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The impact of steroid sulfatase (STS) activity in the circulating levels of both sulfated and unconjugated steroids is only partially known. In addition, the sulfated steroid pathway, a parallel pathway to the one for unconjugated steroids, which uses the same enzymes, has never been characterized in detail before. Patients with steroid sulfatase deficiency (STSD) are unable to enzymatically convert sulfated steroids into their unconjugated forms, and are a good model to elucidate how STS affects steroid biosynthesis and to study the metabolism of sulfated steroids. We quantified unconjugated and sulfated steroids in STSD serum, and compared these results with data obtained from serum of healthy controls. Most sulfated steroids were increased in STSD. However, androstenediol-3-sulfate and epiandrosterone sulfate showed similar levels in both groups, and the concentrations of androsterone sulfate were notably lower. Hydroxylated forms of DHEAS and of pregnenolone sulfate were found to be increased in STSD, suggesting a mechanism to improve the excretion of sulfated steroids. STSD testosterone concentrations were normal, but cholesterol and DHEA were significantly decreased. Additionally, serum bile acids were three-fold higher in STSD. Correlations between concentrations of steroids in each group indicate that 17α-hydroxy-pregnenolone-3-sulfate in men is mainly biosynthesized from the precursor pregnenolone sulfate and androstenediol-3-sulfate from DHEAS. These findings confirm the coexistence of two steroidogenic pathways: one for unconjugated steroids and another one for sulfated steroids. Each pathway is responsible for the synthesis of specific steroids. The equal levels of testosterone, and the reduced level of unconjugated precursors in STSD, support that testosterone is primarily synthesized from sulfated steroids. In consequence, testosterone synthesis in STSD relies on an enzyme with sulfatase activity other than STS. This study reveals that STS is a key player of steroid biosynthesis regulating the availability of circulating cholesterol.

(+)-Nootkatone is a natural ingredient that occurs in grapefruit and certain other plants and is responsible for the characteristic smell of grapefruit. Due to its versatile applications in the flavor and fragrance industry as well as its application in some medical uses it recruits the interests of academic research along with industrial biotechnology. In the current work we present the application of a novel short chain dehydrogenase from Bacillus megaterium in an in vivo whole-cell biocatalyst system for the conversion of the intermediate nootkatol into the industrially valuable (+)-nootkatone. The newly identified dehydrogenase converted nootkatol selectively and efficiently into the final product. The conversion ratio of about 100% was achieved within 40 min yielding about 44 mg/L (+)-nootkatone. Furthermore, the herein identified dehydrogenase provides a new tool to overcome the limitation of the two-step enzymatic biotechnological process for the production of (+)-nootkatone.

Cytochromes P450 (P450s) require electron transfer partners to catalyze substrate conversions. With regard to biotechnological approaches, the elucidation of novel electron transfer proteins is of special interest, as they can influence the enzymatic activity and specificity of the P450s. In the current work we present the identification and characterization of a novel soluble NADPH-dependent diflavin reductase from Bacillus megaterium with activity towards a bacterial (CYP106A1) and a microsomal (CYP21A2) P450 and, therefore, we referred to it as B. megaterium cytochrome P450 reductase (BmCPR). Sequence analysis of the protein revealed besides the conserved FMN-, FAD- and NADPH-binding motifs, the presence of negatively charged cluster, which is thought to represent the interaction domain with P450s and/or cytochrome c. BmCPR was expressed and purified to homogeneity in Escherichia coli. The purified BmCPR exhibited a characteristic diflavin reductase spectrum, and showed a cytochrome c reducing activity. Furthermore, in an in vitro reconstituted system, the BmCPR was able to support the hydroxylation of testosterone and progesterone with CYP106A1 and CYP21A2, respectively. Moreover, in view of the biotechnological application, the BmCPR is very promising, as it could be successfully utilized to establish CYP106A1- and CYP21A2-based whole-cell biotransformation systems, which yielded 0.3g/L hydroxy-testosterone products within 8h and 0.16g/L 21-hydroxyprogesterone within 6h, respectively. In conclusion, the BmCPR reported herein owns a great potential for further applications and studies and should be taken into consideration for bacterial and/or microsomal CYP-dependent bioconversions.

17α,20β-Dihydroxy-4-pregnen-3-one (17α,20βDiOH-P) and 17α,20β,21α-trihydroxy-4-pregnen-3-one (20βOH-RSS) are the critical hormones required for oocyte maturation in fish. We utilized B. megaterium's endogenous 20β-hydroxysteroid dehydrogenase (20βHSD) for the efficient production of both progestogens after genetically modifying the microorganism to reduce side-product formation. First, the gene encoding the autologous cytochrome P450 CYP106A1 was deleted, resulting in a strain devoid of any steroid hydroxylation activity. Cultivation of this strain in the presence of 17α-hydroxyprogesterone (17αOH-P) led to the formation of 17α,20α-dihydroxy-4-pregnen-3-one (17α,20αDiOH-P) as a major and 17α,20βDiOH-P as a minor product. Four enzymes were identified as 20αHSDs and their genes deleted to yield a strain with no 20αHSD activity. The 3-oxoacyl-(acyl-carrier-protein) reductase FabG was found to exhibit 20βHSD-activity and overexpressed to create a biocatalyst yielding 0.22g/L 17α,20βDiOH-P and 0.34g/L 20βOH-RSS after 8h using shake-flask cultivation, thus obtaining products that are at least a thousand times more expensive than their substrates.

