A novel short chain dehydrogenase from Bacillus megaterium for the conversion of the sesquiterpene nootkatol to (+)-nootkatone.

(+)-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.

Functionalized poly(3-hydroxybutyric acid) bodies as new in vitro biocatalysts.

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.

Role of steroid sulfatase in steroid homeostasis and characterization of the sulfated steroid pathway: Evidence from steroid sulfatase deficiency.

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.

A Novel NADPH-dependent flavoprotein reductase from Bacillus megaterium acts as an efficient cytochrome P450 reductase.

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.

Genetic engineering of Bacillus megaterium for high-yield production of the major teleost progestogens 17α,20ß-di- and 17α,20ß,21α-trihydroxy-4-pregnen-3-one.

17α,20ß-Dihydroxy-4-pregnen-3-one (17α,20ßDiOH-P) and 17α,20ß,21α-trihydroxy-4-pregnen-3-one (20ßOH-RSS) are the critical hormones required for oocyte maturation in fish. We utilized B. megaterium's endogenous 20ß-hydroxysteroid dehydrogenase (20ßHSD) 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,20ßDiOH-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 20ßHSD-activity and overexpressed to create a biocatalyst yielding 0.22g/L 17α,20ßDiOH-P and 0.34g/L 20ßOH-RSS after 8h using shake-flask cultivation, thus obtaining products that are at least a thousand times more expensive than their substrates.

Human CYP27A1 catalyzes hydroxylation of ß-sitosterol and ergosterol.

ß-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. ß-Sitosterol was found to be converted into 26-hydroxy-ß-sitosterol and 29-hydroxy-ß-sitosterol, whereas ergosterol was converted into 24-hydroxyergosterol, 26-hydroxyergosterol and 28-hydroxyergosterol.

Expression of human CYP27A1 in B. megaterium for the efficient hydroxylation of cholesterol, vitamin D3 and 7-dehydrocholesterol.

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.

Functionalized PHB granules provide the basis for the efficient side-chain cleavage of cholesterol and analogs in recombinant Bacillus megaterium.

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-ß-cyclodextrin and Quillaja saponin as solubilizing agents, the zoosterols cholesterol and 7-dehydrocholesterol, as well as the phytosterol ß-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.