Bioconversion Of Phytosterols Research Articles

Functional characterization of two reductase KshBs in Mycobacterium fortuitum and their applications to C9 non-hydroxylated steroid production

3-ketosteroid-9α-hydroxylase (KSH), a two-component monooxygenase consisting of a Rieske oxygenase KshA and a ferredoxin reductase KshB, is a crucial enzyme involved in C-9 hydroxylation and is indispensable in the microbial catabolism of sterols. However, the in vivo function of KshB remains unclear. In this study, two reductase subunits, KshB1 and KshB2 from Mycobacterium fortuitum ATCC 35855, were first characterized and then engineered in the strain MFΔkstD, which produces 9-OHAD (9α-hydroxy-4-androstene-3,17-dione), to construct the strains producing AD (4-androstene-3,17-dione). The transcriptional level and specific enzyme activity of KshB1 under phytosterols induction was significantly higher compared to KshB2. After knocking out kshB1, AD peaked at 1.77 g/L at 48 h, subsequently being fully converted to 9-OHAD. Furthermore, we developed the stable AD-producer MFΔkstDΔkshB by null mutation both kshB1 and kshB2, although no AD was detected by single knocking out kshB2. Through gene complementation and the bioconversion of phytosterols, KshB1 emerged as the primary reductase in the KSH system, with KshB2 serving a complementary role. The stable AD-producer MFΔkstDΔkshB could produce 5.18 g/L, 6.91 g/L, and 9.03 g/L AD from the transformation of 10 g/L, 15 g/L, and 20 g/L phytosterols, respectively. In conclusion, these findings highlight a new strategy for the metabolic engineering of Mycobacterium fortuitum, involving the inactivation of the reductase KshB to facilitate the production of C9 non-hydroxylated steroidal intermediates.

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.

Novel approach for downstream processing of phytosterol bioconversion

AbstractBACKGROUNDDownstream processing is an important part of industrial steroid biotechnology. The traditional recovery methods require significant amounts of the organic solvents which are associated with environmental risks. There is a need for environmentally friendly downstream processing which also may open the prospects for regeneration and repeated use of the costly materials.RESULTSMicrobial conversion of phytosterol to C17‐ketosteroids in methylated cyclodextrin (mCD) solutions was studied as a model bioprocess. Quantitative aspects of competitive complexation of mCD and natural β‐CD (β‐CD) with steroids were studied under different temperature modes and CD concentrations. The method of steroid recovery based on precipitation of insoluble β‐CD complexes ensured the complete isolation of androst‐4‐ene‐3,17‐dione, androsta‐1,4‐diene‐3,17‐dione, 9α‐hydroxyandrost‐4‐ene‐3,17‐dione, 20‐hydroxymethyl‐pregna‐1,4‐dien‐3‐one and 3β‐hydroxyandrost‐5‐en‐17‐one from aqueous media. Analysis of the 1H nuclear magnetic resonance (NMR) spectra showed a constant molar ratio of β‐CD to steroid equal to 2 in the obtained steroid β‐CD complexes. The extraction of the steroid from the β‐CD complex by small volumes of organic solvent provided a quantitative yield. The method developed ensures regeneration and repeated use of the mCDs without decrease of the basic bioconversion outputs, as was demonstrated in a series of phytosterol to androsta‐1,4‐diene‐3,17‐dione bioconversions by Mycolicibacterium neoaurum VKM Ac‐1816D.CONCLUSIONSDownstream processing based on manipulation of steroid solubility in aqueous cyclodextrin solutions is a promising alternative to traditional steroid extraction with organic solvents. This environmentally friendly approach ensures re‐use of materials without decrease in bioconversion outputs. © 2023 Society of Chemical Industry (SCI).

Functional analysis of acyl-CoA dehydrogenases and their application to C22 steroid production

Acyl-CoA dehydrogenase (ChsE) is involved in the steroid side-chain degradation process. However, their function in vivo remains unclear. In this study, three ChsE, ChsE1-ChsE2, ChsE3, and ChsE4-ChsE5, were identified in Mycolicibacterium neoaurum, and their functions in vivo are studied and compared with those from Mycobacterium tuberculosis in vitro. By gene knockout, complementation, and the bioconversion of phytosterols, the function of ChsE was elucidated that ChsE4-ChsE5 could utilize C27, C24, and C22 steroids in vivo. ChsE3 could utilize C27 and C24 steroids in vivo. ChsE1-ChsE2 could utilize C27, C24, and C22 steroids in vivo. What is more, the production strain of a C22 steroid, 3-oxo-4,17-pregadiene-20-carboxylic acid methyl ester (PDCE), is constructed with ChsE overexpression. This study improved the understanding of the steroid bioconversion pathway and proposed a method of the production of a new C22 steroid. KEY POINTS: • Three ChsE paralogs from M. neoaurum are identified and studied. • The function of ChsE is overlapped in vivo. • A C22 steroid (PDCE) producer was constructed with ChsE overexpression.

