Engineering of phenylalanine dehydrogenase from Thermoactinomyces intermedius for the production of a novel homoglutamate
The dramatic increase in healthcare costs has become a significant burden to this era. Many patients are unable to access medication because of the high price of drugs. Genetic engineering has made advances to increase the yield, titer, and productivity in the bio-based production of chemicals, mate...
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| Veröffentlicht in: | PloS one 2022-03, Vol.17 (3), p.e0263784-e0263784 |
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| creator | Tariq, Muhammad Israr, Muhammad Raza, Muslim Ahmad, Bashir Azizullah, Azizullah Ur Rehman, Shafiq Faheem, Muhammad Sun, Xinxiao Yuan, Qipeng |
| description | The dramatic increase in healthcare costs has become a significant burden to this era. Many patients are unable to access medication because of the high price of drugs. Genetic engineering has made advances to increase the yield, titer, and productivity in the bio-based production of chemicals, materials of interest, and identification of innovative targets for drug discovery. Currently, the production of homoglutamate (α-Aminoadipic acid) involves petrochemical routes that are costly with low yield and often not suitable for industrial production. Here, we established the development of NADH-dependent homoglutamate by engineering NADH-dependent phenylalanine dehydrogenase (PDH) from Thermoactinomyces intermedius, which provides a novel tool for in-vivo metabolic engineering and in-vitro catalysis. Based on computational insight into the structure, we proposed the site-specific directed mutagenesis of the two important residues of PDH through docking simulations by AutoDock Vina which elucidated the binding mode of PDH with α-Ketoadipic acid and ligands. Our results demonstrated that the catalytic efficiency Km/Kcat of the final mutant Ala135Arg showed a 3-fold increase amination activity towards the ketoadipic acid as compared to the other mutant Gly114Arg, a double mutant Gly114Arg/Ala135Arg, and wild type TiPDH. Furthermore, we have introduced formate dehydrogenase as a cofactor regenerative system in this study which further made this study economically viable. Our study unfolds the possibility of biosynthesis of other non-proteinogenic amino acids that might be valuable pharmaceutical intermediaries. |
| doi_str_mv | 10.1371/journal.pone.0263784 |
| format | Article |
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Many patients are unable to access medication because of the high price of drugs. Genetic engineering has made advances to increase the yield, titer, and productivity in the bio-based production of chemicals, materials of interest, and identification of innovative targets for drug discovery. Currently, the production of homoglutamate (α-Aminoadipic acid) involves petrochemical routes that are costly with low yield and often not suitable for industrial production. Here, we established the development of NADH-dependent homoglutamate by engineering NADH-dependent phenylalanine dehydrogenase (PDH) from Thermoactinomyces intermedius, which provides a novel tool for in-vivo metabolic engineering and in-vitro catalysis. Based on computational insight into the structure, we proposed the site-specific directed mutagenesis of the two important residues of PDH through docking simulations by AutoDock Vina which elucidated the binding mode of PDH with α-Ketoadipic acid and ligands. Our results demonstrated that the catalytic efficiency Km/Kcat of the final mutant Ala135Arg showed a 3-fold increase amination activity towards the ketoadipic acid as compared to the other mutant Gly114Arg, a double mutant Gly114Arg/Ala135Arg, and wild type TiPDH. Furthermore, we have introduced formate dehydrogenase as a cofactor regenerative system in this study which further made this study economically viable. Our study unfolds the possibility of biosynthesis of other non-proteinogenic amino acids that might be valuable pharmaceutical intermediaries.