CONVERSION OF ISOLEUCINE TO 4-HYDROXYISOLEUCINE BY MICROORGANISMS

FIELD OF INVENTION

[0001] This invention relates to the field of conversion of isoleucine to 4-hydroxyisoleucine by microorganisms.

BACKGROUND OF THE INVENTION

[0002] 4-hydroxyisoleucine is a polar, non-charged amino acid not found in mammalian tissues but present in other biological materials, for example, the highly toxic γ-amanitin peptide produced by the Amanita phalloide mushroom as well as in some specific plants, especially Tήgonella species including, for example, T. foenum-graecum (commonly known as fenugreek) an annual herbaceous plant widely distributed across Asia, Africa, and Europe.

[0003] 4-Hydroxyisoleucine (4-HIL) may comprise any or all of eight possible configurational isomers having the following absolute stereochemical configurations: (2S.3K.4S), (2R,3R,4S), (2S.3RAR), (2S,3S,4R), (2R,3S,4R), (2R3S.4S), {2R,3R,4R), (2S,3S,4S) as well as salts and/or the lactone forms of such isomers.

[0004] 4-hydroxyisoleucine accounts for about 80% of the total content of free amino acids in fenugreek seeds (Sauvaire et al., Herbs, Botanicals and Teas (2000), Edited by G. Mazza and B. P. Oomah, p. 107-129). In the fenugreek seed, 4-hydroxyisoleucine is mainly present as two diastereoisomers: the major one having the (2S,3f?,4S) configuration (approximately 90% of the total amount) and the minor one (approximately 10%) having the (2R,3R,λS) configuration.

[0005] In fenugreek, the major (2S,3R,4S) isomer of 4-HIL has been shown to have insulinotropic and insulin sensitizing activities (Broca et al., Am. J. Physiol. 277:E617-E623, 1999; Broca et al., Eur. J. Pharmacol. 390:339-345, 2000; Broca et al., Endocrinol. Metab. 287:E463-E471 , 2004) and this compound has been developed for the treatment of diabetes (PCT publication Nos. WO 97/32577 and WO 01/15689). 4-hydroxyisoleucine shows great potential as both a therapeutic agent and as an ingredient in health foods.

[0006] Extraction of 4-hydroxyisoleucine from fenugreek seeds can require very large masses of the seeds and large volumes of solvents and water which can be costly and can lead to waste disposal problems which can further increase the cost of the extraction process. There is thus a need for alternative methods for obtaining 4-hydroxyisoleucine. The present invention seeks to meet this and other needs.

[0007] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

[0008] The present invention relates to a method for the conversion (converting) of isoleucine to 4-hydroxyisoleucine by a microorganism. The method may comprise culturing a microorganism in medium comprising isoleucine. The method may further comprise detecting 4-hydroxyisoleucine. The method may also further comprise the step of isolating and/or substantially purifying 4-hydroxyisoleucine. Isoleucine may initially be present in the medium (present and/or added at the time of inoculation of a microorganism in medium). Alternatively, isoleucine may also be added at a later time following inoculation. In addition, isoleucine may be continuously and/or repeatedly added. The present invention also relates to the use of a microorganism for converting isoleucine to 4-hydroxyisoleucine. The present invention further relates to a method of identifying a microorganism capable of converting isoleucine to 4- hydroxyisoleucine comprising culturing a microorganism in medium containing isoleucine and/or detecting 4-hydroxyisoleucine.

BRIEF DESCRIPTION OF DRAWINGS

[0009] In drawings which illustrates non-limitative exemplary embodiments of the present invention,

[0010] Figure 1 shows exemplary embodiments of isoleucine and 4-hydroxyisoleucine described herein, including the lactone form; and

[0011] Figure 2 shows yield quantification of microorganism cultures which converted isoleucine to 4-hydroxyisoleucine .

DETAILED DESCRIPTION

[0012] In the present description, the terms 4-hydroxyisoleucine and 4-HIL are used interchangeably.

[0013] The present invention relates to a method for converting isoleucine to 4- hydroxyisoleucine by a microorganism. The method may comprise cultuhng a microorganism in medium which contains isoleucine, for example, L-isoleucine, D-isoleucine and/or allo- isoleucine (isoleucine is meant to include any stereoisomers of isoleucine). According to the present invention, the isoleucine may be a mixture of L-isoleucine, D-isoleucine and/or allo- isoleucine and such mixture may be a non-racemic or a racemic mixture of L- and D- isoleucine. Isoleucine may initially be present in the medium and/or may be added to the medium. Isoleucine may also be continuously and/or repeatedly added to the medium. The method may further comprise the steps of detecting 4-hydroxyisoleucine and/or isolating 4- hydroxyisoleucine. The method may further comprise a step of purifying and/or substantially purifying 4-hydroxyisoleucine.

