Androstadienedione Research Articles

Rerouting phytosterol degradation pathway for directed androst-1,4-diene-3,17-dione microbial bioconversion

Steroid-based drugs are now mainly produced by the microbial transformation of phytosterol, and a two-step bioprocess is adopted to reach high space–time yields, but byproducts are frequently observed during the bioprocessing. In this study, the catabolic switch between the C19- and C22-steroidal subpathways was investigated in resting cells of Mycobacterium neoaurum NRRL B-3805, and a dose-dependent transcriptional response toward the induction of phytosterol with increased concentrations was found in the putative node enzymes including ChoM2, KstD1, OpccR, Sal, and Hsd4A. Aldolase Sal presented a dominant role in the C22 steroidal side-chain cleavage, and the byproduct was eliminated after sequential deletion of opccR and sal. Meanwhile, the molar yield of androst-1,4-diene-3,17-dione (ADD) was increased from 59.4 to 71.3%. With the regard of insufficient activity of rate-limiting enzymes may also cause byproduct accumulation, a chromosomal integration platform for target gene overexpression was established supported by a strong promoter L2 combined with site-specific recombination in the engineered cell. Rate-limiting steps of ADD bioconversion were further characterized and overcome. Overexpression of the kstD1 gene further strengthened the bioconversion from AD to ADD. After subsequential optimization of the bioconversion system, the directed biotransformation route was developed and allowed up to 82.0% molar yield with a space–time yield of 4.22 g·L−1·day−1. The catabolic diversion elements and the genetic overexpression tools as confirmed and developed in present study offer new ideas of M. neoaurum cell factory development for directed biotransformation for C19- and C22-steroidal drug intermediates from phytosterol.Key points• Resting cells exhibited a catabolic switch between the C19- and C22-steroidal subpathways.• The C22-steroidal byproduct was eliminated after sequential deletion of opccR and sal.• Rate-limiting steps were overcome by promoter engineering and chromosomal integration.

Identification of a Gene Encoding a New Fungal Steroid 7-Hydroxylase and Its Functional Characterization in Pichia pastoris Yeast.

The hydroxylation of steroids in the C7β position is one of the rare reactions that allow the production of value-added precursors in the synthesis of ursodeoxycholic acid and other pharmaceuticals. Recently, we discovered this activity in the ascomycete Curvularia sp. VKM F-3040. In this study, the novel gene of 7-hydroxylase (P450cur) was identified as being heterologously expressed and functionally characterized in Pichia pastoris. Transcriptome data mining and differential expression analysis revealed that 12 putative genes in Curvularia sp. mycelia significantly increased their expression in response to dehydroepiandrosterone (DHEA). The transcriptional level of the most up-regulated cytochrome P450cur gene was increased more than 300-fold. A two-gene construct with a candidate P450cur gene and the gene of its natural redox partner, NADPH-cytochrome P450 reductase (CPR), which is interconnected by a T2A element, was created. Using this construct, recombinant P. pastoris strains co-expressing fungal P450cur and CPR genes were obtained. The functional activity of the recombinant P450cur was studied in vivo during the bioconversion of androstane steroids. The fungal 7-monooxygenase predominantly catalyzed the 7β-hydroxylation of androstadienedione (ADD), DHEA, and androstenediol, whereas 1-dehydrotestosterone was hydroxylated by P450cur mainly at the C7-Hα position. To our knowledge, this is the first report of a recombinant yeast capable of catalyzing the 7α/β-hydroxylation of ADD and DHEA.

Process development of transforming phytosterols to steroidal pharmaceutical intermediates by Mycolicibacterium neoaurum with L-carnitine

The process of phytosterol microbial transformation in aqueous phase is the most important pathway to prepare steroidal pharmaceutical intermediates. However, the low solubility of phytosterols and difficult contact between substrate and cells become the main obstacles in practical application. In this study, a comparison of commonly used vegetable oils, surfactants, cosolvents and ionic liquids identified L-carnitine as a novel zwitterionic additive to enhance phytosterol bioconversion by growing and resting cells of Mycolicibacterium neoaurum LLZ2. The addition of L-carnitine had significance in obtaining more biomass and increasing glucose metabolic velocity. Moreover, the surface of cells cultivated with L-carnitine was smoother, which probably enhanced the contact of phytosterols and cells. In other aspects, the dispersion of substrate and several key enzymes involving in the synthesis of steroidal intermediates were not influenced by L-carnitine. Through the optimization of reaction parameters, the total yield of androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD) was improved by 39%. This study provides a green approach for reaction engineering of Mycolicibacterium-mediated phytosterol bioconversion, and new insights into possible mechanism.