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0263784</identifier><identifier>PMID: 35353822</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Amination ; Amino Acid Oxidoreductases - genetics ; Amino acids ; Analysis ; Biology ; Biology and Life Sciences ; Biosynthesis ; Catalysis ; Cloning ; Computer applications ; Dehydrogenase ; Dehydrogenases ; DNA polymerase ; Drug discovery ; E coli ; Enzymes ; Formate dehydrogenase ; Genes ; Genetic aspects ; Genetic engineering ; Glutamic Acid - biosynthesis ; Industrial production ; Life sciences ; Metabolic engineering ; Mutagenesis ; Mutants ; NAD - metabolism ; NADH ; Nicotinamide adenine dinucleotide ; Petrochemicals ; Phenylalanine ; Phenylalanine dehydrogenase ; Physical Sciences ; Plasmids ; Protein Engineering ; Raw materials ; Thermoactinomyces ; Thermoactinomyces - enzymology</subject><ispartof>PloS one, 2022-03, Vol.17 (3), p.e0263784-e0263784</ispartof><rights>COPYRIGHT 2022 Public Library of Science</rights><rights>2022 Tariq et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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Many patients are unable to access medication because of the high price of drugs. Genetic engineering has made advances to increase the yield, titer, and productivity in the bio-based production of chemicals, materials of interest, and identification of innovative targets for drug discovery. Currently, the production of homoglutamate (α-Aminoadipic acid) involves petrochemical routes that are costly with low yield and often not suitable for industrial production. Here, we established the development of NADH-dependent homoglutamate by engineering NADH-dependent phenylalanine dehydrogenase (PDH) from Thermoactinomyces intermedius, which provides a novel tool for in-vivo metabolic engineering and in-vitro catalysis. Based on computational insight into the structure, we proposed the site-specific directed mutagenesis of the two important residues of PDH through docking simulations by AutoDock Vina which elucidated the binding mode of PDH with α-Ketoadipic acid and ligands. Our results demonstrated that the catalytic efficiency Km/Kcat of the final mutant Ala135Arg showed a 3-fold increase amination activity towards the ketoadipic acid as compared to the other mutant Gly114Arg, a double mutant Gly114Arg/Ala135Arg, and wild type TiPDH. Furthermore, we have introduced formate dehydrogenase as a cofactor regenerative system in this study which further made this study economically viable. Our study unfolds the possibility of biosynthesis of other non-proteinogenic amino acids that might be valuable pharmaceutical intermediaries.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>35353822</pmid><doi>10.1371/journal.pone.0263784</doi><tpages>e0263784</tpages><orcidid>https://orcid.org/0000-0002-7630-0400</orcidid><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2022-03, Vol.17 (3), p.e0263784-e0263784 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_2645449668 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Public Library of Science (PLoS); PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Amination Amino Acid Oxidoreductases - genetics Amino acids Analysis Biology Biology and Life Sciences Biosynthesis Catalysis Cloning Computer applications Dehydrogenase Dehydrogenases DNA polymerase Drug discovery E coli Enzymes Formate dehydrogenase Genes Genetic aspects Genetic engineering Glutamic Acid - biosynthesis Industrial production Life sciences Metabolic engineering Mutagenesis Mutants NAD - metabolism NADH Nicotinamide adenine dinucleotide Petrochemicals Phenylalanine Phenylalanine dehydrogenase Physical Sciences Plasmids Protein Engineering Raw materials Thermoactinomyces Thermoactinomyces - enzymology |
| title | Engineering of phenylalanine dehydrogenase from Thermoactinomyces intermedius for the production of a novel homoglutamate |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-21T08%3A04%3A14IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Engineering%20of%20phenylalanine%20dehydrogenase%20from%20Thermoactinomyces%20intermedius%20for%20the%20production%20of%20a%20novel%20homoglutamate&rft.jtitle=PloS%20one&rft.au=Tariq,%20Muhammad&rft.date=2022-03-30&rft.volume=17&rft.issue=3&rft.spage=e0263784&rft.epage=e0263784&rft.pages=e0263784-e0263784&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0263784&rft_dat=%3Cgale_plos_%3EA698800466%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=2645449668&rft_id=info:pmid/35353822&rft_galeid=A698800466&rft_doaj_id=oai_doaj_org_article_f070b9f8508c4474882d0117dd94e8c9&rfr_iscdi=true |