Repurposing Styrene Catabolic Enzymes for the Synthesis of Penicillins

重新利用苯乙烯分解代谢酶来合成青霉素

• 批准号: 10411114
• 负责人: George T. Gassner
• 金额: $ 15.5万
• 依托单位: SAN FRANCISCO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-20 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10411114
• 关键词: Acids; Active Sites; Acyl Coenzyme A; Acylation; Acyltransferase; Aldehydes; Amino Acid Substitution; Amino Acids; Antibiotics; Bacterial Meningitis; Biological Assay; C-terminal; Carboxylic Acids; Chemicals; Chemistry; Chimeric Proteins; Coenzyme A; Coenzyme A Ligases; Development; Disease; Engineering; Environmental Impact; Enzymes; Fermentation; Flavins; Fluorescence; Future; Gonorrhea; Gram-Positive Bacterial Infections; High Pressure Liquid Chromatography; Histidine; In Vitro; Isomerase; Kinetics; Laboratories; Ligase; Methods; Mixed Function Oxygenases; Monobactams; Mutation; Natural regeneration; Outcome; Oxidoreductase; Pathway interactions; Penicillins; Persons; Pharyngeal structure; Preparation; Process; Production; Protein Engineering; Pseudomonas; Reaction; Recombinants; Resolution; Route; Side; Specificity; Structure; Styrenes; Substrate Specificity; Sulfhydryl Compounds; Synthetic Genes; Syphilis; System; Transferase; United States; Work; Yaws; aldehyde dehydrogenases; amidase; amidation; aptamer; base; beta-Lactams; catalase; chemical synthesis; cost; detection assay; fungus; improved; mutant; novel strategies; phenylacetaldehyde; polypeptide; prototype; pyridine nucleotide; styrene oxide; thioester

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.

抽象的 青霉素代表用于分辨革兰氏阳性细菌的最具影响力的抗生素之一 感染包括细菌性脑膜炎，白喉，链球菌喉咙，梅毒，淋病和偏航病 每年有超过600万人。这些抗生素经常供应不足并改善生产 需要方法，既可以提高可访问性，同时降低效率低下和环境影响 与当前的生产方法相关联。青霉素商业生产的主要途径开始 通过真菌的批处理发酵，金氏菌作为一种生物合成途径，通向6-氨基核酸（6-APA）， 随后，它用作合成B-内酰胺抗生素的起始材料。作为替代方案 目前采用有机合成途径来鉴定基于6-APA的化学酶合成方法 青霉素胺（PAS）或等苯二甲酸转移酶（IAT）的6-APA酰化可提供竞争力 基于青霉素的抗生素的绿色化学方法。每种化学酶方法都带来了独特的挑战。 6-APA的酰胺酶催化的酰化需要化学活化的羧酸衍生物作为 底物和进行较低的转化效率。另一方面，IAT取决于苯乙酰基 - 辅酶A连接酶，其稳定性较低且底物特异性有限。 在目前的工作中，我们针对一种替代化学酶策略，用于合成青霉素 通过连接IAT的活性和工程化的硫醇醛醛脱氢酶（TAD）来加入醛。第一个 这项工作的具体目的将针对选择性引入突变的催化活性位点 来自假单胞菌（S12）的苯基乙醛脱氢酶（NPADH），其具有宽阔的醛 特异性。突变将针对NPADH转化为TAD，以合成N-乙酰囊这汀 （SNAC）硫代植物。在我们的第二个目标中，将引入6-- apa在IAT催化的青霉素合成中。预计该策略将导致高产品 在消除与苯乙酰基CoA连接酶相关的局限性的同时。发展这个过程 将作为典型的绿色化学途径，可以进一步扩展到生产平台 现有和新类别的B-内酰胺抗生素。