The catabolism of sterols in mycobacteria is highly important due to its close relevance in the pathogenesis of pathogenic strains and the biotechnological applications of nonpathogenic strains for steroid synthesis. However, some key metabolic steps remain unknown. In this study, the hsd4A gene from Mycobacterium neoaurum ATCC 25795 was investigated. The encoded protein, Hsd4A, was characterized as a dual-function enzyme, with both 17β-hydroxysteroid dehydrogenase and β-hydroxyacyl-CoA dehydrogenase activities in vitro. Using a kshAs-null strain of M. neoaurum ATCC 25795 (NwIB-XII) as a model, Hsd4A was further confirmed to exert dual-function in sterol catabolism in vivo. The deletion of hsd4A in NwIB-XII resulted in the production of 23,24-bisnorcholenic steroids (HBCs), indicating that hsd4A plays a key role in sterol side-chain degradation. Therefore, two competing pathways, the AD and HBC pathways, were proposed for the side-chain degradation. The proposed HBC pathway has great value in illustrating the production mechanism of HBCs in sterol catabolism and in developing HBCs producing strains for industrial application via metabolic engineering. Through the combined modification of hsd4A and other genes, three HBCs producing strains were constructed that resulted in promising productivities of 0.127, 0.109 and 0.074 g/l/h, respectively.

/pdf/unraveling-and-engineering-the-production-of-23-24-2dao6giea2.pdf

Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism.

Mycolicibacterium cell factory for the production of steroid-based drug intermediates.

The bioconversion of phytosterols into high value-added steroidal intermediates, including the 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC), is the cornerstone in steroid pharmaceutical industry. However, the low transportation efficiency of hydrophobic substrates into mycobacterial cells severely limits the transformation. In this study, a robust and stable modification of the cell wall in M. neoaurum strain strikingly enhanced the cell permeability for the high production of steroids. The deletion of the nonessential kasB, encoding a β-ketoacyl-acyl carrier protein synthase, led to a disturbed proportion of mycolic acids (MAs), which is one of the most important components in the cell wall of Mycobacterium neoaurum ATCC 25795. The determination of cell permeability displayed about two times improvement in the kasB-deficient strain than that of the wild type M. neoaurum. Thus, the deficiency of kasB in the 9-OHAD-producing strain resulted in a significant increase of 137.7% in the yield of 9α-hydroxy-4-androstene-3,17-dione (9-OHAD). Ultimately, the 9-OHAD productivity in an industrial used resting cell system was reached 0.1135 g/L/h (10.9 g/L 9-OHAD from 20 g/L phytosterol) and the conversion time was shortened by 33%. In addition, a similar self-enhancement effect (34.5%) was realized in the 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) producing strain. The modification of kasB resulted in a meaningful change in the cell wall mycolic acids. Deletion of the kasB gene remarkably improved the cell permeability, leading to a self-enhancement of the steroidal intermediate conversion. The results showed a high efficiency and feasibility of this construction strategy.

/pdf/enhancing-the-bioconversion-of-phytosterols-to-steroidal-4rzj46h3sy.pdf

Enhancing the bioconversion of phytosterols to steroidal intermediates by the deficiency of kasB in the cell wall synthesis of Mycobacterium neoaurum.

Androstenedione (AD) is a key intermediate in the body’s steroid metabolism, used as a precursor for several steroid substances, such as testosterone, estradiol, ethinyl estradiol, testolactone, progesterone, cortisone, cortisol, prednisone, and prednisolone. The world market for AD and ADD (androstadienedione) exceeds 1000 tons per year, which stimulates the pharmaceutical industry’s search for newer and cheaper raw materials to produce steroidal compounds. In light of this interest, we aimed to investigate the progress of AD biosynthesis from phytosterols by prospecting scientific articles (Scopus, Web of Science, and Google Scholar databases) and patents (USPTO database). A wide variety of articles and patents involving AD and phytosterol were found in the last few decades, resulting in 108 relevant articles (from January 2000 to December 2021) and 23 patents of interest (from January 1976 to December 2021). The separation of these documents into macro, meso, and micro categories revealed that most studies (articles) are performed in China (54.8%) and in universities (76%), while patents are mostly granted to United States companies. It also highlights the fact that AD production studies are focused on “process improvement” techniques and on possible modifications of the “microorganism” involved in biosynthesis (64 and 62 documents, respectively). The most-reported “process improvement” technique is “chemical addition” (40%), which means that the addition of solvents, surfactants, cofactors, inducers, ionic liquids, etc., can significantly increase AD production. Microbial genetic modifications stand out in the “microorganism” category because this strategy improves AD yield considerably. These documents also revealed the main aspects of AD and ADD biosynthesis: Mycolicibacterium sp. (basonym: Mycobacterium sp.) (40%) and Mycolicibacterium neoaurum (known previously as Mycobacterium neoaurum) (32%) are the most recurrent species studied. Microbial incubation temperatures can vary from 29 °C to 37 °C; incubation can last from 72 h to 14 days; the mixture is agitated at 140 to 220 rpm; vegetable oils, mainly soybean, can be used as the source of a mixture of phytosterols. In general, the results obtained in the present technological prospecting study are fundamental to mapping the possibilities of AD biosynthesis process optimization, as well as to identifying emerging technologies and methodologies in this scenario.

