Creation and Characterization of Mycolicibacterium Smegmatis mc2155 with Deletions in Genes Encoding Sterol Oxidation Enzymes
The fast-growing saprotrophic strain Mycolicibacterium smegmatis mc2155 is capable of utilizing plant and animal sterols and can be used for creation of genetically engineered strains producing biologically active steroids. Oxidation of the 3β-hydroxyl group and Δ5(6)→Δ4(5) double bond isomerization...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
EDP Sciences
2023-01-01
|
| Series: | BIO Web of Conferences |
| Online Access: | https://www.bio-conferences.org/articles/bioconf/pdf/2023/02/bioconf_itsm2023_03004.pdf |
| _version_ | 1797951235852075008 |
|---|---|
| author | Byakov Artem Karpov Mikhail Strizhov Nikolai Donova Marina |
| author_facet | Byakov Artem Karpov Mikhail Strizhov Nikolai Donova Marina |
| author_sort | Byakov Artem |
| collection | DOAJ |
| description | The fast-growing saprotrophic strain Mycolicibacterium smegmatis mc2155 is capable of utilizing plant and animal sterols and can be used for creation of genetically engineered strains producing biologically active steroids. Oxidation of the 3β-hydroxyl group and Δ5(6)→Δ4(5) double bond isomerization followed by formation of stenones from sterols are considered as the initial stage of steroid catabolism in some actinobacteria. The study of the mechanism of steroid nucleus 3β-hydroxyl group oxidation is relevant for the creation of a method of the microbiological production of valuable 3β-hydroxy-5-en-steroids. A mutant strain of M. smegmatis with deletions in three genes (MSMEG_1604, MSMEG_5228 and MSMEG_5233) encoding known enzymes exhibiting 3β-hydroxysteroid dehydrogenase activity was constructed by homologous recombination coupled with double selection. The resulting mutant retained macromorphological properties and the ability to convert cholesterol. 3-Keto-4-en-steroids were found among the sterol catabolism intermediates. Experimentally obtained data indicate the presence of a previously undetected intracellular enzyme that performs the function of 3β-hydroxysteroid dehydrogenase/Δ5(6)→Δ4(5) isomerase. |
| first_indexed | 2024-04-10T22:27:27Z |
| format | Article |
| id | doaj.art-7831cc84ff96416aae8c11e1f4fc461e |
| institution | Directory Open Access Journal |
| issn | 2117-4458 |
| language | English |
| last_indexed | 2024-04-10T22:27:27Z |
| publishDate | 2023-01-01 |
| publisher | EDP Sciences |
| record_format | Article |
| series | BIO Web of Conferences |
| spelling | doaj.art-7831cc84ff96416aae8c11e1f4fc461e2023-01-17T09:11:28ZengEDP SciencesBIO Web of Conferences2117-44582023-01-01570300410.1051/bioconf/20235703004bioconf_itsm2023_03004Creation and Characterization of Mycolicibacterium Smegmatis mc2155 with Deletions in Genes Encoding Sterol Oxidation EnzymesByakov Artem0Karpov Mikhail1Strizhov Nikolai2Donova Marina3G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences; Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences; Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences; Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences; Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”The fast-growing saprotrophic strain Mycolicibacterium smegmatis mc2155 is capable of utilizing plant and animal sterols and can be used for creation of genetically engineered strains producing biologically active steroids. Oxidation of the 3β-hydroxyl group and Δ5(6)→Δ4(5) double bond isomerization followed by formation of stenones from sterols are considered as the initial stage of steroid catabolism in some actinobacteria. The study of the mechanism of steroid nucleus 3β-hydroxyl group oxidation is relevant for the creation of a method of the microbiological production of valuable 3β-hydroxy-5-en-steroids. A mutant strain of M. smegmatis with deletions in three genes (MSMEG_1604, MSMEG_5228 and MSMEG_5233) encoding known enzymes exhibiting 3β-hydroxysteroid dehydrogenase activity was constructed by homologous recombination coupled with double selection. The resulting mutant retained macromorphological properties and the ability to convert cholesterol. 3-Keto-4-en-steroids were found among the sterol catabolism intermediates. Experimentally obtained data indicate the presence of a previously undetected intracellular enzyme that performs the function of 3β-hydroxysteroid dehydrogenase/Δ5(6)→Δ4(5) isomerase.https://www.bio-conferences.org/articles/bioconf/pdf/2023/02/bioconf_itsm2023_03004.pdf |
| spellingShingle | Byakov Artem Karpov Mikhail Strizhov Nikolai Donova Marina Creation and Characterization of Mycolicibacterium Smegmatis mc2155 with Deletions in Genes Encoding Sterol Oxidation Enzymes BIO Web of Conferences |
| title | Creation and Characterization of Mycolicibacterium Smegmatis mc2155 with Deletions in Genes Encoding Sterol Oxidation Enzymes |
| title_full | Creation and Characterization of Mycolicibacterium Smegmatis mc2155 with Deletions in Genes Encoding Sterol Oxidation Enzymes |
| title_fullStr | Creation and Characterization of Mycolicibacterium Smegmatis mc2155 with Deletions in Genes Encoding Sterol Oxidation Enzymes |
| title_full_unstemmed | Creation and Characterization of Mycolicibacterium Smegmatis mc2155 with Deletions in Genes Encoding Sterol Oxidation Enzymes |
| title_short | Creation and Characterization of Mycolicibacterium Smegmatis mc2155 with Deletions in Genes Encoding Sterol Oxidation Enzymes |
| title_sort | creation and characterization of mycolicibacterium smegmatis mc2155 with deletions in genes encoding sterol oxidation enzymes |
| url | https://www.bio-conferences.org/articles/bioconf/pdf/2023/02/bioconf_itsm2023_03004.pdf |
| work_keys_str_mv | AT byakovartem creationandcharacterizationofmycolicibacteriumsmegmatismc2155withdeletionsingenesencodingsteroloxidationenzymes AT karpovmikhail creationandcharacterizationofmycolicibacteriumsmegmatismc2155withdeletionsingenesencodingsteroloxidationenzymes AT strizhovnikolai creationandcharacterizationofmycolicibacteriumsmegmatismc2155withdeletionsingenesencodingsteroloxidationenzymes AT donovamarina creationandcharacterizationofmycolicibacteriumsmegmatismc2155withdeletionsingenesencodingsteroloxidationenzymes |