β-Sitosterol and ergosterol are the equivalents of cholesterol in plants and fungi, respectively, and common sterols in the human diet. In the current work, both were identified as novel CYP27A1 substrates by in vitro experiments applying purified human CYP27A1 and its redox partners adrenodoxin (Adx) and adrenodoxin reductase (AdR). A Bacillus megaterium based biocatalyst recombinantly expressing the same proteins was utilized for the conversion of the substrates to obtain sufficient amounts of the novel products for a structural NMR analysis. β-Sitosterol was found to be converted into 26-hydroxy-β-sitosterol and 29-hydroxy-β-sitosterol, whereas ergosterol was converted into 24-hydroxyergosterol, 26-hydroxyergosterol and 28-hydroxyergosterol.

The biosynthesis of steroid hormones in vertebrates is initiated by the cytochrome P450 CYP11A1, which performs the side-chain cleavage of cholesterol thereby producing pregnenolone. In this study, we report a direct stimulatory effect of the estrogens estradiol and estrone onto the pregnenolone formation in a reconstituted in vitro system consisting of purified CYP11A1 and its natural redox partners. We demonstrated the formation of new products from 11-deoxycorticosterone (DOC), androstenedione, testosterone and dehydroepiandrosterone (DHEA) during the in vitro reaction catalyzed by CYP11A1. In addition, we also established an Escherichia coli-based whole-cell biocatalytic system consisting of CYP11A1 and its redox partners to obtain sufficient yields of products for NMR-characterization. Our results indicate that CYP11A1, in addition to the previously described 6β-hydroxylase activity, possesses a 2β-hydroxylase activity towards DOC and androstenedione as well as a 16β-hydroxylase activity towards DHEA. We also showed that CYP11A1 is able to perform the 6β-hydroxylation of testosterone, a reaction that has been predominantly attributed to CYP3A4. Our results are the first evidence that sex hormones positively regulate the overall production of steroid hormones suggesting the need to reassess the role of CYP11A1 in steroid hormone biosynthesis and its substrate-dependent mechanistic properties.

Cytochromes P450 play a key role in the drug and steroid metabolism in the human body. This leads to a high interest in this class of proteins. Mammalian cytochromes P450 are rather delicate. Due to their localization in the mitochondrial or microsomal membrane, they tend to aggregate during expression and purification and to convert to an inactive form so that they have to be purified and stored in complex buffers. The complex buffers and low storage temperatures, however, limit the feasibility of fast, automated screening of the corresponding cytochrome P450-effector interactions, which are necessary to study substrate-protein and inhibitor-protein interactions. Here, we present the production and isolation of functionalized poly(3-hydroxybutyrate) granules (PHB bodies) from Bacillus megaterium MS941 strain. In contrast to the expression in Escherichia coli, where mammalian cytochromes P450 are associated to the cell membrane, when CYP11A1 is heterologously expressed in Bacillus megaterium, it is located on the PHB bodies. The surface of these particles provides a matrix for immobilization and stabilization of the CYP11A1 during the storage of the protein and substrate conversion. It was demonstrated that the PHB polymer basis is inert concerning the performed conversion. Immobilization of the CYP11A1 onto the PHB bodies allows freeze-drying of the complex without significant decrease of the CYP11A1 activity. This is the first lyophilization of a mammalian cytochrome P450, which allows storage over more than 18days at 4°C instead of storage at -80°C. In addition, we were able to immobilize the cytochrome P450 on the PHB bodies in vitro. In this case the expression of the protein is separated from the production of the immobilization matrix, which widens the application of this method. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

In the current work the ability of Bacillus megaterium to take up hydrophobic substrates and efficiently express eukaryotic membrane proteins was utilized for establishing a CYP27A1-based biocatalyst. The human mitochondrial cytochrome P450CYP27A1 was co-expressed with its redox partners adrenodoxin reductase (Adr) and adrenodoxin (Adx). CYP27A1 could be localized at the cell's polyhydroxybutyrate (PHB) granules, carbon storage serving organelle-like vesicles that can take up cholesterol, resulting in bioreactor-like structures in B. megaterium . The resulting whole cell system allowed the efficient biotechnological conversion of the CYP27A1 substrates cholesterol, 7-dehydrocholesterol (7-DHC) and vitamin D3. After 48 h, nearly 100% of cholesterol was metabolized producing a final concentration of 113.14 mg/l 27-hydroxycholesterol (27-HC). Moreover, 70% of vitamin D3 was converted into 25-hydroxyvitamin D3 (25-OH-D3) with a final concentration of 80.81 mg/l. Also more than 97% of 7-DHC were found to be metabolized into two products, corresponding to 26/27-hydroxy-7-dehydrocholesterol (P1) and 25-hydroxy-7-dehydrocholesterol (P2). To our knowledge this is the first CYP27A1-based whole-cell system, allowing the efficient and low-cost production of pharmaceutically interesting metabolites of this enzyme from relatively cheap substrates.