/pdf/biotransformation-of-phytosterols-into-androstenedione-a-3ukaebkp.pdf

Biotransformation of Phytosterols into Androstenedione—A Technological Prospecting Study

Biotransformation of androstenedione and androstadienedione by selected Ascomycota and Zygomycota fungal strains

Based on the hydroxypropyl-β-cyclodextrin-resting cells reaction system, high substrate concentration and improved reaction rate were achieved in a bioprocess of microbial side-chain cleavage of phytosterols to 9-OH-AD. Investigation of the kinetics of biotransformation showed that the initial reaction rate was strongly inhibited by a substrate concentration greater than 8 g/L. The oxygen transfer rate was also a critical parameter that could influence the rate of reaction. The results highlighted the importance of dissolved oxygen, particularly at high substrate concentration. Finally, analysis of metabolites showed that the main loss of process yield was due to by-product (16.3%) and nucleus degradation (8–11%) in the biotransformation. Investigation of the kinetics of metabolite formation provided fundamental knowledge on the biochemical reaction which was critical for understanding how to develop the biotransformation into an industrial application.

Investigation of factors affecting biotransformation of phytosterols to 9-hydroxyandrost-4-ene-3,-17-dione based on the HP-β-CD-resting cells reaction system

The invention provides a method for preparing a steroid drug intermediate employing bioconversion phytosterol. The method comprises the following steps: 1) providing turbid liquid of phytosterol; 2) providing a resting cell of a microbial strain for converting the phytosterol into a steroid drug intermediate; 3) mixing the turbid liquid of the phytosterol with the resting cell, and adding beta-cyclodextrin or chemically modified beta-cyclodextrin to evenly mix, and cultivating to finish conversion; 4) centrifuging the conversion liquid obtained in the step 3), separating supernatant from thallus, separating to obtain the steroid drug intermediate from the supernatant by solvent extraction. By adopting the method provided by the invention, the sterol feeding concentration is improved, the target product quantity is increased, simple separation of the thallus, a cosolvent and a product is achieved when the conversion lasting time is shortened, repeated use of the thallus and the cosolvent for a plurality of times is achieved, the production cost is greatly saved, and the method is applicable to industrial production.

Method for preparing steroid drug intermediate employing bioconversion phytosterol

The steroid metabolites 9-hydroxy-androstenedione (9-OH-AD), androstadienedione (ADD) and androstenedione (AD) are important steroidal pharmaceuticals. In order to raise the production of steroid metabolites, an efficient resting cell phytosterol bioconversion process was developed to produce 9-OH-AD in the presence of hydroxypropyl-b-cyclodextrin (HP-b-CD). Cell growth medium containing phytosterol as an inducer positively improved cell activity. Under aerobic conditions, bioconversion proceeded at 70 g L –1 phytosterol in the presence of HP-b-CD (the optimized molar ratio of HP-b-CD/phytosterol was 1:1) with 30 g L –1 resting Mycobacterium neoaurum NwIB-yV cells (cell dry mass) in a 5-L bioreactor, where 9-OH-AD production and space-time yield reached 36.4 g L –1 and 9.1 g L –1 d –1 , respectively. The recycling of cells and HP-b-CD enables cost-saving and industrial applications. This bioprocess was also applied for the production of ADD and AD. The production of these steroid metabolites was much higher than that reported in previous studies.

/pdf/enhanced-steroid-metabolites-production-by-resting-cell-304g9l5bqy.pdf

Enhanced Steroid Metabolites Production by Resting Cell Phytosterol Bioconversion

UNLABELLED One of the steroid intermediates, 4-androstene-3, 17-dione (AD), in the biotransformation of phytosterols is valuable for the production of steroid medicaments. However, its degradation during the conversion process is one of the main obstacles to obtain high yields. In this study, the effect of temperature on nucleus degradation during microbial biotransformation of phytosterol was investigated. The results indicated that microbial degradation of phytosterol followed the AD-ADD-'9-OH-ADD' pathway, and that two important reactions involved in nucleus degradation, conversions of AD to ADD and ADD to 9-OH-ADD, were inhibited at 37°C. With a change in the culture temperature from 30 to 37°C, nucleus degradation was reduced from 39·9% to 17·6%, due to inhibition of the putative KstD and Ksh. These results suggested a simple way to decrease the nucleus degradation in phytosterol biotransformation and a new perspective on the possibilities of modifying the metabolism of strains used in industrial applications. SIGNIFICANCE AND IMPACT OF THE STUDY Nucleus degradation of products is one of the main problems encountered during phytosterol biotransformation. To solve this problem, the effect of temperature on nucleus degradation was investigated in the industrial production of steroid intermediates. The results are also helpful to the genetic modification of sterol-producing strains.

Influence of temperature on nucleus degradation of 4-androstene-3, 17-dione in phytosterol biotransformation by Mycobacterium sp.

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Investigation of factors affecting biotransformation of phytosterols to 9-hydroxyandrost-4-ene-3,-17-dione based on the HP-β-CD-resting cells reaction system

Method for preparing steroid drug intermediate employing bioconversion phytosterol

Enhanced Steroid Metabolites Production by Resting Cell Phytosterol Bioconversion

Influence of temperature on nucleus degradation of 4-androstene-3, 17-dione in phytosterol biotransformation by Mycobacterium sp.