Title of Invention

PROCESS FOR FERMENTING PHYTOSTEROL COMPOSITIONS TO ANDROSTENEDIONE AND ANDROSTADIENEDIONE

Abstract ABSTRACT 1677/CHENP/2004 "Process for fermenting phytosterol compositions to androstenedione and androstadienedione" This invention relates to a process of fennenting a phytosterol composition to produce androstenedione (androst-4-ene-3, I 7-dione) and subsequently androstadienedione (androsta-1 ,4- diene,3,I 7-dione) which comprises: propagating a microbial culture of the genus Mycobacterium in a first nutrient medium; placing the microbial culture and the phytosterol composition in a bioreactor for a sufficient time to transform the composition substantially to androstenedione (the "AD solution"); propagating a fungal culture of the genus Fusarium in a second nutrient medium; injecting one or more of 1) Fusarium sp; and 2) the fungal culture medium into the bioreactor for a sufficient time to transform the AD solution to androstadienedione.
Full Text

TITLE: PROCESS FOR THE FERMENTATION OF PI YTOSTEROLS TO
ANDROSTADIENEDIONE
FIELD OF THE INVENTION
This invention relates in general to fermentation processes, and ir particular to tlie bio-conversion of phytosterol compositions to androstenedione and/or androstadienedione.
BACKGROUND OF THE INVENTION
At present there are two main routes for the Industrial production >f sterokj drugs. One is the diosgenin route and the other is the microbiologicaf transfc innation route. Diosgenln Is a spirok^ai type steroidal a giycone isolated from a Composite plant {DIoscorea sp.). In the late 1940's and early 1950's the route ^/as developed using mainiy chemical reactions:


Afttioughthis route remained importantfor the commercial proiuctior> of steroids, alternative methods came under consideration in the 1970's. Th a price of diosgenin was significantly increased particutarty by the Mexican govemmen t and due, in part, to decreased content of diosgenin in the dry plant mass, and incref sed labor cost, etc. Therefore, an alternative methodology was required for future AC production.
It Is well known from extensh/e research in the pharmaceutical Industry in the ig60's and 70'9 that numerous mictoarganlams (e.g., those of the g anera Arthrobacter. Brevfbacterfum, Aiffcrobacferlum, Profam/nobscfBr, Sacfffus, Noardia, Streptomyces and, In partioilar, Mycobacterium) possess the natural ability of degrading naturally occurring Sp-hydroxy^A^-sterols (such as cholesterol, |3 sitosterol or a-sitostensl) or a mixture of sterols (as shown below) to cadson dioxide and water, ar d that 4-andrc»tene-3,17-dione (androstenedlone) and 1,4-androstadiene-3,17-dIone (indrostadienedione) are tbrmad as intermediates during the degradation.
Phytosterol Compounds Used as Substrates lor Microbial E liotransfomnation.


One of the present commercial sources of phytasterols are vegetable-derived oils. The microbial conversion of these available sterols to AD and ADD provides a highly important route to the steroid drug industry.
ADD isanessential rawmateiial In the manufacture of various synthetic steroids induding anti-Inflammatory agents, oral contraceptives, synthetic adrenal steroids, anabolic agents (growth stimulating), and other synthetic steroids.
In general, the known fermentation process involves the propagation of a mutant of Mycobact&Hum in an appropriate nutrient medium, transfer of the culture to a bioreactor containing the phytasterols, and then allowing the biotranfonnation to AD and/or ADD over a period of approximately 120 hours. Harvesting of the fermentafion bre>th, extraction of the latter vinth an organic solvent, and subsequent crystallization in an organic solvent, generally provides the products AD and/or ADD as wtiite ciystalline

powders. Pertinent references that discuss ttie knovm process and summarize the earlier studies are as follows:
S. Kraychy, and R.D. Mulr, U.S. Patent No. 3,684,657 (1972).
W.J. Marsheck. S. Kraychy and R.D. Mulr, Appl. Microbfol., 2^, 72 (1072).
A.H. Conner, M. Nagaol 32,310(1976).
K. Kiesllch, J. Basic Microbiol., aS. 461 (1986).
One of the problems associated with the known phytosterol blo-converslon process (whk:h problem plagues the entire sterokl Industry) Involves the poor solubility of the substrate, in this case a phytosterol composltton, In ttie aqueous nutrient medium. Inadequate solubility dictates the presence of only relatively low concentrations of substrate in the nutrient medium, resulting in poor contact witfi the micro-organism and generally leading to low yields of end products. Long femnentation times are also typically needed to achieve any satisfactory degree of blo-conversbn. To afford an alternative route to AD and ADD in which the Issue of substrate solubility was addressed, the present applicants demonstrated that the phytosterols (0-sitosterol, campesteroi and stigmastanol) obtained from 'soap", available In multi-tonne quantities from the forest Industry, could be solubillzed and biotransformed quantitatively by Mycobacterium mutants to ADD (PCT/CAgo/00267).
Another problem associated with the known phytosterol bk>-oonverslon process is thatthe end-product of the bk)-conversion from phytosterols (or compositions of phytosterols) typically contains significant amounts of both AD and ADD. Given the similar chemical stoictiJFe of AD and ADD, it is difficult and expensive to subsequently separate Uiese two sterokl products from one another.
Accordingly in order to pnxluce ADD, the product of the inttial fermentation (AD) must be extracted, purified and then subjected to a second biotransfonnatiQn In order to produce ADD. Naturally, this pn>cess Is e}^enslve and time-consuming as it involves the interruption of the AD fermentation, and tt\e removal and purification of AD prior to the second biotransfbmiatlon.

