Conversion of phytosterols into androstenedione by Mycobacterium neoaurum

Optimum conditions for transformation of phytosterols by Mycobacterium neoaururm, required for selective cleavage of the lateral chain into androstenedione, were shown to differ from the known conditions of animal sterol (cholesterol) transformation. Complete conversion of phytosterols into androstenedione at a substrate load of no less than 20 g/l was achieved on increasing the amount of the inoculum and the concentration of glucose (by 2 and 4 times, respectively, relative to cholesterol) and performing the fermentation under conditions of turbulent mixing. Under these conditions, both the rate of the transformation and the yield of the reaction product were high, due to the saturation of the culture liquid with hydrocarbonate. Data from the literature show that this ion is involved in cleavage of the branched lateral chain at carbon in position 24.     

Preparation of androsta-1,4-diene-3,17-dione from sterols using <Emphasis Type="Italic">Mycobacterium neoaurum</Emphasis> VKPM Ac-1656 strain

A product of microbiological cleavage of the sterols side chain, androsta-1,4-diene-3,17-dione, is toxic for bacteria, in particular, actinobacteria of the genera Mycobacterium and Arthrobacter. Sterols were transformed into androsta-1,4-diene-3,17-dione by culturing the M. neoaurum VKPM Ac-1656 strain in a high yield, provided that a sorbent was used for elimination of contact between the bacterial cells and the product. Unlike the cholesterol side chain, the more branched chains of phytosterols were cleaved in the presence of M. neoaurum at a high rate only under turbulent stirring of the culture medium, which intensified the formation of hydrocarbonate ion from NaNI₃ in situ.     

Studies on Baeyer–Villiger oxidation of steroids: DHEA and pregnenolone d-lactonization pathways in Penicillium camemberti AM83

Penicillium camemberti AM83 strain is able to carry out effective Baeyer–Villiger type oxidation of DHEA, pregnenolone, androstenedione and progesterone to testololactone. Pregnenolone and DHEA underwent oxidation to testololactone via two routes: through 4-en-3-ketones (progesterone and/or androstenedione respectively) or through 3β-hydroxy-17a-oxa-d-homo-androst-5-en-17-one.Analysis of transformation progress of studied substrates as function of time indicates that the 17β-side chain cleavage and oxidation of 17-ketones to d-lactones are catalyzed by two different, substrate-induced, BVMOs. In the presence of a C-21 substrate (pregnenolone or progesterone) induction of the enzyme catalyzing cleavage at 17β-acetyl chain was observed, whereas DHEA and androstenedione induced activity of the BVMO responsible for the ring-D oxidation; 5-en-3β-alcohol was a more effective inducer that the respective 4-en-3-ketone.     

Preparation of androsta-1,4-diene-3,17-dione from sterols using Mycobacterium neoaurum VKPM As-1656 strain

A product of microbiological cleavage of the sterols side chain, androsta-1,4-diene-3,17-dione, is toxic for bacteria, in particular, actinobacteria of the genera Mycobacterium and Arthrobacter. Sterols were transformed into androsta-1,4-diene-3,17-dione by culturing the M. neoaurum VKPM An-1656 strain in a high yield, provided that a sorbent was used for elimination of contact between the bacterial cells and the product. Unlike the cholesterol side chain, the more branched chains of phytosterols were cleaved in the presence of M. neoaurum at a high rate only under turbulent stirring of the culture medium, which intensified the formation of hydrocarbonate ion from NaNI3 in situ.     

Modulation of plant mitochondrial VDAC by phytosterols

We have investigated the effect of cholesterol and two abundant phytosterols (sitosterol and stigmasterol) on the voltage-dependent anion-selective channel (VDAC) purified from mitochondria of bean seeds (Phaseolus coccineus). These sterols differ by the degree of freedom of their lateral chain. We show that VDAC displays sensitivity to the lipid-sterol ratio and to the type of sterol found in the membrane. The main findings of this study are: 1), cholesterol and phytosterols modulate the selectivity but only stigmasterol alters the voltage-dependence of the plant VDAC in the range of sterol fraction found in the plant mitochondrial membrane; 2), VDAC unitary conductance is not affected by the addition of sterols; 3), the effect of sterols on the VDAC is reversible upon sterol depletion with 10 μM methyl-β-cyclodextrins; and 4), phytosterols are essential for the channel gating at salt concentration prevailing in vivo. A quantitative analysis of the voltage-dependence indicates that stigmasterol inhibits the transition of the VDAC in the lowest subconductance states.     