[0014] A microorganism of the present invention may be a bacterium and/or a fungus. As used herein, the term "bacterium" includes archaebacterium and eubacterium (Woese, C. R., et al., Proc. Natl. Acad. Sci. USA 87:4576-4579 (1990)). Exemplary eubacterium may be a Gram-negative and/or a Gram-positive organism. Exemplary embodiment of eubacterium encompassed by the present invention may include the genus Streptomyces. Exemplary species may be, for example, but without limitation, Streptomyces albus, and/or Streptomyces rimosus. Exemplary strains may include for example Streptomyces albus BCC003, and Streptomyces rimosus BCC073. BCC strains are from Biocatalytics Inc. (129 N. Hill Avenue, Suite 103, Pasadena, CA, 91 106 USA).

[0015] Exemplary fungi encompassed by the present invention may include the genera Neurospora, Rhizopus, Beauveria, Syncephalastrum, Mortierella, Umbelopsis, Gluconobacter oxydans, Cunninghamella, Aspergillus, Mucor, Rhodotorula, Syncephalastrum and/or Phanerochaete. Exemplary fungi species may be, for example, but without limitation, Neurospora crassa, Rhizopus arrhizus , Beauveria bassiana, Syncephalastrum racemosus, Mortierella isabellina, Umbelopsis vinacea, Gluconobacter oxydans, Cunninghamella echinulata, Aspergillus ochraceus, Mucor ramannianus, Rhizopus

stolonifer, Rhodotorula mucilaginosa and/or Phanerochaete chrysosporium. Exemplary strains of fungus encompassed by the present invention may include, for example, Aspergillus ochraceus strain ATCC 22947, Cunninghamella echinulata strain ATCC 36190, Mucor ramannianus strain 9628, Rhizopus stolonifer strain ATCC 24795, Rhodotorula mucilaginosa strain ATCC 20129, Neurospora crassa BCC068 and BCC61, Rhizopus arrhizus BCC069, Beauveria bassiana BCC078, Syncephalastrum racemosus BCC082, Mortierella isabellina BCC109, Umbelopsis vinacea BCC114, Gluconobacter oxydans BCCOU, Cunninghamella echinulata BCC111, Aspergillus ochraceus BCC141, and/or Phanerochaete chrysosporium BCC149. The BCC strains were from Biocatalytics Inc. (129 N. Hill Avenue, Suite 103, Pasadena, CA, 91106 USA).

[0016] In accordance with the present invention, "culturing" means allowing a microorganism (bacterium and/or fungus) to grow and/or survive in medium. For example, allowing a microorganism to remain alive and/or healthy without necessarily being in a growing phase. The method of the present invention may comprise providing a microorganism with media comprising isoleucine for a sufficient period of time to allow the conversion of isoleucine to 4-hydroxyisoleucine and/or detecting 4-hydroxyisoleucine. llsoleucine may be provided to a microorganism in excess of the usual concentration used to allow for the growth and/or survival of a microorganism. Alternatively, a microorganism may be grown in medium, separated from that medium and subsequently contacted with isoleucine.

[0017] In accordance with the present invention, '"medium"' may be any liquid, semi-solid and/or solid preparation that allows the growth and/or maintenance and/or survival of a microorganism. Standard medium are known in the art and may include, for example, Luria- Bertani Broth, Mannitol Salt Broth, Trypticase Soy Broth, Brain Heart Infusion, Dubos Medium, Dextrose Blood Agar, Peptone-Yeast Extract Broth, Staphylococcus Broth, Mannitol Salt Agar, Thioglycolate Medium, Brewer Modified and Peptone Glucose Yeast Extract Agar. In an exemplary embodiment, the culture may be a liquid containing the necessary nutrients for growth of the microorganism and those additives which may also be necessary for enhancing the ability of the microorganism to perform the desired conversion. Such additives may include, but are not limited to, iron salts, ascorbic acid, vitamins, etc.

[0018] 4-hydroxyisoleucine may encompass any and/or all of eight possible configurational isomers having the following absolute stereochemical configurations:

(2S,3R,4S), (2R.3R.4S), {2S,3R,4R), {2S,3S,4R), (2R,3S,4R), (2R3S.4S), (2R,3R,4R), (2S.3S.4S), salts and/or the lactone forms of such isomers. In an exemplary embodiment of the present invention, 4-hydroxyisoleucine is in the (2S.3R.4S) configuration. In another exemplary embodiment of the present invention, 4-hydroxyisoleucine is in the (2S,3R,4R) configuration. Commonly the (2S,3ft,4S) isomer may be called 4-L-HIL, and the (2R,3S,4R) isomer may be called 4-D-HIL.