New Insights into the Modification of the Non-Core Metabolic Pathway of Steroids in Mycolicibacterium and the Application of Fermentation Biotechnology in C-19 Steroid Production

Androsta-4-ene-3,17-dione (AD), androsta-1,4-diene-3,17-dione (ADD), and 9α-hydroxy-4-androstene-3,17-dione (9-OHAD), which belong to C-19 steroids, are critical steroid-based drug intermediates. The biotransformation of phytosterols into C-19 steroids by Mycolicibacterium cell factories is the core step in the synthesis of steroid-based drugs. The production performance of engineered mycolicibacterial strains has been effectively enhanced by sterol core metabolic modification. In recent years, research on the non-core metabolic pathway of steroids (NCMS) in mycolicibacterial strains has made significant progress. This review discusses the molecular mechanisms and metabolic modifications of NCMS for accelerating sterol uptake, regulating coenzyme I balance, promoting propionyl-CoA metabolism, reducing reactive oxygen species, and regulating energy metabolism. In addition, the recent applications of biotechnology in steroid intermediate production are summarized and compared, and the future development trend of NCMS research is discussed. This review provides powerful theoretical support for metabolic regulation in the biotransformation of phytosterols.

Bioconversion of Phytosterols into Androstenedione by Mycolicibacterium.

The chapter describes the bioconversion of phytosterols into androstenedione (AD) by Mycolicibacterium spp. in shake flasks and fermenters, as well as LC-MS-based methods for analysis of phytosterols and steroid products. Phytosterols are derived as by-products of vegetable oil refining and manufacture of wood pulp. They contain the same four-ring nucleus as steroids and may be converted to high-value steroids by removing the sidechain at C17 and minor changes at other sites in the ring structure. Many bacteria, including Mycolicibacterium spp., can degrade phytosterols. Mutants of Mycolicibacterium spp. unable of ring cleavage can, when growing on phytosterols, accumulate the steroid intermediates androstenedione (AD) and androstadienedione (ADD). The practical challenge with microbial conversion of phytosterols to steroids is that both the substrate and the product are virtually insoluble in water. In addition, some steroids, notably ADD, may be toxic for the cells. Two main strategies have been employed to overcome this challenge: the use of two-phase systems and the addition of chemically modified cyclodextrins. The latter method is used here. Defined cultivation and bioconversion media for both shake flask and fermenter are given, as well as hints how to minimize the practical problems due to the water-insoluble phytosterol. Sampling, sample extraction, and quantification of substrates and products using LC-MS analysis are described.

Biotransformation of Phytosterols into Androstenedione-A Technological Prospecting Study.

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.

Biotransformation Enables Innovations Toward Green Synthesis of Steroidal Pharmaceuticals.

Steroids have been widely used in birth-control, prevention, and treatment of various diseases, representing the largest sector after antibiotics in the global pharmaceutical market. The steroidal active pharmaceutical ingredients (APIs) have been produced via partial synthetic processes first mainly from sapogenins, which was converted into 16-dehydropregnenolone by the famous "Marker Degradation". Traditional mutation and screening, and process engineering have resulted in the industrial production of 4-androstene-3,17-dione (AD), androst-1,4-diene-3,17-dione (ADD), 9α-hydroxy-androsta-4-ene-3,17-dione (9α-OH-AD), and so on, which serve as the key intermediates for the synthesis of steroidal APIs. Recently, genetic and metabolic engineering have generated highly efficient microbial strains for the production of these precursors, leading to the replacement of sapogenins with phytosterols as the starting materials. Further advances in synthetic biology hold promise in the design and construction of microbial cell factories for the industrial production of steroidal intermediates and/or APIs from simple carbon sources such as glucose. Integration of biotransformation into the synthesis of steroidal APIs can greatly reduce the number of reaction steps, achieve lower waste discharge and higher production efficiency, thus enabling a greener steroidal pharmaceutical industry.