Studies on Baeyer–Villiger oxidation of steroids: DHEA and pregnenolone d-lactonization pathways in Penicillium camemberti AM83

Penicillium camemberti AM83 strain is able to carry out effective Baeyer–Villiger type oxidation of DHEA, pregnenolone, androstenedione and progesterone to testololactone. Pregnenolone and DHEA underwent oxidation to testololactone via two routes: through 4-en-3-ketones (progesterone and/or androstenedione respectively) or through 3β-hydroxy-17a-oxa-d-homo-androst-5-en-17-one.Analysis of transformation progress of studied substrates as function of time indicates that the 17β-side chain cleavage and oxidation of 17-ketones to d-lactones are catalyzed by two different, substrate-induced, BVMOs. In the presence of a C-21 substrate (pregnenolone or progesterone) induction of the enzyme catalyzing cleavage at 17β-acetyl chain was observed, whereas DHEA and androstenedione induced activity of the BVMO responsible for the ring-D oxidation; 5-en-3β-alcohol was a more effective inducer that the respective 4-en-3-ketone.     

Phytosterols in low- and nonfat beverages as part of a controlled diet fail to lower plasma lipid levels

Dietary phytosterols have been shown to reduce plasma cholesterol concentrations when consumed in different food matrices, but their effectiveness in nonfat or low-fat beverages has not been established. The objective of this study was to examine whether phytosterols alter plasma lipid levels when incorporated into nonfat or low-fat beverages. Fifteen moderately hypercholesterolemic men and women consumed three precisely controlled diets for periods of 21 days each in random order. Diets contained either a nonfat placebo beverage (NF), a beverage that is nonfat with added phytosterols (NFPS), or a beverage that is low in fat with added phytosterols (LFPS). Total cholesterol concentrations were not different between groups at endpoint, decreasing (P < 0.05) equally by 8.5%, 11.6%, and 10.1% with NF, NFPS, and LFPS consumption, respectively. There was no effect of dietary treatment on LDL cholesterol concentrations, which decreased over time (P < 0.05) by 5%, 10.4%, and 8.5% with NF, NFPS, and LFPS, respectively. HDL cholesterol and triacylglycerol concentrations were unaffected by the diets. Provision of phytosterols as part of nonfat and low-fat beverages did not exert any greater hypocholesterolemic effect than a nonfat placebo beverage. These results show that intake of phytosterols in a low-fat beverage format is not efficacious for lipid level modification.     

The stereospecific biosynthesis of plant sterols and α- and β-amyrin

1. A preparation of pea seedlings has been obtained that will incorporate [2-(14)C]mevalonate into squalene, alpha- and beta-amyrin and the phytosterols. 2. The (14)C/(3)H ratio in alpha- and beta-amyrin biosynthesized in the presence of [2-(14)C,4R-(3)H]-mevalonate is the same as in the starting material and in squalene; this gives experimental support to the mechanism for the cyclization of squalene proposed by Ruzicka for the formation of these pentacyclic triterpenoids. 3. The (14)C/(3)H ratio for beta-sitosterol was 5:3, the same as that in cholesterol in liver. 4. As the absence of (3)H from C-3 in beta-sitosterol was demonstrated (3)H must be present in the side chain and thus the H at C-24 is not lost during alkylation of the side chain; it probably migrates to C-25.     

Polyphosphoinositide activation of cholesterol side chain cleavage with purified cytochrome P-450scc

