Repurposing Styrene Catabolic Enzymes for the Synthesis of Penicillins

摘要 青霉素代表用于解决革兰氏阳性细菌感染的最有效的抗生素之一。 包括细菌性脑膜炎、白喉、链球菌性喉炎、梅毒、淋病和雅司病在内的感染， 每年超过600万人。这些抗生素经常供应短缺， 需要既提高可达性又降低效率和环境影响的方法 与目前的生产方式有关。青霉素商业化生产的主要途径开始于 利用产黄青霉菌的分批发酵作为6-氨基青霉烷酸（6-阿帕）的生物合成途径， 其随后用作合成β-内酰胺抗生素的起始原料。作为替代 目前采用的酰胺化6-阿帕的有机合成路线，基于 青霉素酰胺酶（PA）或异青霉烷酸转移酶（IAT）对6-阿帕酰胺化提供了竞争性的 青霉素类抗生素的绿色化学方法。每种化学酶方法都提出了独特的挑战。 酰胺酶催化的6-阿帕酰化需要使用化学活化的羧酸衍生物， 底物，并以低转化效率进行。另一方面，IAT依赖于苯乙酰基- 辅酶A连接酶，其具有低稳定性和有限的底物特异性。 在目前的工作中，我们的目标是一个替代的化学酶的策略，从青霉素的合成 通过结合IAT和工程改造的巯基酰化醛脱氢酶（thiol-acylating aldehyde dehydrogenase，简称DHD）的活性，第一 这项工作的具体目标将针对选择性地引入突变的催化活性位点， 来自恶臭假单胞菌（S12）的苯乙醛脱氢酶（NPADH），其具有宽的醛 的特异性突变将靶向NPADH转化为用于合成N-乙酰半胱胺的底物 （SNAc）硫酯。在我们的第二个目标中，将引入作为酰基-CoA的替代物的SNAc硫代酯，其中6- 阿帕辅助底物在IAT催化青霉素合成中的作用。这一战略预计将导致高产品 产率，同时消除了与苯乙酰辅酶A连接酶相关的限制。这个过程的发展 将作为原型绿色化学途径，可以进一步扩展为生产平台， 现有的和新的B-内酰胺类抗生素。

Abstract Penicillins represent one of the most impactful antibiotics in use for the resolution of gram-positive bacterial infections including bacterial meningitis, diptheria, strep throat, syphilis, gonorrhea, and yaws disease that afflict more than 6 million people annually. These antibiotics are frequently in short supply and improved production methods are needed that both increase the accessibility while reducing inefficiency and environmental impacts associated with current production methods. The primary route to the commercial production of penicillins begins with batch fermentation of the fungus, P. chrysogenum as a biosynthetic route to 6-aminopenicillanic acid (6-APA), which is subsequently used as a starting material for the synthesis of b-lactam antibiotics. As an alternative to currently employed organic synthetic routes to amidation of 6-APA, chemoenzymatic synthetic methods based on the amidation of 6-APA by penicillin amidases (PAs) or isopenicillanic acid transferases (IATs) provide a competitive green chemical approach to penicillin-based antibiotics. Each chemoenzymatic approach poses unique challenges. The amidase-catalyzed acylation of 6-APA requires the use of chemically activated carboxylic acid derivatives as substrates and proceeds with low transformation efficiency. IATs on the other hand are dependent on phenylacetyl- Coenzyme A ligases, which have low stability and limited substrate specificity. In the present work we target an alternate chemoenzymatic strategy for the synthesis of penicillins from aldehydes by joining the activities of IAT and an engineered thiol-acylating aldehyde dehydrogenase (TAD). The first specific aim of this work will target the selective introduction of mutations in the catalytic active site of phenylacetaldehyde dehydrogenase (NPADH) from Pseudomonas putidia (S12), which has a broad aldehyde specificity. Mutations will target the transformation of NPADH into a TAD for the synthesis of the N-acetylcysteamine (SNAc) thioesters. In our second aim, SNAc thiosesters, which are surrogates of acyl-CoA will be introduced with 6- APA co-substrates in the IAT-catalyzed synthesis of penicillins. This strategy is expected to result in a high product yield while eliminating the limitations associated with the phenylacetyl CoA ligases. The development this process will serve as prototypical green-chemistry pathway that can be further expanded into a platform for the production of existing and new classes of b-lactam antibiotics.