Engineering Monobactam Biosynthesis towards the Generation of Novel β-Lactam Antibiotics

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β-lactam antibiotics are included in the WHO model list of essential medicines and represent an indispensible tool for the treatment of bacterial infections. Yet, emerging bacterial resistance constitutes a serious threat towards the medical community and calls for the development of new β-lactam derivatives. Monobactams , a subclass of β-lactam antibiotics, stand out due to their intrinsic stability against metallo-β-lactamases that render most of the clinically used β-lactam antibiotics ineffective. All monobactams (approved or in clinical studies) are manufactured completely synthetic. The recent discovery of the first bacterial biosynthetic gene cluster that is responsible for the production of the monobactam sulfazecin offers a very promising target towards the generation of novel monobactams through bioengineering and semi-synthetic chemistry. The backbone of sulfazecin is made from three amino acid building blocks by two multi-domain enzymes and further modified to yield the final β-lactam product. Preliminary studies show that the biosynthetic machinery is also capable to process a structural analog of the third amino acid building block, producing a sulfacezin analog that features an additional methyl-substituent at the β-lactam core (from synthetic monobactams it is known that substitution at this position dramatically increases hydrolytic stability). Promiscuity of each enzyme of the biosynthetic machinery towards this structural substrate analog harbours a tremendous potential towards the generation of novel monobactams by green chemistry (fermentation). Structure guided mutations within defined parts of the involved biosynthetic enzymes are purposed to extend and manipulate the spectrum of incorporated building blocks, yielding the desired monobactam products. Subsequent chemical side chain modification is intended to increase the structural variety even more. The substrate promiscuity/specificity of the mutant enzymes can be either assessed in vivo or in vitro. Bioassays will evaluated potential of novel monobactams.