Highly purified beef adrenal cytochrome P-450 specific for cholesterol side chain cleavage (P-450-scc) has been reconstituted with sonicated vesicles containing cholesterol and either dimyristoyl phosphatidylcholine (DMPC) or dioleoyl phosphatidylcholine (DOPC). When cholesterol was present in DMPC vesicles at 1:15 molar ratio, cardiolipin and L-alpha-phosphatidylinositol 4-monophosphate (DPI) increased side chain cleavage by at least 5-fold (0.7 min-1-3.5 min-1). In DOPC vesicles, a smaller increase was observed (2.8 min-1-5.0 min-1). Activator phospholipids increased the rate of transference of cholesterol both to and from the cytochrome when, respectively, cholesterol-free P-450scc and cholesterol-P-450scc complex are combined with either DMPC or DOPC vesicles. Transfer of cholesterol to and from cytochrome P-450 occurred with similar first order rate constants and was also independent of the concentrations of cholesterol vesicles and P-450. It is suggested that transfer in both directions is limited by the rate of insertion of P-450scc into the membrane. Phospholipid stimulatory effects for both cholesterol transfer and for activation of side chain cleavage occurred with the same ranking, even though cholesterol transfer, following reconstitution, was 5-10 times slower than the turnover of side chain cleavage. DPI increased Vmax for side chain cleavage in both DMPC and DOPC vesicles to the same rate (12 min-1) without effect on the Km for cholesterol, while cardiolipin both produced a similar increase in Vmax and decreased Km (cholesterol). This activation by DPI is attributed to more favorable incorporation of P-450scc in these membranes and is consistent with previously reported effects of acidic phospholipids on other mitochondrial proteins.     

Phytosterols,phytostanols, and their conjugates in foods: structural diversity,quantitative analysis,and health-promoting uses

Phytosterols (plant sterols) are triterpenes that are important structural components of plant membranes, and free phytosterols serve to stabilize phospholipid bilayers in plant cell membranes just as cholesterol does in animal cell membranes. Most phytosterols contain 28 or 29 carbons and one or two carbon–carbon double bonds, typically one in the sterol nucleus and sometimes a second in the alkyl side chain. Phytostanols are a fully-saturated subgroup of phytosterols (contain no double bonds). Phytostanols occur in trace levels in many plant species and they occur in high levels in tissues of only in a few cereal species. Phytosterols can be converted to phytostanols by chemical hydrogenation. More than 200 different types of phytosterols have been reported in plant species. In addition to the free form, phytosterols occur as four types of “conjugates,” in which the 3β-OH group is esterified to a fatty acid or a hydroxycinnamic acid, or glycosylated with a hexose (usually glucose) or a 6-fatty-acyl hexose. The most popular methods for phytosterol analysis involve hydrolysis of the esters (and sometimes the glycosides) and capillary GLC of the total phytosterols, either in the free form or as TMS or acetylated derivatives. Several alternative methods have been reported for analysis of free phytosterols and intact phytosteryl conjugates. Phytosterols and phytostanols have received much attention in the last five years because of their cholesterol-lowering properties. Early phytosterol-enriched products contained free phytosterols and relatively large dosages were required to significantly lower serum cholesterol. In the last several years two spreads, one containing phytostanyl fatty-acid esters and the other phytosteryl fatty-acid esters, have been commercialized and were shown to significantly lower serum cholesterol at dosages of 1–3 g per day. The popularity of these products has caused the medical and biochemical community to focus much attention on phytosterols and consequently research activity on phytosterols has increased dramatically.     

Efficient biotransformation of cholesterol to androsta-1,4-diene-3,17-dione by a newly isolated actinomycete <Emphasis Type="Italic">Gordonia neofelifaecis</Emphasis>

A newly isolated actinomycete, Gordonia neofelifaecis (NRRL B-59395) from the faeces of Neofelis nebulosa, was used to selectively degrade the side-chain of cholesterol. The intermediates were purified and characterized. Quantitative analysis of the accumulated metabolites from cholesterol side-chain cleavage was conducted during the biotransformation. The results showed that the profile of accumulated intermediates was different from those of other reported microorganisms. Among the five metabolites, androsta-1,4-diene-3,17-dione (ADD) was the main product of the side-chain degradation, with a high conversion rate (87.2%), indicating its potential for industrial production of ADD. At the end of transformation, the substrate cholesterol was completely consumed. The effect of some factors on the bioconversion was also investigated. To our best knowledge, this is the first report regarding cholesterol side-chain cleavage using bacteria belonging to Gordonia.     