It ]& an object of the present invention to obviate or mitigate the above disadvantages.
SUMMARY OF THE INVENTION
The present invention provides, In one aspect, a process of femienting a phytosterol composition to produce androstenedlone (androst-4-ene-3,17- propagating a microbial culture of the genus Mycobactarium In a first nutrient medium; placing the microbial c^ilture and the phytosterol composition in a bloreactorfor a sufficient time to transft^rn the composition substantially to androstenedlone (the "AD solution"); propagating a fungal culture of the genus Fusarfum In a second nutrient medium; and ttien injecting one or more of 1) Fusarium sp; or 2) the fungal culture medium comprising Fusarium sp. into the bioreactor for a suffldent time to transfonnn the AD solution to androsUHJienedlone.
The present Invention further comprises a process of preparing androstadlenedione from androstenedlone which comprises fermenting AD In a bioreactor in the presence of 1) Fusa/ium sp; or 2) a Fuaarium ^fungal culture medium for a sufficient time to transform ^e AD Into ADD.
The present invention further comprises compositions of ADD prepared by the aforementioned processes.
Importantly, and a significant advantage of the process of the present invention. Is that the biotransformation of AD to ADD by Fusarium sp occurs in the same bioreactor as the prior bacterial transfbrmab'on i.e. it is the same one in which AD is produced from the phytostenal composition. In other words, the AD produced from the initial 'fermentation step is not removed from the bioreactor (as Is Known and practised In the art) but ratiier, the bioreactor is inoculated (as described fn more detail below) wtth either 1) the fungal culture, or 2) the fungal culture medium In order to transform the AD solution irvto ADD. It has been found thaX, surprisingly, Fusarium sp and in particular the preened strain as disclosed hereinbeiow retains Its capacity to transform AD to ADD despite the presence of the residual bacterial cultuns

Accordingly, the process of the present Invention does not invoive the interruption of the initial AD fermentation, or the removal and purification of AD pilor to the second biotransformation. Significant time, energy and materials are saved.
The present Invention also provides a novel microorganisni of the genus Fusaiium, hereinafter referred to as PTA-3947 or FMI33, which was deposited at ATCC as Fusaiium so/an/FMi 33 on Decemt)ar21, 2001.
DESCRIPTION OF THE DRAWINGS
The present invention Is Hlustrated by the following non-^imlting drawings in which:
Figure 1 is a schematic showing themicrobiaiconversionofphytosterolstoADDin accordance with the present invention; and
Figure 2 Is a flow diagram shoving the prefened procedure for biotransformation of AD to ADD by Fusarium so/on/ in accordance vi^h the present invention.
DETAII^D DESCRIPTION OF THE INVENTION
The following dialled description is provided to aid those skilled in the art \n practising the preswit invention. Hcwsuer, this detailed desorlption should not be constmed so as to unduly limit the scope of the present invention. Modifications and variations to the embodlmente discusswJ hwein may be made by those with ordinary skiH in the art without departing from the spirit or scope of the present invention.
in a first step of the present invention, a microbial culture of the genus Mycobacterium is propagated in a nutrient medium. There are many suitable cultivation media and conditions known and available in the art and the present invention is not Intended to be limited to any one. Nonetheless, in a preferred form, the Mycobacteiia sp. are grown in a seed medium comprising refiner's molasses, sodium nitrate, ammonium phosphate and glucose. The

contents of US Patent No 6,071,714 to Kutney et al., in which appropriate cuiture media are described. Is incorporated herein by reference.
Generally, the nutrient media required for cultivation of the Mycobacteria sp and the Fusarjum sp should contain assimilable nitrogen and cart»n and an adequate supply of sterile air maintained therein, for example by exposing a large surface of the media to air or pas^ng it through the media in quantities sufficient to support submerged growth.
Suitable nitrogen sources are those normally employed for the purpose, including soybean meal, cottonseed meal, pancreatic digest of casein, fish meal, com steep liquor, meat extract, protein, autoiyzed brewer's yeast with milk solids, peptone, yeast extract, casein hydrol^ate, ntbate and/or ammonium compounds. Most of these may also be used as the cart>on source. Other carbon containing substeinces include cartxjhydrates such as glycerol, glucose, fructose, sucnsse, lactose, maltose. Inositol, dextrin, molasses, starch and whey. Combinations of these carison and nitrogen sources may be used advantageously.
Phosphate, magnesium and/or ferrous ions may be Incorporated as growth promoting factors if desired; buffers may be added to assure that growth is initiated as substantially neutral pH. Anti-foaming agents may be beneficial. Where isolated cells or enzymes are used to Induce fennentation rather than tfie Intact and growing microorganism, nutrients need not be present but in either event the medium is usually mostiy aqueous.
In a Geocmd s^ of the present invention, the substrate (a phytosterol composition), dissolved in a ajitable solvent or in emulsified form, and the microbial culture are added to a btoreactor. Fermentation Is allowed to proceed until maximum substrate conversion (phytostensis to AD solution) is achieved.
Any emuisl^ing agent may be used as long as it ^cilitates mixing of the phytosterol substrate in ^e solvent thereby oF^miang availability of the substrate to the miottolal cuiture. Examples include, but are not limited to ethyieneoxy adducts or fetty acid estors of polyglycols and ttiose commerdally avattable under tie names Tegin™, Tvireen™ and Span™.
In the alternative, soiubaizlng agente may be used to allow^e phytosterol composition to

