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.

摘要