Injected phytosterols/stanols suppress plasma cholesterol levels in hamsters

Although plant sterols are known to suppress intestinal cholesterol absorption, whether plasma and hepatic lipid levels are influenced through non-gut related internal mechanisms has not been established. To examine this question 50 male hamsters were divided into 5 groups and fed semi-purified diets containing 20% energy as fat and 0.25% (w/w) cholesterol ad libitum for 60 days. The control group (i) received diet alone, while four additional groups consumed the diet plus one of four equivalent phytosterol mixtures (5 mg/kg/day) given either as (ii) tall oil phytosterols/stanols mixed with diet (oralSA), (iii) tall oil phytosterols/stanols subcutaneously injected (subSA), (iv) soybean oil phytosterols alone mixed with diet (oralSE), or (v) soybean oil subcutaneous injected phytosterols alone (subSE). The control group and both orally supplemented groups also received placebo subcutaneous sham injections. Neither food consumption, body weight, nor liver weight differed across treatment groups. Subcutaneous administration of SA and SE decreased plasma total cholesterol levels by 21% and 23% (p < 0.0001) and non-apolipoprotein-A cholesterol concentrations by 22% and 15% (p < 0.0002), respectively, compared to control. HDL cholesterol and TG concentrations remained unchanged across all groups, except for a decline of 25% (p < 0.0001) in HDL concentration in the subSE group versus control. Plasma campesterol levels were lower (p < 0.05) in the subSA group relative to all other groups. Plasma campesterol:cholesterol and campesterol:sitosterol ratios were, however, higher (p < 0.0001) for both the oral and subSE groups. Hepatic cholesterol levels were higher (p < 0.0001) in the oral and subSE phytosterol groups by 30% and 31%, respectively, relative to control. We conclude that low doses of subcutaneously administered plant sterols reduce circulating cholesterol levels through mechanisms other than inhibiting intestinal cholesterol absorption.     

Oxidation of cholesterol and cholesterol analogues by mitochondrial preparations of steroid-hormone-producing tissue.

The rate of oxidation of cholesterol and its analogues to pregnenolone (3beta-hydroxypregn-5-en-20-one) by various mitochondrial preparations was measured. Sterols with the cholest-5-en-3beta-ol ring system and saturated side chains of different lengths were converted into pregnenolone rat rates similar to that of cholesterol. This marked lack of mitochondrial specificity towards the steroid side chains is in direct contrast with the rat liver microsomal cholesterol 7alpha-hydroxylase, which has a high specificity for the side chain. Steroids that retain the ring system, but contain hydroxyl groups at various points in the side chain, are converted into pregnenolone at rates three to eight times higher than in cholesterol. The results are discussed with reference to current ideas on the mechanism of the side-chain cleavage of cholesterol. The results are discussed with reference to current ideas on the mechanism of the side-chain cleavage of cholesterol.     

Protein kinase stimulation of a reconstituted cholesterol side chain cleavage enzyme system in the bovine corpus luteum.

A solubilized preparation of cytochrome P-450, obtained by treatment of mitochondria from bovine corpora lutea with phospholipase A, contained all of the necessary components for the cholesterol side chain cleavage activity. The solubilized cytochrome -450 preparation could be isolated essentially free of endogenous cholesterol side chain cleavage activity by various fractionation techniques. A cholesterol side chain cleavage enzyme system was reconstituted using the isolated cytochrome P-450 preparation and purified adrenodoxin and adrenodoxin reductase (components of the enzyme system purified from the adrenal cortex). Protein kinase was partially purified from the cytosol fraction of bovine corpora lutea. It was purified 43-fold and the activity was highly dependent on cyclic adenosine 3:5-monophosphate (cyclic AMP). When ATP and this partially purified cyclic AMP-dependent protein kinase were added to the reconstituted cholesterol side chain cleavage enzyme assay in which cytochrome P-450 was limiting, a stimulation (20 to 74%) of the conversion of cholesterol into pregnenolone was observed. This stimulation was statistically significant with p value less than 0.001. The stimulatory effect of the protein kinase appeared to be dependent on ATP and was not mimicked by bovine serum albumin, indicating that the effect was specific for protein kinase. Protein kinase caused a phosphorylation of the cytochrome P-450 preparation when large amounts of this preparation were used in the assay. It is concluded from these results that the direct activation of the cytochrome P-450 component of the cholesterol side chain cleavage by protein kinase may be one of the ways by which cyclic AMP mediates the effect of luteinizine.     