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.

Bioproduction of testosterone from phytosterol by Mycolicibacterium neoaurum strains: “one-pot”, two modes

The main male hormone, testosterone is obtained from cheap and readily available phytosterol using the strains of Mycolicibacterium neoaurum VKM Ac-1815D, or Ac-1816D. During the first “oxidative” stage, phytosterol (5–10 g/L) was aerobically converted by Ac-1815D, or Ac-1816D to form 17-ketoandrostanes: androstenedione, or androstadienedione, respectively. At the same bioreactor, the 17-ketoandrostanes were further transformed to testosterone due to the presence of 17β-hydroxysteroid dehydrogenase activity in the strains (“reductive” mode). The conditions favorable for “oxidative” and “reductive” stages have been revealed to increase the final testosterone yield. Glucose supplement and microaerophilic conditions during the “reductive” mode ensured increased testosterone production by mycolicibacteria cells. Both strains effectively produced testosterone from phytosterol, but highest ever reported testosterone yield was achieved using M. neoaurum VKM Ac-1815D: 4.59 g/l testosterone was reached from 10 g/l phytosterol thus corresponding to the molar yield of over 66%. The results contribute to the knowledge on phytosterol bioconversion by mycolicibacteria, and are of significance for one-pot testosterone bioproduction from phytosterol bypassing the intermediate isolation of the 17-ketoandrostanes.Graphical

Bioconversion of Phytosterols to 9-Hydroxy-3-Oxo-4,17-Pregadiene-20-Carboxylic Acid Methyl Ester by Enoyl-CoA Deficiency and Modifying Multiple Genes in Mycolicibacterium neoaurum.

Steroid drug precursors, including C19 and C22 steroids, are crucial to steroid drug synthesis and development. However, C22 steroids are less developed due to the intricacy of the steroid metabolic pathway. In this study, a C22 steroid drug precursor, 9-hydroxy-3-oxo-4,17-pregadiene-20-carboxylic acid methyl ester (9-OH-PDCE), was successfully obtained from Mycolicibacterium neoaurum by 3-ketosteroid-Δ1-dehydrogenase and enoyl-CoA hydratase ChsH deficiency. The production of 9-OH-PDCE was improved by the overexpression of 17β-hydroxysteroid dehydrogenase Hsd4A and acyl-CoA dehydrogenase ChsE1-ChsE2 to reduce the accumulation of by-products. The purity of 9-OH-PDCE in fermentation broth was improved from 71.7% to 89.7%. Hence, the molar yield of 9-OH-PDCE was improved from 66.7% to 86.7%, with a yield of 0.78 g/L. Furthermore, enoyl-CoA hydratase ChsH1-ChsH2 was identified to form an indispensable complex in Mycolicibacterium neoaurum DSM 44704. IMPORTANCE C22 steroids are valuable precursors for steroid drug synthesis, but the development of C22 steroids remains unsatisfactory. This study presented a strategy for the one-step bioconversion of phytosterols to a C22 steroid drug precursor, 9-hydroxy-3-oxo-4,17-pregadiene-20-carboxylic acid methyl ester (9-OH-PDCE), by 3-ketosteroid-Δ1-dehydrogenase and enoyl-CoA hydratase deficiency with overexpression of 17β-hydroxysteroid dehydrogenase acyl-CoA dehydrogenase in Mycolicibacterium. The function of the enoyl-CoA hydratase ChsH in vivo was revealed. Construction of the novel C22 steroid drug precursor producer provided more potential for steroid drug synthesis, and the characterization of the function of ChsH and the transformation of steroids further revealed the steroid metabolic pathway.

Production of 9,21-dihydroxy-20-methyl-pregna-4-en-3-one from phytosterols in Mycobacterium neoaurum by modifying multiple genes and improving the intracellular environment

BackgroundSteroid drugs are essential for disease prevention and clinical treatment. However, due to intricated steroid structure, traditional chemical methods are rarely implemented into the whole synthetic process for generating steroid intermediates. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HP) is a novel steroid drug precursor, suitable for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain.ResultsThe Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. Hsd4A, encoding a β-hydroxyacyl-CoA dehydrogenase, and fadA5, encoding an acyl-CoA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains were able to accumulate 0.59 g L−1 and 0.47 g L−1 9-OH-4-HP from 1 g L−1 phytosterols, respectively. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 deficient strain was 11.9% higher than that of the Hsd4A deficient strain and 40.4% higher than that of the strain with FadA5 deficiency strain, respectively. The purity of 9-OH-4-HP obtained from the Hsd4A and FadA5 deficient strain has reached 94.9%. Subsequently, the catalase katE from Mycobacterium neoaurum and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment, leading to a higher 9-OH-4-HP production. Ultimately, 9-OH-4-HP production reached 3.58 g L−1 from 5 g L−1 phytosterols, and the purity of 9-OH-4-HP improved to 97%. The final 9-OH-4-HP production strain showed the best molar yield of 85.5%, compared with the previous reported strain with 30% molar yield of 9-OH-4-HP.ConclusionKstD, Hsd4A, and FadA5 are key enzymes for phytosterol side-chain degradation in the C19 pathway. Double deletion of hsd4A and fadA5 contributes to the blockage of the C19 pathway. Improving the intracellular environment of Mycobacterium neoaurum during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.