be dissolved bs form a clear solution, thereby affording excellent contact wl^ the micro¬organism utilized in the bio-conversion. These selected suitable solubilizing agents, which Include members of the glycol ^mlly and members of the silicone family, allow high concentrations of phytosterols to be dissolved in the nutrient medium. This provides for excellent contact with the micro-organism, reduces fermentation times, and affords relatively high yields of end-products. Prior-Known solubllzing agents such as sunflower oil are preferred.
In a third step of the present Inventlo-i, a fun^l culture of ^e genus Fusarfum Is propagated In a second nutrient medium. As above, there are many suitable cultivation media and conditions knovm and availalsle In the art and the present invention Is not Intended to be limited to any one. Nonetheless, In a prefen-ed form, the Fusailum sp. are grown in a seed medium comprising com steep liquor and glucose.
In a fourth step of the preserrt invention, the bioreactor comprising the AD solution is Injected with one or more of: 1) the FusaHum, or 2) the fungal culture medium (comprising fungal enzymes) In order to transform the AD soluton Into ADD. In a preferred embodiment, the tNoreactor is sterilized (for example by heating at around 120° C) prior to this injection to Idll the bacteria.
The simplest and most economical way of proceeding wKh this "Injection" step Is to inoculate the sterilized bioreactor with some of the fungal culture medium. Alternatively, however, it Is possible to separate the fungal cells from the culture medium by, forexampie, centrifugation and Just Inject these cells. Moreover, the cells can be ruptured, ultrasonicaBy or othenvise, to teciHtate access to the enzymes present, which can be isolated by filtration or extracted with a solvent such as acetone and vrater and then injected Into Vne bioreactor instead of the medium or fungal celts.
The optimal subsb^te conc«itration, substrate addition time and femientation period in each of the two "phases" (phytosterols to AD; AD to ADD) depends on tiie type of microorganism used and the exact fermentation conditions. The precise conditions could readily be detemnined without undue experimentation by anyone sidlled In this field.
In a most preferred forni of the present invention, the bloconversion of the phytosterol composition to AD Is achieved by the use of MycobactBrium MB 3683. In a most

preterred form of the present invention, the biooonversion of AD to ADO is achievod by ttiB use of ^her Fusarfum solan! or Fusarium oxysporum.
Within one embodiment of the present Invention, mutation of F. solani, using nitrosoguanldlne has resulted in the production of a novel mutant which Is especially effective in transforming AD to ADD in the same reaction vessel as the transformation of the steroids to AD i.e.wlthout the necessity of separating AD torn the first fermentatjon step. This mutant microoi^anlsm has been given the patent deposit designation PTA-3947 by the American Type Culture Collection ("ATCC"), 10801 University Blvd., Manassas VA, USA, 20110-2209, where It lias been deposited in a pennanent collection. A subculture of this microorganism Is available upon request thereto, however, It should be understood that such availability does not constitute a license to practise the subject invention.
Figure 1 outlines the microbial and fungal fermentation steps of one embodiment of the present Invention wherein phytostenais are transformed by Mycobacterium sp. to AD and subsequently the AD Is transformed by Fusarium sp to ADD and boldenona.
Figure 2 Illustrates a fuller embodiment of the present invention wherein ADD is prepared dlredlv from an AD source. A Fusarium sp. culture is propagated and the Inoculum added to a vessel comprising AD under femientation conditions. Thereafter ADD is isolated and purified.
Phytostsrotfl/Phytostanols
As us^ herain, the term "phytasterol composition" includes all sterols without llmi^tlon, for example: sitosterol, campesterol, stigmasterol, Ijrasslcastavl (Induding dihydrobraasicasterol), desmostorol, chalinostefol, portferasterol, cjionasterol, ergostero). coprosterol, codisterol, isofucosterol, fuoosterol, derosteiol, norvisterol, lathosterol, stellastepd, spinasteroi, chondrtUastenol, peposterol, avenastorot, isoavenasterol, fecosterol, pollinastasterol, cholesterol and all natural or synthesized forms and derivatives thereof, including isomers, and includes all hydrog^ated counterparts ("phytostanols"). Phytostanols indudes all saturated or hydrogenated phytosterols and all natural or synthesized fomis and derivatives thereof, Including Isomers. It Is also to be understood that, when in doubt thnxjghout the specification, the term 'phytosterol composition' may encompass both phytosterols and phytostanols.