Inhibition of bovine adrenocortical cytochrome P-450scc by 3,3'-dimethoxybenzidine

The effect of 3,3'-dimethoxybenzidine ($\text{o}$-dianisidine) on the conversion of cholesterol to pregnenolone was investigated in a reconstituted side chain cleavage system using enzymes purified from bovine adrenal cortex; d-$\text{p}$-aminoglutethimide was also assayed under similar conditions for comparison. 3,3'-Dimethoxybenzidine was found to be a potent inhibitor of pregnenolone formation, causing 50% inhibition at a concentration of 1.5 μM when using 70 μM cholesterol — this dose is approximately one fourth that required of 3-methoxybenzidine and one twentieth that required of benzidine for equal inhibition. In the same system, d-$\text{p}$-aminoglutethimide exhibited an I₅₀ value of about 55 μM. No effects of 3,3'-dimetoxybenzidine on adrenodoxin reductase or adrenodoxin activities could be detected, and inhibition of side chain cleavage could be relieved by dilution suggesting that the inhibitor acts by reversibly binding to cytochrome P-450scc.     

A moderate intake of phytosterols from habitual diet affects cholesterol metabolism

Cholesterol metabolism homeostasis is the result of a balance between synthesis, degradation and intestinal absorption. It is well established that intestinal cholesterol absorption efficiency can be modified by the intake of phytosterol-enriched food and, therefore, have a serum cholesterol-lowering effect. Recent epidemiological and clinical studies have shown that presence of phytosterols at normal diet levels could also be effective on lowering total and LDL serum cholesterol since they affect whole-body cholesterol metabolism even at those moderate doses. The aim of this study was to analyze the effect of the levels of the naturally-occurring phytosterols in the diet on cholesterol metabolism parameters. In order to do that a group of 99 healthy volunteers was studied for their dietary habits and surrogate markers of cholesterol synthesis and absorption. The mean daily dietary intake of phytosterols, measured by a food semiquantitative frequency questionnaire, was found to be 494 mg being β-sitosterol the major contributor to it. Subjects were classified into tertiles according to their total phytosterol intake and comparisons were done between subgroups. No statistical differences were observed for surrogate markers of intestinal cholesterol absorption, but a significant increase in the cholesterol synthesis surrogate marker lathosterol-to-cholesterol ratio associated to highest dietary phytosterol intake was observed. Regardless of this, only a non significant trend toward a less atherogenic lipid profile was observed in the upper tertile. In conclusion, the intake of moderate amounts of phytosterols naturally present in habitual diet may affect cholesterol metabolism and specially the rate of cholesterol synthesis as estimated by the surrogate marker lathosterol-to-cholesterol ratio in serum.     

Inhibition of cholesterol transport into skin cells in cultures by phytosterol-loaded microemulsion

Cholesterol and plant phytosterols are lipophilic compounds solubilized by intestinal micelles in a competitive manner. In this work, we used radioactive cholesterol- and phytosterol-loaded oil-in-water microemulsions to follow their incorporation and mutual competition in HaCaT keratinocytes, SZ95 sebocytes, and skin pieces in cultures. Dynamic light scattering showed homogenous nanostructures of 10.5+/-1.5 nm diameter and cryo-transmission electron microscopy confirmed the presence of uniform spherical droplets of 7.0+/-1.0 nm diameter. Up to 320 nmol/ml of cholesterol can be solubilized and transported into cells with minimal toxic effect by 0.5 wt% nanodroplets in a cell medium. Phytosterols inhibit incorporation of cholesterol into cells, in vitro, at molar ratios (phytosterols/cholesterol) of 4 and above. The loaded nanodroplets accumulate in intracellular vesicles (presumably endosomes). No metabolic conversion of cholesterol or phytosterols was found in these cells, in vitro, after 24 h, at 37 degrees C.     

Conjugated and free sterols from black bean (Phaseolus vulgaris L.) seed coats as cholesterol micelle disruptors and their effect on lipid metabolism and cholesterol transport in rat primary hepatocytes

Phytosterols have been widely studied for their cholesterol-lowering effect. Conjugated phytosterol forms have been found more active than free moieties. There are no reports about the sterol profile of black bean seed coats neither its effects on cholesterol metabolism. The aim of this research was to identify and quantify phytosterols from black bean seed coats and to determine their effects on cholesterol micellar solubility and on mRNA and key protein levels involved in lipid/cholesterol metabolism and cholesterol transport in primary rat hepatocytes. Free phytosterols, acylated steryl glycosides, and steryl glycosides were extracted from black bean seed coats. They were identified through HPLC–MS–TOF and quantified through HPLC equipped with UV–visible and evaporative light-scattering detectors. Free and conjugated phytosterols from the coats significantly increased the inhibitory effect of cholesterol micelle formation compared with stigmasterol, which was used as control (P < 0.05). In addition, phytosterols of black bean seed coat decreased lipogenesis by the downregulation of lipogenic proteins such as sterol regulatory element-binding protein 1 and fatty acid synthesis (FAS) in primary rat hepatocytes. Regarding β-oxidation, phytosterols upregulated the expression of carnitine palmitoyltransferase I and promoted the β-oxidation of long-chain fatty acids. Phytosterols inhibited cholesterol micellar solubility and reduced the activation of the liver X receptor, decreasing hepatic FAS and promoting hepatic β-oxidation of long-chain fatty acids.     

