{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T09:19:21Z","timestamp":1763025561909,"version":"3.45.0"},"reference-count":57,"publisher":"MDPI AG","issue":"22","license":[{"start":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T00:00:00Z","timestamp":1762992000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012190","name":"Ministry of Science and Higher Education of the Russian Federation","doi-asserted-by":"publisher","award":["122040500054-3","125041005029-5"],"award-info":[{"award-number":["122040500054-3","125041005029-5"]}],"id":[{"id":"10.13039\/501100012190","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>In this study, for the first time, the genes encoding the mammalian steroidogenesis system\u2014cytochrome P450scc (CYP11A1), and its native redox partners adrenodoxin and adrenodoxin reductase\u2014were successfully expressed in the methylobacterium Methylorubrum extorquens. The advantage of using methylobacteria as an expression chassis is that they grow on inexpensive mineral media, use methanol as a carbon and energy source, and do not possess their own sterol catabolism systems. Using recombinant methylobacteria, the valuable steroid pregnenolone was obtained as a sole metabolite from cholesterol. The effect of media composition, bioconversion conditions such as methanol and N-sources content, modes of substrate addition, detergents, methyl-\u03b2-cyclodextrin, biomass, and aeration on pregnenolone accumulation was investigated. Under optimized conditions, its yield exceeded 100 mg\/L. The results demonstrate a proof of concept relating to the use of bacteria lacking their own steroid degradation systems as microbial chassis for heterologous steroidogenesis systems, including mammalian cytochrome CYP11A1.<\/jats:p>","DOI":"10.3390\/ijms262210975","type":"journal-article","created":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T09:10:45Z","timestamp":1763025045000},"page":"10975","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Pregnenolone Bioproduction in Engineered Methylobacteria: Design and Elaboration"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4780-0905","authenticated-orcid":false,"given":"Daria","family":"Tekucheva","sequence":"first","affiliation":[{"name":"G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms (IBPM), Russian Academy of Sciences, Federal Research Center \u201cPushchino Scientific Center for Biological Research of the Russian Academy of Sciences\u201d, Prospekt Nauki 5, Pushchino, Moscow 142290, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3415-1929","authenticated-orcid":false,"given":"Veronika","family":"Poshekhontseva","sequence":"additional","affiliation":[{"name":"G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms (IBPM), Russian Academy of Sciences, Federal Research Center \u201cPushchino Scientific Center for Biological Research of the Russian Academy of Sciences\u201d, Prospekt Nauki 5, Pushchino, Moscow 142290, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7338-7929","authenticated-orcid":false,"given":"Dmitry","family":"Fedorov","sequence":"additional","affiliation":[{"name":"G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms (IBPM), Russian Academy of Sciences, Federal Research Center \u201cPushchino Scientific Center for Biological Research of the Russian Academy of Sciences\u201d, Prospekt Nauki 5, Pushchino, Moscow 142290, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9162-7196","authenticated-orcid":false,"given":"Mikhail","family":"Karpov","sequence":"additional","affiliation":[{"name":"G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms (IBPM), Russian Academy of Sciences, Federal Research Center \u201cPushchino Scientific Center for Biological Research of the Russian Academy of Sciences\u201d, Prospekt Nauki 5, Pushchino, Moscow 142290, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-1563-5723","authenticated-orcid":false,"given":"Ludmila","family":"Novikova","sequence":"additional","affiliation":[{"name":"Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1\/40, Moscow 119991, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7524-8877","authenticated-orcid":false,"given":"Alexey","family":"Zamalutdinov","sequence":"additional","affiliation":[{"name":"G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms (IBPM), Russian Academy of Sciences, Federal Research Center \u201cPushchino