The sterols and stands for use in the biotransfonnation of the present invention may be procured from a variety of natural sources. For example, they may be obtained from the processing erf plant cite (Including aquaUc plants) such as com oil and other vegetable oils, wheat genn oil, soy extract rice extract, rice bran, rapeseed oil, suntloweroil. sesame oH and fish (and other marine-source) oHs. They may also be derived from fungi, for example ergosierol. Accordingly, the present invention is not to be Kmltad to any one source of sterols. US Patent Serial No. 4,420,427 teaches the preparation of sterols frun vegetable ofl sludge using solvents such as methanol. Alternatively, phytosterols and phytostanols may be (kitalned irom tall oil pltc^ or pulping soap, by-products of fores&y practises as descnbed in US Patent Serial No.5,770,749. Incorporated herein by reference.
During the fermentation process, it is pretered that the temperature be maintained in the range of alaout 30-37'C, more preferably from 25-35" C. tvftwiltoring of the pH during the fermentation reveals that the pH can vary in the range of appnsximately 7.0 in the initial period to approximatety 4.7 at harvest time. Upon completion of the fermentation or transformation process, the ADD is recovered by means well known and practised in the art.
The following examples represent laboratory trials. However, each can be extrapolated to industrial-scale application using icnown industrial techniques to implement the invention as described herein.
EXAMPLE 1 BIOTRANSFORMATION OF PHYTOSTEROLS TO ADD WITH MYCOBACTERIUM SP. AND FUSARfUM SOLANI
Biotransformation with Mycobacterium sp. (Stage 1) Preparation of Seed CuKure of A^obacterium sp.
Inoculum for biotransfonnation is prepared in liquid culture. The Erienmeyer flasks are inoculated ^m an agar slant (grown In a glass test tube). Forworidng \H:4umes up to 40 L, 1 L Erienmeyer flasks with 200 mL seed culUire medium each were used, while for 50 L and larger volumes 2 L Erienmeyer flasks witti 400 mL medium each were utilized. The inoculation Is performed under sterile conditions (in a Bic^azard Hood], Sterile water (5 mL) is pipetted onto the agar slant. TTte bacterial culture Is gently scraped with the end of the pipette. Then the suspended culture Is pipetted and released into the Erienmeyer flasks. The bacteria Is propagated on a rotary shaker (200 rpm, 1" throw, SO'C ambient temperatura). After 72 hours of incubation the seed culture is ready for transfer to the

bioreactor. Prolonged storage of the seed culture before inoculation is not recommended. A summary of the Seed Culture protocol is given below:
medium: eegd culture fnedjum (per L)
54 mL Refiners molasses (BC sugar)
5.4 g NaNOa
0.6 g NH4H2PO4
6.0 g glucose
ad|U8ttopH7.0
medium volume: 200 mUflask
Inoculum volume; 3ml cells suspension from agar slant per 200 mL
new medium
vessel: 1000 ml Erlenmeyer flask
agttation: shaker {V throw), 200 rpm
temperature: 30^
time of growth: 72 h incubation
Comment Good culluregrowth Is indicated ifayellowcolouredcelleedimentfomns at the bottom.
Preparation of Blotransformatioii Medium for 3 L Experiments wHh Mycobacterium sp.
This pracedure of medium preparation is utilized only for 3 L working volume experiments. At a larger scale the procedure described below. The composition of the biotransformatkin medium is as follows:
medium: biotransformation medium
(for 3 Liters of medium)
162 mL sterilized molasses [BC Sugar) (5.4% v/v)
16.2gNaN03 (0.54%)
1.8gNH4H2P04 (0.06%)
substrate: 30.Q g vegeteible phytc^tsrol (1.0%)
emulsifien 480 mL sunflower oil (16%)
The necessary amount of Reflners molasses (162 mL) Is mixed with water (300 ml) and sterilized (12rCfbr20mln) in an Erlenmeyer flask one day before use.
In a flask or a t}eaker water (2.0 L) and the medium components (except the phytosterols and the sunflower oil) are mixed. The pi-l of the medium is adjusted to 8.1 with 1 M NaOH solution (40 g/L NaOH), the medium is transfened to the bioreador and then heated to 60-70'C.
Phytosterols (30 g) are weighed in a beaker, sunflower oil (480 mL) is added and the

mixture (s stirred under heating on a hot plate until the oil becomes clear. This is usually reached at --120'C. The clear yellow phytosteroi solution in oil is poured Into the already warmed liquid medium (60-70'C) in the bioreactor without stinlng.
The fermentor is sterilized (121X for 30 min) and cooled to SCC.
The atjove procedure of medium preparation Is utilized only for 3 L working volumes.
Commertts: The Uotransformatlon medium has 7 % dry substance (refractometrically measured).
Preparation of Biotransformation Medium for Large Scale Experiments with Mycobacterium sp.
This procedure of medium preparation is utilized for 40 L and larger working volumes.


The necessary volume of Refiners molasses (54 mUL medium) Is mixed with water (100 mUL medium) and sterilized one day before use.
All mmponents (indudlng the necessary volume of water, but excluding the phytosterola and the sunflower oil) are mixed under stlning in the fermentor to obtain a clear solution. Thenecessary volumeofwaterlscfdculated by substracting the volumes of nnolasses, oil and Inoculum from the de^red final \^lume. The pH of the medium is adjusted to 8.1 with aqueous NaOH solution (20%). The phytosterols and the sunfloweroil are added to the medium and the fermentor is sterilized (121X, 30-60 min).
Comments: The blotransfonmation medium has 7 % dry substance (refractometrlcaliy measured).
Biotransfbrmatlon with Mycobactertum In a Fermentor
The bloconverslon experiments were performed in stainless steel fermentors with efficient bottom stinlng. The main parameters of the utilized fermantors are shown in Table 2. The temperature of the medium in the fermentor is adjusted to 30°C and the inoculum Is introduced under sterile conditions. The entire process is performed at 30'C. The pH and the sterility of the process are monitored from the beginning until the end of this stage. The monitoring of the substrate (phytosterols) and the product (AD) Is Initiated after 40 hours. Details on the analyticai procedures (HPLC, GC) and resufts are given below. The piH of the biotransformation mixture usually varies within 1 pH unit and is not corrected during the process. The biotransformation Is considered completed when maximal product (AD) concentration in the foam layer is achieved or when the substrate Oihytcstensl) concentration reaches 1/20-th of Its initial concentration.