Steroid modification by filamentous fungus Drechslera sp.: Focus on 7-hydroxylase and 17β-hydroxysteroid dehydrogenase activities

Fungal strain Drechslera sp. Ph F-34 was shown to modify 3-oxo- and 3-hydroxy steroids of androstane series to form the corresponding allylic 7-alcohols and 17β-reduced derivatives thus evidencing the presence of 7α-, 7β-hydroxylase and 17β-hydroxysteroid dehydrogenase (17β-HSD) activities. The growing mycelium predominantly hydroxylated androsta-1,4-diene-3,17-dione (ADD) at the 7β-position, while much lower 7α-hydroxylation was observed. Along with 7β-hydroxy-ADD and its corresponding 7α-isomer, their respective 17β-alcohols were produced.In this study, transformation of ADD, androst-4-en-17β-ol-3-one (testosterone, TS) and 3β-hydroxyandrost-5-en-17-one (dehydroepiandrosterone, DHEA) by resting mycelium of Drechslera sp. have been estimated in different conditions with regard to the inducibility and functionality of the 17β-HSD and 7-hydroxylase enzyme systems. Steroids of androstane, pregnane and cholane series were evaluated as inducers. The inhibitory analysis was provided using cycloheximide (CHX). Steroids were assayed using TLC and HPLC methods, and the structures were confirmed by mass-spectrometry, 1H and 13C NMR spectroscopy data.17β-HSD of the mycelium constitutively reduced 17-carbonyl group of ADD and DHEA to form the corresponding 17β-alcohols, namely, androsta-1,4-diene-17β-ol-3-one (1-dehydro-TS), and androst-5-ene-3β,17β-diol. Production of the 7α- and 7β-hydroxylated derivatives depended on the induction conditions. The inducer effect relied on the steroid structure and decreased in the order: DHEA > pregnenolone > lithocholic acid. β-Sitosterol did not induce hydroxylase activity in Drechslera sp. CHX fully inhibited the synthesis of 7-hydroxylase in Drechslera mycelium thus providing selective 17-keto reduction.Results contribute to the diversity of steroid modifying enzymes in fungi and can be used at the development of novel biocatalysts for production of valuable steroid 7(α/β)- and 17β-alcohols.

Enhancing the sustainability of KsdD as a biocatalyst for steroid transformation by immobilization on epoxy support

The Δ1-dehydrogenation of 3-ketosteroid substrates is a crucial reaction in the production of steroids. Although 3-ketosteroid Δ1-dehydrogenase (KsdD) catalyzes this reaction with high efficiency and selectivity, the low stability and high cost of the purified enzyme catalyst have limited its industrial application. In this study, an epoxy support was used to evaluate the covalent immobilization of KsdD from Pimelobacter simplex, and the best androsta-1,4-diene-317-dione (ADD) production was achieved after optimized immobilization of KsdD enzyme in 1.5 M NaH2PO4- Na2HPO4 buffer (pH 6.5) for 12 h at 25 °C. The immobilized KsdD exhibited higher tolerance toward 20 % methanol. The dehydrogenation reaction reached a conversion efficiency of up to 90.0 % in 2 h when using 0.6 mg/mL of 4-androstene-317-dione (AD). The W299A and W299 G mutants of KsdD were also immobilized, and both showed the better catalytic performance with higher kcat/KM values compared with the wild type (WT). The immobilized W299A, W299 G and WT KsdD respectively maintained 70.5, 65.7 and 38.7 % of their initial activity at the end of 15 reaction cycles. Furthermore, the W299A retained 66.3 % of the initial activity after 30 days of incubation at 4 °C, and was more stable than free KsdD, Thus, the immobilized W299A is a promising biocatalyst for steroid dehydrogenation. In this study, we investigated the application of immobilized enzymes for the dehydrogenation of steroids, which will be of great importance for improving the development of green technology and sustainable use of biocatalysts in the steroid manufacturing industry.

The Sterol Carrier Hydroxypropyl-β-Cyclodextrin Enhances the Metabolism of Phytosterols by Mycobacterium neoaurum.

Androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD) are valuable steroid pharmaceutical intermediates obtained by soybean phytosterol biotransformation by Mycobacterium Cyclodextrins (CDs) are generally believed to be carriers for phytosterol delivery and can improve the production of AD and ADD due to their effects on steroid solubilization and alteration in cell wall permeability for steroids. To better understand the mechanisms of CD promotion, we performed proteomic quantification of the effects of hydroxypropyl-β-CD (HP-β-CD) on phytosterol metabolism in Mycobacterium neoaurum TCCC 11978 C2. Perturbations are observed in steroid catabolism and glucose metabolism by adding HP-β-CD in a phytosterol bioconversion system. AD and ADD, as metabolic products of phytosterol, are toxic to cells, with inhibited cell growth and biocatalytic activity. Treatment of mycobacteria with HP-β-CD relieves the inhibitory effect of AD(D) on the electron transfer chain and cell growth. These results demonstrate the positive relationship between HP-β-CD and phytosterol metabolism and give insight into the complex functions of CDs as mediators of the regulation of sterol metabolism.IMPORTANCE Phytosterols from soybean are low-cost by-products of soybean oil production and, owing to their good bioavailability in mycobacteria, are preferred as the substrates for steroid drug production via biotransformation by Mycobacterium However, the low level of production of steroid hormone drugs due to the low aqueous solubility (below 0.1 mmol/liter) of phytosterols limits the commercial use of sterol-transformed strains. To improve the bioconversion of steroids, cyclodextrins (CDs) are generally used as an effective carrier for the delivery of hydrophobic steroids to the bacterium. CDs improve the biotransformation of steroids due to their effects on steroid solubilization and alterations in cell wall permeability for steroids. However, studies have rarely reported the effects of CDs on cell metabolic pathways related to sterols. In this study, the effects of hydroxypropyl-β-CD (HP-β-CD) on the expression of enzymes related to steroid catabolic pathways in Mycobacterium neoaurum were systematically investigated. These findings will improve our understanding of the complex functions of CDs in the regulation of sterol metabolism and guide the application of CDs to sterol production.

Microbial bioconversion of phytosterol into important androgen intermediates using Ochrobactrum anthropi strain (ATCC 49188)

Androstenedione (AD) and androstadienedione (ADD) are steroids intermediate valuable for the production of steroid medicaments. Microbial bioconversion of phytosterols to produce AD is a well researched area in synthesis of steroid hormones. The current study focused on bioconversion of β, sitosterol as a plant phytosterol into androstenedione (AD) and androstadienedione (ADD) optimized by Ochrobactrum anthropi strain (ATCC 49188). The study involved the effect of some physiological and biochemical factors affecting on the bioconversion process such as inoculums size, inoculum age, pH of the fermentation medium No.Ⅰ, (g/L) glucose 10 K2HPO4 0.75, KH2PO4 3, (NH4)2SO4 1, MgSO4 .7H2O 1, 8 hydroxyqunioline 0.8), temperature 30as well as substrate concentration. The results showed that the maximum AD (39.29%) output was obtained by using fermentation medium No.Ⅰ at pH 8; inoculum size 2ml/100 ml; 24h inoculum age, temperature 30and 15mg/100ml substrate concentration.

Intracellular Environment Improvement of Mycobacterium neoaurum for Enhancing Androst-1,4-Diene-3,17-Dione Production by Manipulating NADH and Reactive Oxygen Species Levels.

As one of the most significant steroid hormone precursors, androst-1,4-diene-3,17-dione (ADD) could be used to synthesize many valuable hormone drugs. The microbial transformation of sterols to ADD has received extensive attention in recent years. In a previous study, Mycobacterium neoaurum JC-12 was isolated and converted sterols to the major product, ADD. In this work, we enhanced ADD yield by improving the cell intracellular environment. First, we introduced a nicotinamide adenine dinucleotide (NADH) oxidase from Bacillus subtilis to balance the intracellular NAD+ availability in order to strengthen the ADD yield. Then, the catalase gene from M. neoaurum was also over-expressed to simultaneously scavenge the generated H2O2 and eliminate its toxic effects on cell growth and sterol transformation. Finally, using a 5 L fermentor, the recombinant strain JC-12yodC-katA produced 9.66 g/L ADD, which increased by 80% when compared with the parent strain. This work shows a promising way to increase the sterol transformation efficiency by regulating the intracellular environment.