Scientific Center for Biological Research of the Russian Academy of Sciences\u201d, Prospekt Nauki 5, Pushchino, Moscow 142290, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9917-8428","authenticated-orcid":false,"given":"Marina","family":"Donova","sequence":"additional","affiliation":[{"name":"G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms (IBPM), Russian Academy of Sciences, Federal Research Center \u201cPushchino Scientific Center for Biological Research of the Russian Academy of Sciences\u201d, Prospekt Nauki 5, Pushchino, Moscow 142290, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"947","DOI":"10.1210\/er.2003-0030","article-title":"Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones","volume":"25","author":"Payne","year":"2004","journal-title":"Endocr. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1016\/j.pbb.2006.07.009","article-title":"Neuro(active) steroids actions at the neuromodulatory sigma1 (\u03c31) receptor: Biochemical and physiological evidences consequences in neuroprotection","volume":"84","author":"Maurice","year":"2006","journal-title":"Pharmacol. Biochem. Behav."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"951","DOI":"10.1016\/j.bbalip.2010.05.006","article-title":"Allopregnanolone levels are reduced in temporal cortex in patients with Alzheimer\u2019s disease compared to cognitively intact control subjects","volume":"1801","author":"Naylor","year":"2010","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1016\/j.jsbmb.2015.09.030","article-title":"Neurosteroids and potential therapeutics: Focus on pregnenolone","volume":"160","year":"2016","journal-title":"J. Steroid Biochem. Mol. Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1554","DOI":"10.1016\/j.steroids.2011.09.006","article-title":"Pregnenolone derivatives as potential anticancer agents","volume":"76","author":"Choudhary","year":"2011","journal-title":"Steroids"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Lin, Y.C., Cheung, G., Porter, E., and Papadopoulos, V. (2022). The neurosteroid pregnenolone is synthesized by a mitochondrial P450 enzyme other than CYP11A1 in human glial cells. J. Biol. Chem., 298.","DOI":"10.1016\/j.jbc.2022.102110"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1016\/j.antiviral.2015.08.002","article-title":"Synthetic pregnenolone derivatives as antiviral agents against acyclovir-resistant isolates of Herpes Simplex Virus Type 1","volume":"122","author":"Mazaira","year":"2015","journal-title":"Antivir. Res."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1016\/j.steroids.2013.10.010","article-title":"Click chemistry decoration of amino sterols as promising strategy to developed new leishmanicidal drugs","volume":"79","author":"Porta","year":"2014","journal-title":"Steroids"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"4647","DOI":"10.1073\/pnas.81.15.4647","article-title":"Molecular cloning and nucleotide sequence of cDNA for mRNA of mitochondrial cytochrome P-450(SCC) of bovine adrenal cortex","volume":"81","author":"Morohashi","year":"1984","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1482","DOI":"10.1134\/S0006297909130057","article-title":"From structure and functions of steroidogenic enzymes to new technologies of gene engineering","volume":"74","author":"Novikova","year":"2009","journal-title":"Biochemistry (Moscow)"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"376","DOI":"10.1016\/0003-9861(91)90554-V","article-title":"Expression of functional bovine cholesterol side chain cleavage cytochrome P450 (P450scc) in Escherichia coli","volume":"290","author":"Wada","year":"1991","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"173","DOI":"10.4236\/ajmb.2013.34023","article-title":"Functional reconstruction of bovine P450scc steroidogenic system in Escherichia coli","volume":"3","author":"Makeeva","year":"2013","journal-title":"Amer. J. Mol. Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1007\/s12033-019-00158-6","article-title":"Analysis of In Vivo Activity of the Bovine Cholesterol Hydroxylase\/Lyase System Proteins Expressed in Escherichia coli","volume":"61","author":"Efimova","year":"2019","journal-title":"Mol. Biotechnol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"186","DOI":"10.1038\/nbt0298-186","article-title":"Self-sufficient biosynthesis of pregnenolone and progesterone in engineered yeast","volume":"16","author":"Duport","year":"1998","journal-title":"Nat. Biotechnol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2666","DOI":"10.1021\/acssynbio.9b00018","article-title":"Pregnenolone Overproduction in Yarrowia lipolytica by Integrative Components Pairing of the Cytochrome P450scc System","volume":"8","author":"Zhang","year":"2019","journal-title":"ACS Synth. Biol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Strizhov, N., Fokina, V., Sukhodolskaya, G., Dovbnya, D., Karpov, M., Shutov, A., Novikova, L., and Donova, M. (2014). Progesterone biosynthesis by combined action of adrenal steroidogenic and mycobacterial enzymes in fast growing mycobacteria. New Biotechnol., 31.","DOI":"10.1016\/j.nbt.2014.05.1766"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1186\/s12934-024-02385-2","article-title":"Pregnenolone and progesterone production from natural sterols using recombinant strain of Mycolicibacterium smegmatis mc2 155 expressing mammalian steroidogenesis system","volume":"23","author":"Karpov","year":"2024","journal-title":"Microb. Cell Fact."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1186\/s12934-015-0300-y","article-title":"Functionalized PHB granules provide the basis for the efficient side-chain cleavage of cholesterol and analogs in recombinant Bacillus megaterium","volume":"14","author":"Gerber","year":"2015","journal-title":"Microb. Cell Fact."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"660","DOI":"10.1021\/bm801394s","article-title":"Bacterial polyhydroxyalkanoate granules: Biogenesis, structure, and potential use as nano-\/micro-beads in biotechnological and biomedical applications","volume":"10","author":"Grage","year":"2009","journal-title":"Biomacromolecules"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1099\/00221287-93-2-259","article-title":"A biochemical basis for obligate methylotrophy: Properties of a mutant of Pseudomonas AM1 lacking 2-oxoglutarate dehydrogenase","volume":"93","author":"Taylor","year":"1976","journal-title":"J. Gen. Microbiol."},{"key":"ref_21","first-page":"183","article-title":"The Genetics and Molecular Biology of Methanol-Utilizing Bacteria","volume":"Volume 5","author":"Murrell","year":"1992","journal-title":"Methane and Methanol Utilizers"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"601","DOI":"10.1099\/mic.0.25955-0","article-title":"Reconstruction of C3 and C4 metabolism in Methylobacterium extorquens AM1 using transposon mutagenesis","volume":"149","author":"Okubo","year":"2003","journal-title":"Microbiology"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"470","DOI":"10.1042\/bj0810470","article-title":"Microbial growth on C1 compounds. II. Synthesis of cell constituents by methanol-and formate-grown Pseudomonas AM1, and methanol-grown Hyphomicrobium vulgare","volume":"81","author":"Large","year":"1961","journal-title":"Biochem. J."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2980","DOI":"10.1128\/JB.185.10.2980-2987.2003","article-title":"Methylotrophy in Methylobacterium extorquens AM1 from a genomic point of view","volume":"185","author":"Chistoserdova","year":"2003","journal-title":"J. Bacteriol."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Lim, C.K., Villada, J.C., Chalifour, A., Duran, M.F., Lu, H., and Lee, P.K.H. (2019). Designing and Engineering Methylorubrum extorquens AM1 for Itaconic Acid Production. Front. Microbiol., 10.","DOI":"10.3389\/fmicb.2019.01027"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"94","DOI":"10.1016\/j.ab.2008.07.010","article-title":"Quantitative metabolome analysis using liquid chromatography-high-resolution mass spectrometry","volume":"382","author":"Kiefer","year":"2008","journal-title":"Anal. Biochem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"929","DOI":"10.1002\/bit.21652","article-title":"Metabolite profiling analysis of Methylobacterium extorquens AM1 by comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry","volume":"99","author":"Xiaofeng","year":"2008","journal-title":"Biotechnol. Bioeng."