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 青霉素是解决革兰氏阳性细菌最有效的抗生素之一 感染，包括细菌性脑膜炎、白喉、链球菌性咽喉炎、梅毒、淋病和雅司病 每年超过600万人。这些抗生素经常供应短缺并提高产量 需要既增加可达性又减少低效率和环境影响的方法 与当前的生产方式有关。青霉素商业化生产的主要途径开始 通过真菌 P. chrysogenum 的分批发酵作为 6-氨基青霉烷酸 (6-APA) 的生物合成途径， 随后用作合成 β-内酰胺抗生素的起始材料。作为替代方案 目前6-APA酰胺化采用的有机合成路线有基于化学酶法合成的方法 青霉素酰胺酶 (PA) 或异青霉烷酸转移酶 (IAT) 对 6-APA 的酰胺化提供了具有竞争力的 青霉素类抗生素的绿色化学方法。每种化学酶方法都带来独特的挑战。 酰胺酶催化的 6-APA 酰化需要使用化学活化的羧酸衍生物作为 底物和转化效率较低。另一方面，IAT 依赖于苯乙酰基 辅酶 A 连接酶稳定性低，底物特异性有限。 在目前的工作中，我们的目标是采用替代化学酶策略来合成青霉素。 通过结合 IAT 和工程化的硫醇酰化醛脱氢酶 (TAD) 的活性来去除醛。第一个 这项工作的具体目标是在催化活性位点选择性引入突变 来自恶臭假单胞菌 (S12) 的苯乙醛脱氢酶 (NPADH)，具有广泛的醛基 特异性。突变将靶向 NPADH 转化为 TAD，用于合成 N-乙酰半胱胺 (SNAc)硫酯。在我们的第二个目标中，SNAc 硫代酯（酰基辅酶 A 的替代物）将与 6- 一起引入 IAT 催化青霉素合成中的 APA 共底物。该策略预计将带来高产量 产量，同时消除与苯乙酰 CoA 连接酶相关的限制。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 β-内酰胺抗生素。