Biosynthesis of Beta-Lactam Antibiotics

50年来，青霉素和相关的β-内酰胺类抗生素一直是治疗感染的主要药物。然而，与其他已知类别的抗生素一样，它们的有效性受到多重耐药致病菌的日益增长的挑战。已加紧努力了解耐药性的机制并加以克服。通过对抗生素生物合成途径的遗传操作进行结构修饰是一种有前途的方法，可以通过具有成本效益的发酵和半合成方法来生产已知抗生素的变体。在本申请中提出了继续研究β-内酰胺抗生素生物合成的计划。将研究四种已知抗生素中的三种：（1）拉维酸，一种有效的β-内酰胺酶抑制剂/灭活剂和广泛的耐药性来源，（2）诺卡菌素，一种单环β-内酰胺类和代谢相关的单环内酰胺类，和（3）碳青霉烯类，临床上以硫霉素及其衍生物为代表，但大多数是由碳青霉烯-2-烯-3-羧酸引起的。这些主要群体中至少有一个成员的生物合成基因簇的表征导致了当前资助期内使用从有机合成和酶学到分子生物学和大分子结构方法的技术的快速进展。建议通过N2-的机理和结构研究来追求这些发现。（羧乙基）-L-精氨酸合酶，位点特异性突变以检查β-内酰胺合成酶的机制并工程化β-内酰胺合成酶的功能，合作研究以表征棒氨酸合酶的铁中心，以及底物类似物和位点定向突变实验以检查其机制，并研究克拉维酸生物合成的倒数第二步中发生的“对映异构化”。将进行诺卡氏菌素生物合成基因的破坏和过表达以描绘该途径， 了解单环β-内酰胺形成的机制，并确定两种不寻常的非核糖体肽合成酶的作用。将开始对生物合成相关的单环内酰胺类抗生素进行研究。形成碳青霉烯核的三种关键蛋白质的表征将在该组的研究中得到重要的体现，并且将开始对硫霉素进行新的实验，以了解该家族中更复杂的成员及其合成工程。

Penicillin and related beta-lactam antibiotics have been a mainstay in the treatment of infections for 50 years. Their effectiveness, however, like other known classes of antibiotics has come under increasing challenge from the rise of multiply drug-resistant pathogenic bacteria. Efforts have intensified to understand the mechanisms of resistance and to overcome them. Structural modification through genetic manipulation of their biosynthetic pathways is a promising approach to produce variants of known antibiotics by cost-effectivefermentation and semi-synthetic methods. Continuation of a program to investigate beta-lactam antibiotic biosynthesis is proposed in this application. Three of the four known classes of these antibiotics will be studied: (1) lavulanic acid, a potent inhibitor/inactivator of beta-lactamase enzymes and a wide-spread source of resistance, (2) the nocardicins, a family of monocyclic beta-lactams, and the metabolically related monobactams, and (3) the carbapenems, represented clinically by thienamycin and its derivatives, but mostsimply by carbapen-2-em-3-carboxylic acid. Characterization of the biosynthetic gene clusters for at least one member of each of these principal groups has led to rapid advances in the current grant period using techniques ranging from organic synthesis and enzymology to molecular biology and macromolecular structural methods. It is proposed to pursue these discoveries through mechanistic and structural studies of N2-(carboxyethyl)-L-arginine synthase, site-specific mutatagenesis to examine the mechanism and engineer the function of beta-lactam synthetase, collaborative studies to characterize the iron center of clavaminate synthase, and substrate analogue and site-directed mutatagenesis experiments to examine its mechanism, and to investigate the "enantiomerization that occurs in the penultimate step of clavulanic acid biosynthesis. Disruption and over-expression of nocardicin biosynthetic genes will be undertaken to delineate the pathway, understand the mechanism of monocyclic beta-lactam formation and determine the roles of two unusual non-ribosomal peptide synthetases. Investigation of the biosynthetically related monobactams will be initiated. Characterization of three key proteins that form the carbapenem nucleus will figure prominantly in studies of this group, and new experiments with thienamycin will be begun to understand the more complex members of this family and engineering of their synthesis.