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"4846","DOI":"10.1073\/pnas.0810932106","article-title":"Demonstration of the ethylmalonyl-CoA pathway by using 13C metabolomics","volume":"106","author":"Peyraud","year":"2009","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2065","DOI":"10.1099\/00221287-147-8-2065","article-title":"Development of improved versatile broad-hostrange vectors for use in methylotrophs and other Gram-negative bacteria","volume":"147","author":"Marx","year":"2001","journal-title":"Microbiology"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Marx, C.J. (2008). Development of a broad-host-range sacB-based vector for unmarked allelic exchange. BMC Res. Notes, 1.","DOI":"10.1186\/1756-0500-1-1"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1016\/j.celrep.2011.12.003","article-title":"Optimization of gene expression through divergent mutational paths","volume":"1","author":"Chou","year":"2012","journal-title":"Cell Rep."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"4533","DOI":"10.1007\/s00253-013-5456-y","article-title":"Thioesterases for ethylmalonyl-CoA pathway derived dicarboxylic acid production in Methylobacterium extorquens AM1","volume":"98","author":"Sonntag","year":"2014","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Hu, B., and Lidstrom, M.E. (2014). Metabolic engineering of Methylobacterium extorquens AM1 for 1-butanol production. Biotechnol. Biofuels, 7.","DOI":"10.1186\/s13068-014-0156-0"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"3715","DOI":"10.1007\/s00253-013-5490-9","article-title":"Biosynthesis of polyhydroxyalkanoate copolymers from methanol by Methylobacterium extorquens AM1 and the engineered strains under cobalt-deficient conditions","volume":"98","author":"Orita","year":"2014","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1038","DOI":"10.1128\/JB.183.3.1038-1046.2001","article-title":"Connection between Poly-\u03b2-Hydroxybutyrate Biosynthesis and Growth on C1 and C2 Compounds in the Methylotroph Methylobacterium extorquensAM1","volume":"183","author":"Korotkova","year":"2001","journal-title":"J. Bacteriol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"6174","DOI":"10.1128\/JB.184.22.6174-6181.2002","article-title":"Poly-beta-hydroxybutyrate biosynthesis in the facultative methylotroph Methylobacterium extorquens AM1: Identification and mutation of gap11, gap20, and phaR","volume":"184","author":"Korotkova","year":"2002","journal-title":"J. Bacteriol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"3010","DOI":"10.1128\/aem.63.8.3010-3013.1997","article-title":"Positive selection systems for discovery of novel polyester biosynthesis genes based on fatty acid detoxification","volume":"63","author":"Kranz","year":"1997","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"569","DOI":"10.1080\/09168451.2018.1549935","article-title":"Pantothenate auxotrophy of Methylobacterium spp. isolated from living plants","volume":"83","author":"Yoshida","year":"2019","journal-title":"Biosci. Biotechnol. Biochem."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Fujiwara, T., and Harigae, H. (2015). Biology of Heme in Mammalian Erythroid Cells and Related Disorders. Biomed. Res. Int., 2015.","DOI":"10.1155\/2015\/278536"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"80","DOI":"10.1186\/s12934-020-01335-y","article-title":"Enhancing the bioconversion of phytosterols to steroidal intermediates by the deficiency of kasB in the cell wall synthesis of Mycobacterium neoaurum","volume":"19","author":"Xiong","year":"2020","journal-title":"Microb. Cell Fact."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"e00441-20","DOI":"10.1128\/AEM.00441-20","article-title":"The Sterol carrier hydroxypropyl-\u03b2-cyclodextrin enhances the metabolism of phytosterols by Mycobacterium neoaurum","volume":"86","author":"Su","year":"2020","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Tekucheva, D.N., Nikolayeva, V.M., Karpov, M.V., Timakova, T.A., Shutov, A.V., and Donova, M.V. (2022). Bioproduction of testosterone from phytosterol by Mycolicibacterium neoaurum strains: \u201cOne-pot\u201d, two modes. Bioresour. Bioprocess., 9.","DOI":"10.1186\/s40643-022-00602-7"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"303","DOI":"10.1134\/S0026261722300099","article-title":"Cascade biotransformation of phytosterol to testosterone by Mycolicibacterium neoaurum VKM Ac-1815D and Nocardioides simplex VKM Ac-2033D strains","volume":"91","author":"Tekucheva","year":"2022","journal-title":"Microbiology"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"617","DOI":"10.1211\/0022357021778925","article-title":"Estimation of cyclodextrin affinity to steroids","volume":"54","author":"Khomutov","year":"2002","journal-title":"J. Pharm. Pharmacol."