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

Filamentous fungi is a huge phylum of lower eukaryotes with diverse activities towards various substrates, however, their biocatalytic potential towards steroids remains greatly underestimated.In this study, more than forty Ascomycota and Zygomycota fungal strains of 23 different genera were screened for the ability to catalyze structural modifications of 3-oxo-androstane steroids, - androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD). Previously unexplored for these purposes strains of Absidia, Acremonium, Beauveria, Cunninghamella, Doratomyces, Drechslera, Fusarium, Gibberella genera were revealed capable of producing in a good yield valuable 7α-, 7β-, 11α- and 14α-hydroxylated derivatives, as well as 17β-reduced and 1(2)-dehydrogenated androstanes. The bioconversion routes of AD and ADD were proposed based on the key intermediates identification and time courses of the bioprocesses. Six ascomycete strains were discovered to provide effective 7β-hydroxylation of ADD which has not been so far reported. The structures of major products and intermediates were confirmed by HPLC, mass-spectrometry (MS), 1H and 13C NMR analyses.The results contribute to the knowledge on the functional diversity of steroid-transforming filamentous fungi. Previously unexplored fungal biocatalysts capable of effective performing structural modification of AD and ADD can be applied for industrial bioprocesses of new generation.

Overexpression of cytochrome p450 125 in Mycobacterium: a rational strategy in the promotion of phytosterol biotransformation.

Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are generally produced by the biotransformation of phytosterols in Mycobacterium. The AD (D) production increases when the strain has high NAD+/NADH ratio. To enhance the AD (D) production in Mycobacterium neoaurum TCCC 11978 (MNR M3), a rational strategy was developed through overexpression of a gene involved in the phytosterol degradation pathway; NAD+ was generated as well. Proteomic analysis of MNR cultured with and without phytosterols showed that the steroid C27-monooxygenase (Cyp125-3), which performs sequential oxidations of the sterol side chain at the C27 position and has the oxidative cofactor of NAD+ generated, played an important role in the phytosterol biotransformation process of MNR M3. To improve the productivity of AD (D), the cyp125-3 gene was overexpressed in MNR M3. The specific activity of Cyp125-3 in the recombinant strain MNR M3C3 was improved by 22% than that in MNR M3. The NAD+/NADH ratio in MNR M3C3 was 131% higher than that in the parent strain. During phytosterol biotransformation, the conversion of sterols increased from 84 to 96%, and the yield of AD (D) by MNR M3C3 was increased by approximately 18% for 96h fermentation. This rational strain modification strategy may also be applied to develop strains with important application values for efficient production of cofactor-dependent metabolites.

Biochemical and structural characterization of 3‐ketosteroid‐Δ1‐dehydrogenase, a candidate enzyme for efficient bioconversion of steroids

AbstractBackground3‐ketosteroid‐Δ1‐dehydrogenase, a key enzyme involved in microbial catabolism of sterols, catalyzes the 1, 2‐desaturation of steroidal substrates using FAD as a cofactor. Recombinant 3‐ketosteroid‐Δ1‐dehydrogenase from Arthrobacter simplex (KsdD4) was expressed in Escherichia coli BL21 (DE3).ResultsKsdD4 exhibited optimal activity at pH 7.0 and 35°C. KsdD4 had the highest activity toward 4‐androstene‐3,17‐dione (AD) and a moderate activity toward cortisone acetate and hydrocortisone. Structure‐based homology modeling revealed that Y118, Y355, Y528 and G532 are located in the active site, thus the most likely catalytic residues. The substrate‐binding cavity was composed of hydrophobic residues with small side chains, including A49, V328, A390 and A531, which facilitate the recognition of steroidal substrates with large C17 side‐chains. E332 forms a unique hydrogen bond with substrates. Recombinant E. coli resting cells expressing KsdD4 showed high catalytic activity in the presence of various organic solvents. A fed‐batch bioconversion using resting cells resulted in efficient production of 2.66 g L−1 androst‐1,4‐diene‐3,17‐dione (ADD).ConclusionBiochemical data and structural analysis greatly advanced our understanding of the KsdD family enzymes, and the fed‐batch strategy improved the bioconversion of steroids. © 2018 Society of Chemical Industry

Engineered 3-Ketosteroid 9α-Hydroxylases in Mycobacterium neoaurum: an Efficient Platform for Production of Steroid Drugs.

