مورد إلكتروني
Development of a High-Throughput, In Vivo Selection Platform for NADPH-Dependent Reactions Based on Redox Balance Principles.
| العنوان: | Development of a High-Throughput, In Vivo Selection Platform for NADPH-Dependent Reactions Based on Redox Balance Principles. |
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| المؤلفون: | Zhang, Linyue |
| المصدر: | ACS synthetic biology; vol 7, iss 7, 1715-1721; 2161-5063 |
| بيانات النشر: | eScholarship, University of California 2018-07-01 |
| تفاصيل مُضافة: | Zhang, Linyue King, Edward Luo, Ray Li, Han |
| نوع الوثيقة: | Electronic Resource |
| مستخلص: | Bacteria undergoing anaerobic fermentation must maintain redox balance. In vivo metabolic evolution schemes based on this principle have been limited to targeting NADH-dependent reactions. Here, we developed a facile, specific, and high-throughput growth-based selection platform for NADPH-consuming reactions in vivo, based on an engineered NADPH-producing glycolytic pathway in Escherichia coli. We used the selection system in the directed evolution of a NADH-dependent d-lactate dehydrogenase from Lactobacillus delbrueckii toward utilization of NADPH. Through one round of selection, we obtained multiple enzyme variants with superior NADPH-dependent activities and protein expression levels; these mutants may serve as important tools in biomanufacturing d-lactate as a renewable polymer building block. Importantly, sequence analysis and computational protein modeling revealed that diverging evolutionary paths during the selection resulted in two distinct cofactor binding modes, which suggests that the high throughput of our selection system allowed deep searching of protein sequence space to discover diverse candidates en masse. |
| مصطلحات الفهرس: | Escherichia coli, NADP, L-Lactate Dehydrogenase, Oxidation-Reduction, Metabolic Engineering, NADPH-dependent pathways, d-lactate dehydrogenase, high-throughput selection, metabolic engineering, redox balance, D-lactate dehydrogenase, Biotechnology, Medicinal and Biomolecular Chemistry, Biochemistry and Cell Biology, Biomedical Engineering, article |
| URL: | |
| الإتاحة: | Open access content. Open access content CC-BY |
| ملاحظة: | application/pdf ACS synthetic biology vol 7, iss 7, 1715-1721 2161-5063 |
| أرقام أخرى: | CDLER oai:escholarship.org:ark:/13030/qt0m14v4h5 qt0m14v4h5 https://escholarship.org/uc/item/0m14v4h5Test https://escholarship.orgTest/ 1367428231 |
| المصدر المساهم: | UC MASS DIGITIZATION From OAIster®, provided by the OCLC Cooperative. |
| رقم الانضمام: | edsoai.on1367428231 |
| قاعدة البيانات: | OAIster |
| الوصف غير متاح. |