Note on morohoiogv (as Studied using a light microscope):

At every stage, from agar stent to final harvest, the morphology of the cells remained the same, that is, the cells had bipolar nuclei and were rod-shaped.
Biotransfbrmation with Fusarlum solatti (Stage 2) Preparation of Seed Cuitura of Fusarlum solani
The fungal cutture Fusarium solaniia propagated In a liquid niedium (10 g^ com steep liquor, 10 g/L ^ucose, pH6.5). E rianmeyerflaslcs(1 L volunie,0,2 L medium)are inoculated from culture grown on oatmeal agar for 120 hours and at SO'C. The liquid medium culture is propagated on a rotary shaker (26°C, 120 rpm, 1" ttirow) for 72 hours.
Fusarium can also be sub-cultured from liquid to liquid rnedlum for up to 4 - 5 generations or until the next culture becomes pinlc.
The shaKe ffask inoculum could be stored at 4'C for up to 1 month without significant change in its activity.
Preparation for Blotrartsformatlon with Fusartum sotani
The second stage of the biotransfbmiation is performed using the entire biotransformation broth from the first stage.
Upon completion of the transformation of phyttraterois to AD (section 2.3.1.4) the necessary components (10 g/L glucose, 10 g/L com steep liquor) are added to the broth from stage 1 and pH is adjusted to 6.7 with aqueous HCI (4 %). The broth is sterilized (121 "C, 30 min) and the temperature is brought to M'C.
Biotransformation of AD to ADD Utilizing Broth from Stage 1 and Fusarium sofanf
The bloconverslon experiments were perfomied in stainless steel fermentors with efflcierrt bottom stirring. The main parameters of the utilized fenmentOrs are shown in Table 3. The temperature of the medium In the feimentor Is adjusted to ZQ'C and the inoculum Is introduced under sterile conditions.


The entire process is performed at 30°C. The pH and the sterllfty of the process are montored ftom the beginning until the end of this stage. The monitoring of the substrate (AD) and Ihe product (ADD) is tnittsted after 15 hours. DetaDs on the anaiyScat procedures (HPLC, GC) are g(ven in section 4.1. Results from the monitoring of the process are shown In sectiwi 4.2. The pH of the biotransformation mixture usually varies within 1 pH unit and is not corrected during the process. The biotransfomiatton is considered comF^eted when maximal product (ADD) concentration in the foam layer is achieved or when the substrate (AD) concentration reaches 1/20-th of its Initial concentration. The process is terminated by sterilization (lai'C, 30 min).
EXAMPLE 2 ONE STAGE BIOTRANSFORMATION OF AD TO ADD WtTH FUSAKIUM SOLANI
Preparation of Seed Culture of Fusarium solan!
The fungai culture Fusarium sotani is propagated In a liquid medium (10 g/L com steep liquor, 10 g/L glucose, pH 6.5). E rlenmeyer flasks (1 L volume, 0.2 L m edium) a re inoculated from culture grown on oatmeal agar for 120 hours and at SCC. The liquid medium culture is propagated on a rotary shatter (26°C, 120 rpm, 1" throw) for 72 liours.
Fusarium can also t>e sub-cultured from liquid to liquid medium for up to 4 - 5 generations or until the next culture becomes pink.
The shake flask inoculum could be stored at 4°C for up to 1 month without significant change In Its activity.
Preparation of Biotransformation Medium fbr Fusarium solanl
This process is perfomied utilizing already isolated AD.
The composition of the trfotransfomriation medium is as fisliows:
medium: bjotransfomnatton medium
(for 3 Liters of medium)
30 g glucose (10%)
30 mL corn steep liquor (10% v/v)
substrate: 30.0 g AD (1.0%)
emulsifler 480 mL sunflower oil (16%)
In a flaaK or a beaker water (2.6 L) and the medium components (except the AD and the sunflower oil) are mixed. The pH of me medium is adjusted to 6.5 with 1 M NaOH solution (40 g/L I>JaOH), the medium is transfenwl to the bioreactor and tiien heated to 60-70'C.
AO (30 g) is weighed in a beaker, sunflower oil (4B0 ml) is added and the mixture is stirred under heating on a hot plate until the oil becomes clear. The dear yellow AD

solution in oi) is poured into the already warmed liquid medium (eO-TO'C) in tfie bioroactor without stirring.
The fermentor Is sterilized (121 "C for 30 min) and cooled to SO'C.
Blotranstbrmatlon of AD to ADD tn a Stalntess Steel Fermentor
The bloconversion experiments were performed in stainless steel fermentors with efficient bottom stinring. The main parameters of the utilized fermentors are shown In Table 4. The temperature of the medium In the femientor Is adjusted to SCC and the inoculum Is introduced under sterile conditions. The entire process is performed at SO'C, The pH and the sterility of the process are monitored from the beginning until the end of this stage. The monitoring of the substrate (AD) and the product (ADD) is initiated after 15 hours. Details on the analytical procedures (HPLC.GC) and results are given below. The pH of the biotransformation mixture usually varies within 1 pH unit and is not con-ected during the process. The biotransformation Is considansd completed when maximal product (ADD) concentraHon in the toam layer is achieved or when the substrate (AD) concentraSon reaches 1/20-th of its initial concentiBtion.
The process Is terminated by sterilization [121 "C, 30 mIn).