Identification and in situ removal of an inhibitory intermediate to develop an efficient phytosterol bioconversion process using a cyclodextrin-resting cell system.

A classically versatile steroid intermediate, 9α-hydroxyandrost-4-ene-3,17-dione (9α-OH-AD), can be obtained by phytosterol (PS) bioconversion using Mycobacterium. In this study, a cyclodextrin-resting cell reaction system with a high concentration of PS (50 g L−1) was used to produce 9α-OH-AD. However, the inhibitory effect of metabolic intermediates is a key factor limiting production efficiency. After the separation and identification of a series of metabolic intermediates, it was found that 4-ene-3-keto steroids, which are the first metabolites of sterol side-chain degradation, accumulated at the beginning of the bioprocess and had a remarkable inhibitory effect on bioconversion. The bioconversion rate was greatly improved when 5 g L−1 of macroporous adsorbent resin D101 was added to the reaction system in the initial phase. A certain amount of resin acted as a reservoir to remove the inhibitory intermediate in situ and facilitated the bioconversion process, and the 9α-OH-AD space–time yield increased to 8.51 g L−1 d−1, which was 23.15% higher than that without resin addition (6.91 g L−1 d−1) after 72 h bioconversion. In summary, we identified an inhibitory intermediate that limits the bioconversion rate and provided a solution based on resin adsorption for improving 9α-OH-AD production efficiency in a commercial-scale process.

Effect of Cholinium Amino Acids Ionic Liquids As Cosolvents on the Bioconversion of Phytosterols by Mycobacterium sp. Resting Cells

Androst-4-ene-3,17-dione (AD) is an important intermediate of steroid drugs, and the microbial side-chain cleavage of sterols to produce AD by Mycobacterium sp. has attracted much attention. Unfort...

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.

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

BackgroundThe 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.ResultsThe 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.ConclusionsThe 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.

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.

Two-Step Bioprocess for Reducing Nucleus Degradation in Phytosterol Bioconversion by Mycobacterium neoaurum NwIB-R10hsd4A

In order to obtain high 4-androstene-3,17-dione (AD) yield, nucleus degradation needs to be avoided during phytosterol bioconversion process with Mycobacterium neoaurum NwIB-R10hsd4A. 3-Ketosteroid-Δ1-dehydrogenase (KstD) catalyzes 1,2-desaturation of steroids and is a key enzyme involved in steroid nucleus oxidation. Heterogeneous expression and characterization of two KstDs (KstD2, KstD3) from M. neoaurum NwIB-R10hsd4A showed that their activities were inhibited by shifting temperature from 30 to 37°C. However, the total activities of KstD2 and KstD3 were replenished when M. neoaurum NwIB-R10hsd4A was cultured at 37°C because the transcription levels of kstD2 and kstD3 were upregulated 1.61- and 1.43-fold respectively compared with the cultivation at 30°C. As the optimal temperature for cell growth was 30°C, we developed a two-step bioprocess, cell culture at 30°C and bioconversion with resting cells at 37°C avoiding higher transcriptional level of kstD2 and kstD3. This process repressed the activities of KstDs, resulted in the decrease of 1,2-desaturation products, and reduced the nucleus degradation (17.6%). AD production increased to 24.7gl-1 at higher substrate concentration (50gl-1). These results indicated that the two-step bioprocess was potential in phytosterol biotransformation industrially.

Effect of methyl-β-cyclodextrin on gene expression in microbial conversion of phytosterol

Modified β-cyclodextrins are widely used for the enhancement of microbial conversions of lipophilic compounds such as steroids. Multiple mechanisms of cyclodextrin-mediated enhancement of phytosterol bioconversion by mycobacteria had previously been shown to include steroid solubilization, alterations in the cell wall permeability for both steroids and nutrients, facilitation of protein leaking, and activity suppression of some steroid-transforming enzymes.In this work, we studied whether cyclodextrins might affect expression of the genes involved in the steroid catabolic pathway. Phytosterol bioconversion with 9α-hydroxy-androst-4-ene-3,17-dione accumulation by Mycobacterium sp. VKM Ac-1817D in the presence of methylated β-cyclodextrin (MCD) was investigated. RNA sequencing of the whole transcriptomes in different combinations of phytosterol and MCD showed a similar expression level of the steroid catabolism genes related to the KstR-regulon and was responsible for side chain and initial steps of steroid core oxidation; whereas, induction levels of the genes related to the KstR2-regulon were attenuated in the presence of MCD in this strain. The data were attenuated with quantitative real-time PCR.The results contribute to the understanding of cyclodextrin effects on microbial steroid conversion and provide a basis for the use of cyclodextrins as expression enhancers for studies of sterol catabolism in actinobacteria.

