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

β-内酰胺类抗生素被列入世卫组织基本药物标准清单，是治疗细菌感染不可或缺的工具。然而，新出现的细菌耐药性对医学界构成了严重威胁，要求开发新的β-内酰胺衍生物。单奥巴坦是β-内酰胺类抗生素的一个亚类，由于其对金属β-内酰胺酶的固有稳定性而脱颖而出，这使得大多数临床使用的β-内酰胺类抗生素无效。所有单巴坦（已批准或处于临床研究阶段）都是完全人工合成的。最近发现的第一个细菌生物合成基因簇负责生产单巴坦磺胺，为通过生物工程和半合成化学产生新的单巴坦提供了一个非常有希望的目标。磺胺嘧啶的主链由三个氨基酸组成，通过两种多结构域酶进一步修饰生成最终的β-内酰胺产物。初步研究表明，生物合成机制也能够处理第三个氨基酸构建块的结构类似物，产生具有β-内酰胺核心额外甲基取代基的磺胺类类似物（从合成的单菌酰胺中，已知该位置的取代显著提高了水解稳定性）。生物合成机制中每种酶对这种结构底物模拟物的混杂性，为绿色化学（发酵）产生新型单菌菌提供了巨大的潜力。在所涉及的生物合成酶的定义部分内的结构引导突变旨在扩展和操纵纳入的构建块的光谱，产生所需的单巴坦产品。随后的化学侧链修饰旨在进一步增加结构的多样性。突变酶的底物混杂性/特异性可以在体内或体外进行评估。生物测定将评估新型单孢菌的潜力。

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.