The fermentation broth is han/sstsd Into a separatoiy funnel. Ethyl acetate (6.0 L) is added to the broth and shaKen for 2-3 mln. After 15 min the mixture is separated into two layers (ethyl acetate upper layer and aqueous bottom layer). The ethyl acetate layer is removed and transfered to another vessel. The bottom aqueous layer is extracted with a second portion of ethyl acetate (4.0 L), then the upper (ethyl acetate) layer is removed and combined wHti the first ethyl acetate extract. The combined ethyl acetate extracts are concentrated on a rotary evaporator to give a red thit^ oil (-450 ml) which Is used for a further purification.

Each ethyl acetate extract and the aqueous residue are analyzed for steroids by HPLC. The results for two st^e process age shown In Table 5 and the results for one stage process are shown in Table 6.
Partltionlnfl of ADD from the Oil
The oily residue {-450 ml) is mixed well wfth hexanes (1.25 L). The oil-hexanas solution is extracted with MeOH (2.0 L « 2). The two methanol extracts are combined and evaporated until dry to give a beige oily residue. The residue Is vacuum dried. Eachofthe hexanes and mrthanol layers is anafyzeti by HPLCtocontnjIthecompletion of the partitioning.
^ Crystallization of ADD from the Two Stage Fermentation of Phytosterols to ADO First Cryatall&eatlon
in some experiments the residue after the evaporation of methanol is vacuum dried. If so, this residue (-40-50 g, ADD content 17-18 g, purity 3040 % by HPLC) is dissolved in ethyl acetate (100 ml).
If the residue after the evaporation of methanol was not vacuum dried then this residue Is dissolved In ethyl acetate (250 ml), dried over Na2S04 and concentrated to a snnaller volume (100 ml).
Charcoal (4.0 g) is added and iha mixture is refluxed for 1 hr. After cooling, the mixture is ^fered on a sintered glass funnei and the filter Is washed with ethyl acetate. Alternatively, the solution can be filtered througf) a Cellte pad and washed with ethyl acetate. The filtrate Is concentrated on a rotary evaporator until dry. Hexanes (200 m!) are addad, the mixture is stin^d under reflux for 20 min, cooled to roam temperature and left for 30 min to separate. TTie hexanes upper layer Is transferred to another vessel and ailowedtostandovemightforcrystallfaifion.. The ADD cryatete from the hexanes solution are filfered, washed with hsxanes/EtOAc (3/1) and dried under the reduced pressure to give white ADD cn'stala (0.23 g)- The sticky oli is dissolved in ethyl acetate (8ml) and stinBd under heating for 10 min. The solution is cooled to room temperature and allowed to stand overnight The ADD crystals are filtered, washed with hexanes^EtOAc (3/1) and dried underthB reduced presaureto give white ADD cry6te(8(2.51 g).. The crystals and the mother liquor are analyzed by HPLC.
Second Crystallization
The nuther liquid left after ttie flrst crystallization Is concentrated on a rotary

evaporator. Ethyl acetate (4 ml) is added, the mixture is stirred under reflux for 10 min. cooled to room temperature and left overnight for crystallization. The ADD crystals are filtered, washed with hexanes/EtOAc {3/1) and dried under reduced pressure to give white ADD oystals (1.25 g). The product and the mottier liquor are analyzed by HPLC.

Thinl Crystallizsfion
The mother liquid left after the second crysfalllzation is concentrated on a rotary evaporator. A mixture ofethyj acetate (2 ml) and hexanes(1 mL) Is stirred under reflux for 10 min, cooled to room temperature and left overnight fbrcrystallizatton. The ADD crystals are filtered, washed with hexanes/EtOAc (3/1) and then dried under reduced pressure to give white ADD crystals (0.16 g). The pnxluct and the mother liquor are analyzed by HPLC.
Total amount of crystallized ADD is 4.25 g.
B) Isolation of ADD from the One Stage Fennentation of AD to ADD First CrystalllzaUon
If the residue aft«-the evaporation of methanol was vacuum dried, then this residue (-40-50 g, ADD content 17-18 g, purity 30-40 % by HPLC) Is dissolved in ethyl acetate (150 ml).
If the residue after the evaporation of methanol was not vacuum dried, then this residue is dissolved in eth^ acetate (250 ml), dried over Na2S04 and concentrated to a smaller volume (150 ml).