3-Ketosteroid 9α-hydroxylase (Ksh) consists of a terminal oxygenase (KshA) and a ferredoxin reductase and is indispensable in the cleavage of steroid nucleus in microorganisms. The activities of Kshs are crucial factors in determining the yield and distribution of products in the biotechnological transformation of sterols in industrial applications. In this study, two KshA homologues, KshA1N and KshA2N, were characterized and further engineered in a sterol-digesting strain, Mycobacterium neoaurum ATCC 25795, to construct androstenone-producing strains. kshA1N is a member of the gene cluster encoding sterol catabolism enzymes, and its transcription exhibited a 4.7-fold increase under cholesterol induction. Furthermore, null mutation of kshA1N led to the stable accumulation of androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD). We determined kshA2N to be a redundant form of kshA1N Through a combined modification of kshA1N, kshA2N, and other key genes involved in the metabolism of sterols, we constructed a high-yield ADD-producing strain that could produce 9.36 g liter-1 ADD from the transformation of 20 g liter-1 phytosterols in 168 h. Moreover, we improved a previously established 9α-hydroxy-AD-producing strain via the overexpression of a mutant KshA1N that had enhanced Ksh activity. Genetic engineering allowed the new strain to produce 11.7 g liter-1 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) from the transformation of 20.0 g liter-1 phytosterol in 120 h.IMPORTANCE Steroidal drugs are widely used for anti-inflammation, anti-tumor action, endocrine regulation, and fertility management, among other uses. The two main starting materials for the industrial synthesis of steroid drugs are phytosterol and diosgenin. The phytosterol processing is carried out by microbial transformation, which is thought to be superior to the diosgenin processing by chemical conversions, given its simple and environmentally friendly process. However, diosgenin has long been used as the primary starting material instead of phytosterol. This is in response to challenges in developing efficient microbial strains for industrial phytosterol transformation, which stem from complex metabolic processes that feature many currently unclear details. In this study, we identified two oxygenase homologues of 3-ketosteroid-9α-hydroxylase, KshA1N and KshA2N, in M. neoaurum and demonstrated their crucial role in determining the yield and variety of products from phytosterol transformation. This work has practical value in developing industrial strains for phytosterol biotransformation.

A patchwork pathway for oxygenase-independent degradation of side chain containing steroids.

The denitrifying betaproteobacterium Sterolibacterium denitrificans serves as model organism for studying the oxygen-independent degradation of cholesterol. Here, we demonstrate its capability of degrading various globally abundant side chain containing zoo-, phyto- and mycosterols. We provide the complete genome that empowered an integrated genomics/proteomics/metabolomics approach, accompanied by the characterization of a characteristic enzyme of steroid side chain degradation. The results indicate that individual molybdopterin-containing steroid dehydrogenases are involved in C25-hydroxylations of steroids with different isoprenoid side chains, followed by the unusual conversion to C26-oic acids. Side chain degradation to androsta-1,4-diene-3,17-dione (ADD) via aldolytic C-C bond cleavages involves acyl-CoA synthetases/dehydrogenases specific for the respective 26-, 24- and 22-oic acids/-oyl-CoAs and promiscuous MaoC-like enoyl-CoA hydratases, aldolases and aldehyde dehydrogenases. Degradation of rings A and B depends on gene products uniquely found in anaerobic steroid degraders, which after hydrolytic cleavage of ring A, again involves CoA-ester intermediates. The degradation of the remaining CD rings via hydrolytic cleavage appears to be highly similar in aerobic and anaerobic bacteria. Anaerobic cholesterol degradation employs a composite repertoire of more than 40 genes partially known from aerobic degradation in gammaproteobacteria/actinobacteria, supplemented by unique genes that are required to circumvent oxygenase-dependent reactions.

Cofactor engineering to regulate NAD+/NADH ratio with its application to phytosterols biotransformation

