during Antibiotic Synthesis. Biochemistry 57:3353-3358
Li, Rongfeng; Oliver, Ryan A; Townsend, Craig A (2017) Identification and Characterization of the Sulfazecin Monobactam Biosynthetic Gene Cluster. Cell Chem Biol 24:24-34
Townsend, Craig A (2016) Convergent biosynthetic pathways to ?-lactam antibiotics. Curr Opin Chem Biol 35:97-108
Marous, Daniel R; Lloyd, Evan P; Buller, Andrew R et al. (2015) Consecutive radical S-adenosylmethionine methylations form the ethyl side chain in thienamycin biosynthesis. Proc Natl Acad Sci U S A 112:10354-8
Gaudelli, Nicole M; Long, Darcie H; Townsend, Craig A (2015) ?-Lactam formation by a non-ribosomal peptide synthetase during antibiotic biosynthesis. Nature 520:383-7
Gaudelli, Nicole M; Townsend, Craig A (2014) Epimerization and substrate gating by a TE domain in ?-lactam antibiotic biosynthesis. Nat Chem Biol 10:251-8
Li, Rongfeng; Lloyd, Evan P; Moshos, Kristos A et al. (2014) Identification and characterization of the carbapenem MM 4550 and its gene cluster in Streptomyces argenteolus ATCC 11009. Chembiochem 15:320-31
Gaudelli, Nicole M; Townsend, Craig A (2013) Stereocontrolled syntheses of peptide thioesters containing modified seryl residues as probes of antibiotic biosynthesis. J Org Chem 78:6412-26
Phelan, Ryan M; Townsend, Craig A (2013) Mechanistic insights into the bifunctional non-heme iron oxygenase carbapenem synthase by active site saturation mutagenesis. J Am Chem Soc 135:7496-502
Phelan, Ryan M; DiPardo, Benjamin J; Townsend, Craig A (2012) A high-throughput screen for the engineered production of ?-lactam antibiotics. ACS Chem Biol 7:835-40

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