Charcoal (4.0 Q) IS added and the mixture is refluxed for 1 hr. After cooling, the mrxfejre is filtered on a sintered glass funnel and the filter is washed with ethyl acetate. Alternatively, the solution can be filtered through a Celite pad. The filtrate is concentrated on a rotary evaporator until dry, then the residue Is dissolved In ethyl acetate {6 ml) under heating and cooled to room temperature for crystallization. After standing overnight the ADD crystals are filtered, vrashed with haxanes/EfOAc (3/1) and dried under reduced pressure to give white ADD crystals. The crystals and the mother liquor are analyzed by HPLC.
Second Crystallization
The mother liquid left after the first cryatallization is concentrated on a rotary evaporator. Hexanes (50 ml) are added, the mixture Is stimed under reflux for 1 h, cooled to room temperature and left for 30 min to separate. The hexanes upper layer is transferred to another vessel and allowed to stand overnight ftar crystallization. The sticky oil Is mixed with ethyl acetate (2ml), hexanes (1 ml) and stin-ed under heating for 10 min. The solution is cooled to room temperature and allowed to stand overnight. The ADD crystals are filtered, washed with hexanes/EtOAc (3/1) and dried under the reduced pressure to give white ADD crystals. The hexanes layer also gave one portion of ADD crystals. The crystals and the mother liquor are analyzed by HPLC.
Third Crystallization
The mother liquor frtm the second crystallization Is concentrated on a rotary evaporator. The sticky oil is mixed with eth^ acetate (lml), hexanes (3mi) and stirred under heating for 10 min. After cooling, the solutnn is allowed to stand overnight for crystallization. The ADD crystals are filtered, washed with hexanes/EtOAc (3/1) and dried uruler reduced pressure to give ADD crystals. The crystals and the mother liquor are analyzed by HPLC. The results from ADD crystelUzatkin are shown in Table 6.
Table 6. Results from Extraction, Partitioning and Crystallization of ADD from One Stage Process.




EXAMPLE S QUALrTATFVE (TLC) AND QUANTITATIVE (HPLC AND QC) ANALYSIS Quairtativft analysis by TLC
Broth samples (approximately 40 mL each) are taken from the bottom of the fermentor. The broth sample {0.5 mL) is extracted wtth EtOAc (0.5 mL). TLC analysis was carried out on siHca gel pro-cMrted plates [60 F254. thickness 0.2 mm) using 0.050 mL of the EtOAc extract Mobile phase; fiexanes/EtOAc = 3.1; ctilorofbmi/acetone=3;1, ormethylene chloride/acetone - 9:1 were utHized. After developing the plate, one of the following spray (or dip) raagents was used.
Cen-ium reagent containing (for 100 mL): water (94 mL). cerium (IV) sulphate {1.0 g), phosphomolybdic acid hydrate (2.5 g), sulphuric add (6 ml). The components are dissolved in the order of listing. The reagent can be stored for more than 1 year.
Molibdats reagent containing (for 100 mL): water (90 mL), ammonium molitxiate (10.B5 g), sulphurtc add (6 ml).). The components era dissolved in the order of listing. The reagent can be stored for more than 1 year.
The plate was then heated for 3 minutes at 150°C. The resulting blue spots signified the presence of the following compounds (hexanes/EtOAc = 3/1): Rf" 0.75-.sunflowBr oil; Rf = 0,53-phylDsterDl; Rf = 0.4T-androstenedione(AD); and Rf - 0.02-androstadlenedlons (ADD).
Monitoring of AD, ADD and Byproducts by HPLC
A sample (2 g foam or 2 mL liquid or 10 mg crystals) was diluted to 50.0 mL with me^anot, mbwd well and left for 10 min to precipitate the proteins and phytoBterols. An aliquot of the solution was filtered (Miliipore fitter type HV 0.45 ]im). HPLC analysis was perfomied using radialpack column (stationary phase Cts 5^m, 4 x 100 mm. Waters). Mobile phase: water/metianols ratio 4:6 (Isocratic). F)ow1miym1n. Detection at2S0nm.
StarWlard curve for AD (mg) = (8.39 Area + 2033) x 10 '^; in which the area ranges from 0 to 30000.
Standard curve for ADD (mg) = (7.43 Area + 72.22) x 10 "*; in which the area ranges from 0 to 100000.
Standard curves were obtained from the results for pure samples of androstenedione (AD) and androstadlenedione (ADD). The retention times of the products are as follows.
Compound Retention time (mIn)

AD 8.73
ADD 13.20
Monitoring the BUbstratee and products by GC
The sample (50 mg crystals] is dissolved in EtOAc and diUuted to 50.0 mL in a volumetric flask. Altematively, 2 mL biotransformation mixture is extracted wrfth 50 mL EtOAc. The EtOAc solution fs then analyzed for Phytosterols, AD and ADD by GC.
Conditions: SAC-S column, oven temperature 278 "C, injector temperature 300 °C, detector FID. The retention times of the products are as follows.
Compound Retention time (min)
AD 8.06
ADD 9.21
Brassicasterol 20.53
Campesterol 23.20
Campestanol 23.66
Stigmasterol 24.70
p-Sitosterol 27.71
SRostanol (Stigmastanol) 28.27




EXAMPLE 6 IDENTIFICATION OF NEW MUTANT F. 80LANIFMI33
Protocol was used to generate large subunit (LSU)rRNA gene sequence data. Approximately 300 base pairs of the LSU rRNAgene starting at position 3344 was PCR amplified from genomicONAisolatedformtheFMI33fungalcok>nies. Amplication products were purified from excess primers and dNTPs using Microcon 100 (Amicon) mt^ecutar weight cut-off membranes and checked for quality and quantity by rinning a portion of the products on an agarose gel.
Cyde sequencing of the LSU ri?NA amplification products was carried out using AmpllTaq FS DMA polymerase and dRhodamine dye terminators. Excess dye-labeled terminators ware removed from the sequencing reactionfi using s Sephadex G-50 spin column. The products were collected by centrtfugation, dried under vacuum and frozen at -20°C until ready to load. Samples were resuspended in a solution of formamfde/blue dextran/EDTA and denatured prtor to loading. The samples were electrophoresed on a ABI Prism 377 DNA sequencer. Data was analyzed using PE/Applted Blosystems DNA Editing and assembly softuare.
Comparisons were made between sample FMI 33 and other microorganisms in MlcroSeq database using microbial analysis software. FMI 33 was found to have a genetic distance from the next doses! match, F. so/an/, of 0.94%, which is indicative of a separate fungal mutant.