The side-chain cleavage of cholesterol sulfate--II. The effect of phospholipids on the oxidation of the sterol sulfate by inner mitochondrial membranes and by a reconstituted cholesterol desmolase system

This study compares the side-chain cleavage of aqueous suspensions of cholesterol sulfate with the side-chain cleavage of cholesterol sulfate which is incorporated into phospholipid vesicles. Three different cholesterol desmolase systems are examined: the membrane-bound cholesterol side-chain cleavage system present in inner mitochondrial membranes isolated from bovine adrenal mitochondria; a soluble, lipid-depleted, reconstituted side-chain cleavage system prepared from cytochrome P-450scc, adrenodoxin and adrenodoxin reductase; a membrane associated side-chain cleavage system prepared by adding phospholipid vesicles, prepared from adrenal mitochondrial, to the reconstituted system. Soluble cholesterol sulfate, in low concentration, is a good substrate for the lipid-depleted reconstituted side chain cleavage system. However, at concentrations above 2 microM, in the absence of phospholipids, the sterol sulfate appears to bind at a non-productive site on cytochrome P-450scc which leads to substrate inhibition. Phospholipids, while inhibiting the binding of cholesterol sulfate to the cytochrome, also appear to prevent non-productive binding of the sterol sulfate to the cytochrome. Thus the addition of phospholipids to the lipid-depleted enzyme system leads to an activation of side-chain cleavage of high concentrations of the sterol sulfate. Soluble cholesterol sulfate is a good substrate for both the native and reconstituted membrane-bound systems and no substrate inhibition is observed when the membrane bound enzyme systems are employed in the assay of side-chain activity. However, the cleavage of cholesterol sulfate, which is incorporated into phospholipid vesicles, by both membrane bound enzyme systems appears to be competitively inhibited by the phospholipids of the vesicles. The results of this study suggest that the regulation of the side-chain cleavage of cholesterol sulfate may be entirely different than the regulation of the side-chain cleavage of cholesterol, if cholesterol sulfate exists intracellularly as a soluble non-complexed substrate. If, on the other hand, cholesterol sulfate is present in the cell in lipid droplets as a complex with phospholipids, its metabolism may be under the same constraints as the side-chain cleavage of cholesterol.     

Microbial transformation of steroids--II. Transformations of progesterone, testosterone and androstenedione by Phycomyces blakesleeanus

Phycomyces blakesleeanus transformed progesterone, testosterone and androstenedione into mixtures of products. Five monohydroxylated metabolites were obtained in reasonable yields from the progesterone transformation. Only 7 alpha- and 15 beta-hydroxyprogesterone have been reported previously from this organism. We find that it gives these two metabolites and also 6 beta-, 14 alpha- and 15 alpha-hydroxyprogesterone as major products. Five compounds were also purified from testosterone transformation mixtures. Two of these were monohydroxylated, two were ring A dehydrogenation products, and two were oxidised at C-17. The products were identified as 6 beta-hydroxytestosterone, 7 alpha-hydroxytestosterone, androsta-1,4-diene-3,17-dione (1-dehydroandrostenedione), 17 beta-hydroxyandrosta-1,4-diene-3-one (1-dehydrotestosterone) and androstenedione. All five metabolites were produced in reasonable yields, although hydroxylation was the minor transformation in this case. Only two significant products were formed from androstenedione. Both were reduced at C-17; one was also monohydroxylated. They were testosterone and 14 alpha-hydroxytestosterone. The testosterone and androstenedione transformation products have not been reported previously for this organism. We also report for the first time the preparation of P. blakesleeanus cell-free extracts which transformed progesterone reasonably efficiently and faithfully in vitro, although the proportions of each product varied from one extract to another.