BackgroundCofactor engineering is involved in the modification of enzymes related to nicotinamide adenine dinucleotides (NADH and NAD+) metabolism, which results in a significantly altered spectrum of metabolic products. Cofactor engineering plays an important role in metabolic engineering but is rarely reported in the sterols biotransformation process owing to its use of multi-catabolic enzymes, which promote multiple consecutive reactions. Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are important steroid medicine intermediates that are obtained via the nucleus oxidation and the side chain degradation of phytosterols by Mycobacterium. Given that the biotransformation from phytosterols to AD (D) is supposed to be a NAD+-dependent process, this work utilized cofactor engineering in Mycobacterium neoaurum and investigated the effect on cofactor and phytosterols metabolism.ResultsThrough the addition of the coenzyme precursor of nicotinic acid in the phytosterols fermentation system, the intracellular NAD+/NADH ratio and the AD (D) production of M. neoaurum TCCC 11978 (MNR M3) were higher than in the control. Moreover, the NADH: flavin oxidoreductase was identified and was supposed to exert a positive effect on cofactor regulation and phytosterols metabolism pathways via comparative proteomic profiling of MNR cultured with and without phytosterols. In addition, the NADH: flavin oxidoreductase and a water-forming NADH oxidase from Lactobacillus brevis, were successfully overexpressed and heterologously expressed in MNR M3 to improve the intracellular ratio of NAD+/NADH. After 96 h of cultivation, the expression of these two enzymes in MNR M3 resulted in the decrease in intracellular NADH level (by 51 and 67%, respectively) and the increase in NAD+/NADH ratio (by 113 and 192%, respectively). Phytosterols bioconversion revealed that the conversion ratio of engineered stains was ultimately improved by 58 and 147%, respectively. The highest AD (D) conversion ratio by MNR M3N2 was 94% in the conversion system with soybean oil as reaction media to promote the solubility of phytosterols.ConclusionsThe ratio of NAD+/NADH is an important factor for the transformation of phytosterols. Expression of NADH: flavin oxidoreductase and water-forming NADH oxidase in MNR improved AD (D) production. Besides the manipulation of key enzyme activities, which included in phytosterols degradation pathways, maintenance the balance of redox also played an important role in promoting steroid biotransformation. The recombinant MNR strain may be useful in industrial production.

Comparative genomic analysis of Mycobacterium neoaurum MN2 and MN4 substrate and product tolerance.

The microbial bioconversion of sterols can afford valuable steroid precursors, such as 4-androstene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD). The Mycobacterium neoaurum MN4 mutant strain can produce AD in high yield and can tolerate a higher concentration of the substrate phytosterol than the parent strain M. neoaurum MN2. In order to further investigate the mechanisms underlying the enhanced substrate and product tolerance, we performed a genomic analysis of the MN2 and MN4 strains. The genomes were sequenced using a high-throughput approach and analyzed using software for genome assembly, gene prediction and functional annotation, KEGG (Kyoto Encyclopedia of Genes and Genomes) annotation, COG (cluster of orthologous) group cluster analysis, GO cluster analysis, and SNP detection and annotation. Based on comparative genomics, 184 mutations were identified in MN4, the average variant rate of 1 variant every 27,249 bases, with a TS/TV value of 0.5877 and missense mutations in one key sterol degradation genes (ChoM1) and four side chain degradation genes that encode enzymes catalysing β-oxidation. The results suggest the high AD yield might be due to mutation of ChoM and genes encoding FadE, FadB and FadA β-oxidation enzymes. This study provides a theoretical basis for further functional genomics analysis and heterologous production of M. neoaurum MN2 secondary metabolites.

Revealing the influence of glucocorticoid treatment on the excretion of anabolic-androgenic steroids in horses through in vitro digestive simulations and an in vivo case study

Anabolic-androgenic steroids (AAS) are strictly forbidden in equine sports because of their stimulating effect on muscle growth and performance. Nevertheless, low levels of AAS have been found in some horses, untreated with AAS. Glucocorticoids (GC), used as an anti-inflammatory therapy and structurally related to AAS, might play a role in this phenomenon. In order to unravel this possible correlation the influence of glucocorticoid treatment on the excretion of AAS was studied both in vivo and in vitro. In vivo effects were investigated by analysing urine samples collected from a gelding treated with betamethasone. Additionally, multiple in vitro digestion simulations were set up, according to a previously validated protocol, to study the possibility of a direct biotransformation of glucocorticoids to AAS, by the microbiota of the equine hindgut. Urine and in vitro digestion samples were extracted and analysed with UHPLC-MS/MS and UHPLC-Orbitrap-HRMS analytical methods.A significant influence on the urinary excretion of α-testosterone (αT), β-testosterone (βT) and androsta-1,4-diene-3,17-dione (ADD) was seen. αT-concentrations up to 20ng/mL were detected. ADD was not found before treatment but could be detected post-treatment. Cortisone and cortisol also peaked (>30ng/mL) between day 37 and 48 post-treatment. The in vitro digestion results however revealed no direct biotransformation of glucocorticoids to AAS by the microbiota of the equine hindgut. This study shows that a glucocorticoid treatment can disrupt the synthesis and excretion of AAS, not by direct biotransformation upon gastrointestinal digestion, but more likely by influencing the hypothalamic-pituitary-adrenal axis.