Background Cholesterol, the precursor of all steroid hormones, is the most abundant steroid in vertebrates and exhibits highly hydrophobic properties, rendering it a difficult substrate for aqueous microbial biotransformations. In the present study, we developed a Bacillus megaterium based whole-cell system that allows the side-chain cleavage of this sterol and investigated the underlying physiological basis of the biocatalysis. Results CYP11A1, the side-chain cleaving cytochrome P450, was recombinantly expressed in the Gram-positive soil bacterium B. megaterium combined with the required electron transfer proteins. By applying a mixture of 2-hydroxypropyl-β-cyclodextrin and Quillaja saponin as solubilizing agents, the zoosterols cholesterol and 7-dehydrocholesterol, as well as the phytosterol β-sitosterol could be efficiently converted to pregnenolone or 7-dehydropregnenolone. Fluorescence-microscopic analysis revealed that cholesterol accumulates in the carbon and energy storage-serving poly(3-hydroxybutyrate) (PHB) bodies and that the membrane proteins CYP11A1 and its redox partner adrenodoxin reductase (AdR) are likewise localized to their surrounding phospholipid/protein monolayer. The capacity to store cholesterol was absent in a mutant strain devoid of the PHB-producing polymerase subunit PhaC, resulting in a drastically decreased cholesterol conversion rate, while no effect on the expression of the recombinant proteins could be observed. Conclusion We established a whole-cell system based on B. megaterium, which enables the conversion of the steroid hormone precursor cholesterol to pregnenolone in substantial quantities. We demonstrate that the microorganism’s PHB granules, aggregates of bioplastic coated with a protein/phospholipid monolayer, are crucial for the high conversion rate by serving as substrate storage. This microbial system opens the way for an industrial conversion of the abundantly available cholesterol to any type of steroid hormones, which represent one of the biggest groups of drugs for the treatment of a wide variety of diseases. Electronic supplementary material The online version of this article (doi:10.1186/s12934-015-0300-y) contains supplementary material, which is available to authorized users.

This article seeks to make a contribution to comparative history at the level of interpretative concepts. The argument is that the concept of "communalism" provides a lens that is useful for the examination of Japanese history, even as the application of the concept to Japanese material discloses questions that might usefully be pursued by historians of communalism in Europe. In its original historiographical setting (Upper Germany in the late medieval and early modern periods), the concept was put forward as a way of describing how rural and urban communities developed a corporate character and were thus able to take over "state" functions on the local level, such as the making of ordinances, the administration of justice, and preservation of the peace. For purposes of comparison with Upper Germany, this essay focusses on Central Japan. One similarity between the two regions may be seen in the way villages were formed. Both in Europe during the High Middle Ages and in Japan during the thirteenth and fourteenth centuries, changes in the agrarian economy and in rural settlement patterns provided the background for the appearance of village communities. In both areas the fixed peasant household was a foundation stone of the emerging village. While the concept of communalism stresses the importance for Upper Germany of the village assembly of householders, together with the village council and village court, research on Japanese villages has emphasized the importance of cultic associations and the functional division of villagers into age-group associations. Despite such internal divisions, village government in Japan also had a basis in the community as a whole. As in Upper Germany, a vigorous communal organization developed in medieval Japanese villages. It was the community that controlled the "state functions," so important to the proponents of the communalism thesis, as well as certain economic and religious functions. The corporate activity of Central Japanese villages was, in fact, even more impressive than their Upper German counterparts, for in Japan the village could also function-unlike in Germany-as a community of fighting men defending their collective interests with military means. The strong position of Japanese villages was due not least to the upper stratum of village society, which during the wars of the sixteenth century separated itself more and more from ordinary villagers and approximated the status of the warrior estate (samurai). These rural warriors also formed regional associations that transcended village boundaries. In Japan there were no parliaments or representative assemblies at the territorial level, as in Europe and especially in Upper Germany, where village communities often had a voice in such bodies. By contrast, villages in Central Japan formed the basis for regional federations entered into by the warrior upper stratum of village society and the local nobility. These associations represented, in sixteenth-century Japan, a counterpart to the state building efforts of territorial princes. In sum, the village communities of Central Japan and of Upper Germany have a number of features in common that seem adequately to be described by the concept of communalism. By the same token, the distinctive characteristics of village life in Japan-the segmentation into age groupings, the military enterprises of the villages, and a militarized upper stratum building networks across village lines-raise questions that deserve further examination by historians of village communities in Europe.