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Zhou, X., Zhang, Y., Shen, Y., Zhang, X., Zhang, Z., Xu, S., Luo, J., Xia, M., and Wang, M. (2019). Economical production of androstendione and 9\u03b1-hydroxyandrostendione using untreated cane molasses by recombinant mycobacteria. Bioresour. Technol., 290.","DOI":"10.1016\/j.biortech.2019.121750"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1981","DOI":"10.1099\/mic.0.2006\/001636-0","article-title":"Methyl-\u03b2-cyclodextrin alters growth, activity and cell envelope features of sterol-transforming mycobacteria","volume":"153","author":"Donova","year":"2007","journal-title":"Microbiology"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"386","DOI":"10.1080\/10242420701510510","article-title":"The inhibitory effect of cyclodextrin on the degradation of 9\u03b1-hydroxyandrost-4-ene-3,17-dione by Mycobacterium sp. VKM Ac-1817D","volume":"25","author":"Khomutov","year":"2007","journal-title":"Biocatal. Biotransform."},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Tang, R., Ren, X., Xia, M., Shen, Y., Tu, L., Luo, J., Zhang, Q., Wang, Y., Ji, P., and Wang, M. (2021). Efficient one-step biocatalic multienzyme cascade strategy for direct conversion of phytosterol to C17-hydroxylated steroids. Appl. Environ. Microbiol., 87.","DOI":"10.1128\/AEM.00321-21"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1016\/0022-4731(82)90150-9","article-title":"Substrate specificity of adrenocortical cytochrome P-450SCC. II. Effect of structural modification of cholesterol A\/B ring on their side chain cleavage reaction","volume":"16","author":"Morisaki","year":"1982","journal-title":"J. Steroid Biochem."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1016\/j.jsbmb.2011.09.008","article-title":"Cholesterol oxidase ChoD is not a critical enzyme accounting for oxidation of sterols to 3-keto-4-ene steroids in fast-growing Mycobacterium sp. VKM Ac-1815D","volume":"129","author":"Ivashina","year":"2012","journal-title":"Steroid Biochem. Mol. Biol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"392","DOI":"10.1023\/A:1010469708107","article-title":"Aerobic methylobacteria are capable of synthesizing auxins","volume":"70","author":"Ivanova","year":"2001","journal-title":"Microbiology"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1128\/jb.62.3.293-300.1951","article-title":"Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli","volume":"62","author":"Bertani","year":"1951","journal-title":"J. Bacteriol."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"3124","DOI":"10.1002\/jcb.27577","article-title":"Polycistronic expression of the mitochondrial steroidogenic P450scc system in the HEK293T cell line","volume":"120","author":"Efimova","year":"2019","journal-title":"J. Cell Biochem."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"784","DOI":"10.1038\/nbt1183-784","article-title":"A broad host range mobilization system for in vivo genetic engineering: Transposon mutagenesis in Gram negative bacteria","volume":"1","author":"Simon","year":"1983","journal-title":"Nat. Biotechnol."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"680","DOI":"10.1038\/227680a0","article-title":"Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4","volume":"227","author":"Laemmli","year":"1970","journal-title":"Nature"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"175","DOI":"10.1016\/0378-1119(87)90485-9","article-title":"Analysis of alpha-factor secretion signals by fusing with acid phosphatase of yeast","volume":"54","author":"Sidhu","year":"1987","journal-title":"Gene"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1016\/S0021-9258(19)52451-6","article-title":"Protein measurement with the Folin phenol reagent","volume":"193","author":"Lowry","year":"1951","journal-title":"J. Biol. Chem."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/22\/10975\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T09:14:40Z","timestamp":1763025280000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/22\/10975"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,13]]},"references-count":57,"journal-issue":{"issue":"22","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["ijms262210975"],"URL":"https:\/\/doi.org\/10.3390\/ijms262210975","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11,13]]}}}