WE CLAIM:
1. A process of fermenting a phytosterol composition to produce androstenedione
{androst-4-ene-3, I 7-dione) and subsequently androstadienedione (androsta-
1,4- diene,3,I 7-dione) which comprises:
propagating a microbial culture of the genus Mycobacterium in a first nutrient
medium;
placing the microbial culture and the phytosterol composition in a bioreactor
for a sufficient time to transform the composition substantially to
androstenedione (the "AD solution");
propagating a fungal culture of the genus Fusarium in a second nutrient
medium;
injecting one or more of I) Fusarium sp; and 2) the fimgal culture medium into
the bioreactor for a sufficient time to transform the AD solution to
androstadienedione.
2. The process as claimed in claim I, wherein the fungal culture is Fusarium solani.
3. The process as claimed in claim 1, wherein the fimgal culture is Fusarium oxysporum.
4. The process as claimed in claim 1, wherein the microbial culture is Mycobacterium MB 3683.
5. The process as claimed in claim I, wherein the fimgal culture is Fusarium solani FMI33, deposited at ATCC as PTA-3947.

6. The process as claimed in claim i, wherein tlie phytosterol composition is derived from tall oil pitch or pulping soap.
7. The process as claimed in claim 1, wherein the phytosterol composition is derived from a suitable selected vegetable oil.
8. The process as claimed in claun 7, wherein the vegetable oil is selected from the group comprising soy, rapeseed, com, cottonseed, sunflower, olive, linseed, or rice bran.
9. The process as claimed in claim 1, wherein the phytosterol composition comprises one or more of the following: sitosterol, campesterol, stigmasterol, brassicasterol, desmosterol, chalinosterol, poriferasterol, clionasterol, all hydrogenated counterparts and all natural or synthesized forms and derivatives thereof, including isomere.
10. The process as claimed in claim 1, wherein the first nutrient medium comprises Refiners molasses and inorganic salts.
11. The process as claimed in claim 1, wherein the second nutrient medium comprises glucose and com steep liquor.
12. The process as claimed in claim 1, wherein the fermentations are carried out aerobically.
13. The process as claimed in claim 1, wherein the phytosterol composition is dissolved into a solution using a solubilizing agent and then added to the bioreactor.

14. The process as claimed in claim 13, wherein the solubilizing agent is a member
of the glycol family.
15. The process as claimed in claim 13, wherein the solubilizing agent is
polypropylene glycol.
16. The process as claimed in claim 13, wherein the solubilizing agent is a member of the silicone family.
17. The process as claimed in claim 1, wherein the bioreactor is sterilized and then cooled prior to the injection of 1) the fiingal culture or 2) the fimgal culture medium.
18. Fusarium solani FMI 33, deposited at ATCC as PTA-3947.


Documents:

1677-chenp-2004 abstract-duplicate.pdf

1677-chenp-2004 abstract.pdf

1677-chenp-2004 assigment.pdf

1677-chenp-2004 claims-duplicate.pdf

1677-chenp-2004 claims.pdf

1677-chenp-2004 complete specification-duplicate.pdf

1677-chenp-2004 complete specification.pdf

1677-chenp-2004 correspondences others.pdf

1677-chenp-2004 correspondences po.pdf

1677-chenp-2004 drawings-duplicate.pdf

1677-chenp-2004 drawings.pdf

1677-chenp-2004 form-1.pdf

1677-chenp-2004 form-18.pdf

1677-chenp-2004 form-26.pdf

1677-chenp-2004 form-3.pdf

1677-chenp-2004 form-5.pdf

1677-chenp-2004 form-6.pdf

1677-chenp-2004 others.pdf

1677-chenp-2004 pct search report.pdf

1677-chenp-2004 pct.pdf

1677-chenp-2004 petition.pdf


Patent Number 229009
Indian Patent Application Number 1677/CHENP/2004
PG Journal Number 12/2009
Publication Date 20-Mar-2009
Grant Date 13-Feb-2009
Date of Filing 29-Jul-2004
Name of Patentee N.V. ORGANON
Applicant Address KLOOSTERSTRAAT 6, NL-5349 AB OSS,
Inventors:
# Inventor's Name Inventor's Address
1 KUTNEY, JAMES, P 403-1777 BAYSHORE DRIVE, VANCOUVER, BRITISH COLUMBIA V6G 3H2,
2 HERRINGTON, EDWARD, J 3894 WEST 11TH AVENUE, VANCOUVER, BRITISH COLUMBIA V6R 2K9,
3 SPASSOV, GRIGOR 207-1749 ROBSON STREET, VANCOUVER, BRITISH COLUMBIA V6G E1E,
PCT International Classification Number C12P 33/16
PCT International Application Number PCT/CA03/00131
PCT International Filing date 2003-02-03
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 10/066,395 2002-02-01 U.S.A.