Bioconversion of Phytosterols into Androstenedione by Mycobacterium.

The chapter describes the bioconversion of phytosterols to androstenedione (AD) with Mycobacterium spp. in shake flasks and fermenters, as well as LC-MS based methods for analysis of phytosterols and steroid products.Phytosterols are derived as a by-product of vegetable oil refining and of manufacture of wood pulp. Phytosterols contain the same four-ring nucleus as steroids, and may be converted to high-value steroids by removing the side chain at C17 and minor changes at other sites in the ring structure.Many bacteria, including Mycobacterium spp., are able to degrade phytosterols. Mutants of Mycobacterium spp. unable of ring cleavage can, when growing on phytosterols, accumulate the steroid intermediates androstenedione (AD) and/or 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 to 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 suggestions to minimize the practical problems caused by the water-insoluble phytosterol. Sampling, sample extraction, and quantification of substrates and products using LC-MS analysis are described.

Bioconversion of Phytosterols into Androstadienedione by Mycobacterium smegmatis CECT 8331.

The C19 steroid 1,4-androstadiene-3,17-dione (androstadienedione, ADD) is an added value product used as a synthon in the pharmaceutical industry for the commercial production of corticosteroids, mineralocorticoids, oral contraceptives, and other pharmaceutical steroids. Phytosterol biotransformation catalyzed by microbial whole cells is actually a very well-established research area in white biotechnology. The protocol below provides detailed information on ADD production by the mutant CECT 8331 of Mycobacterium smegmatis mc2155 using phytosterols as raw material in a lab scale. This protocol describes the bioconversion of phytosterols into ADD in a single fermentation step.

English

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.

Sterol production and phytosterol bioconversion in two species of heterotrophic protists, Oxyrrhis marina and Gyrodinium dominans

The kinetics and efficiency of sterol production and bioconversion of phytosterols in two heterotrophic protists Oxyrrhis marina and Gyrodinium dominans were examined by feeding them two different algal species (Rhodomonas salina and Dunaliella tertiolecta) differing in sterol profiles. R. salina contains predominantly brassicasterol (≅99%) and <2% cholesterol. The major sterols in D. tertiolecta are ergosterol (45–49%), 7-dehydroporiferasterol (29–31%) and fungisterol (21–26%). O. marina fed R. salina metabolized dietary brassicasterol to produce 22-dehydrocholesterol and cholesterol. O. marina fed D. tertiolecta metabolized dietary sterols to produce cholesterol, 22-dehydrocholesterol, brassicasterol and stigmasterol. G. dominans fed either R. salina or D. tertiolecta metabolized dietary sterols to make cholesterol, brassicasterol and a series of unknown sterols. When protists were fed R. salina, which contains cholesterol, the levels of cholesterol were increased to a magnitude of nearly 5- to 30-fold at the phytoplankton-heterotrophic protist interface, equivalent to a production of 172.5 ± 16.2 and 987.7 ± 377.7 ng cholesterol per mg R. salina carbon consumed by O. marina and G. dominans, respectively. When protists were fed D. tertiolecta, which contains no cholesterol, a net production of cholesterol by the protists ranged from 123.2 ± 30.6 to 871.8 ± 130.8 ng per mg algal C consumed. Cholesterol is not only the dominant sterol, but a critical precursor for many physiologically functional biochemicals in higher animal. As intermediates, these heterotrophic protists increase the amount of cholesterol at the phytoplankton–zooplankton interface available to higher trophic levels relative to zooplankton feeding on algae directly.

Bioconversion of phytosterols to androstanes by mycobacteria growing on sugar cane mud

Direct sterol conversion of sugar cane mud (residue) by Mycobacterium sp. was demonstrated to be possible technologically, thus avoiding sugar cane oil extraction and further processes of extraction and purification of phytosterols from this oil. Indeed, mycobacterial cells were able to convert phytosterols from sugar cane mud into 4-androstene-dione (AD) and 1,4 androsta-diene-3,17-dione (ADD). For the various concentrations assayed, concomitant higher yields for both androstanes were achieved at 20% (w/w) sugar cane mud in media. Furthermore, conversions were similar to those from other substrates, such as a mixture of phytosterols. The results suggest that the mycobacterial cell is able to easily access and bioconvert sugar cane mud phytosterols.