[0019] To increase the production of 4-hydroxyisoleucine and/or to assist in the isolation of 4-hydroxyisoleucine from the medium, and also to assist in further purification of the 4- hydroxyisoleucine, it may be useful to protect the isoleucine. The isoleucine of the present invention may therefore have a protecting group at the amino and/or carboxylic acid function thereof.

[002O]By "protecting group" it is meant a chemical moiety used to protect a functional group already present in isoleucine from undesired chemical reaction and/or salt formation. Isoleucine possesses two functional groups, an amine and a carboxylic acid, which may be protected by a number of known methods. In an exemplary embodiment of the present invention, the amine functional group of isoleucine is protected. Protecting groups for such a purpose are well known in the art and include those listed, for example, in Greene's Protective Groups in Organic Synthesis, 4th Edition. Any amine protecting group known in the art may be used. In an exemplary embodiment of the present invention, the amine- protecting group may be N-carboxyethoxy group, commonly abbreviated as 'E'. In another exemplary embodiment of the present invention, the amine-protection group may be N- benzyloxy group, commonly abbreviated as '71. The protecting group(s) may be removed after completion of the conversion by the microorganism by procedures well known to those skilled in the art. Exemplary drawings of E- and Z- protected L-isoleucine and hydroxyisoleucine are shown in FIGURE 1.

[0021] In the instance that a protecting group is used, the processes of isolating and/or purifying 4-hydroxyisoleucine may comprise a step for removing the protecting group. Such a step is typically termed a "deprotection step".

[0022] The present invention may further generate unprotected 4-hydroxyisoleucine from protected isoleucine in the case in which the microorganism possesses the ability to remove

the protecting group in addition to the ability to perform the desired conversion. The desired conversion, namely, the formal hydroxylation of the 4-position of the protected and/or unprotected isoleucine, may occur before or after the removal of the protecting group.

[0023] Detection of 4-hydroxyisoleucine and/or protected 4-hydroxyisoleucine may be performed by several means and/or processes known in the art. In an exemplary embodiment of the present invention, 4-hydroxyisoleucine and/or protected A- hydroxyisoleucine may be detected using liquid chromatography/ mass spectrometry) analysis (LC/MS). Furthermore, the product of the conversion may also be subjected to appropriate reaction conditions so as to convert any lactone form of 4-HIL into the corresponding linear form.

[0024] By "isolating steps" it is meant, without limitation, filtration of 4-hydroxyisoleucine, adsorption of 4-hydroxyisoleucine, extraction of 4-hydroxyisoleucine and/or other isolation methods such as, for example, chromatography and crystallization.

[0025] As used herein, the term "substantially purifying" or "substantially pure"' means that isolating steps are performed so as to obtain a product and/or a composition enriched in A- hydroxyisoleucine such as, for example, a composition having a content of A- hydroxyisoleucine in the range of about 10% to 100% such as 15%-100%, 20%-100%, 20%- 95%, 25%-95%, etc. A "range" as mentioned herein relates to and explicitly incorporates herein each and every specific member and combination of sub-ranges whatsoever. Thus, any specified range is to be understood as a shorthand way of referring to each and every member of a range individually as well as each and every possible sub-ranges encompassed therein.

[0026] The present invention also relates to the product obtained from a method for the conversion of isoleucine to 4-hydroxyisoleucine by microorganisms as described herein.

[0027] The present invention further relates to the use of a microorganism for the conversion of isoleucine to 4-hydroxyisoleucine.

[0028] The present invention further relates to a method of identifying a microorganism capable of converting isoleucine to 4-hydroxyisoleucine, the method which may comprise

culturing a microorganism in medium containing isoleucine and/or detecting 4- hydroxyisoleucine.

[0029] The following examples illustrate potential applications of the invention and are not intended to limit its scope. Modifications and variations may be made therein without departing from the spirit and scope of the invention.

EXAMPLES

Example 1 : Synthesis of Standards

[0030] L-isoleucine and Z-L-lsoleucine (isoleucine with a benzyloxy group) were purchased from Aldrich Chemical (St. Louis, MO). E-L-isoleucine (isoleucine with a carboxyethoxy group) was synthesized. The structure 4-HIL, E-L-isoleucine, Z-L-isoleucine, 4S-Hydroxy-E- He, 4S-Hydroxy-Z-lle and their corresponding lactone are shown in FIGURE 1. 4S-hydroxy-E- L-isoleucine and 4S-hydroxy-Z-L-isoleucine standards were synthesized. Quality control was performed on all compounds by NMR Spectra.

Synthesis of E-L-lsoleucine

[0031] In a 5OmL round bottom flask, L-isoleucine (2g, 17.6mmol) was dissolved in 18 mL of 2M sodium hydroxide solution, and the solution cooled to 0 ⁰ C. Ethyl chloroformate (2.1 g, 19.4mmol) was slowly added, and the reaction was allowed to stir at 0 ⁰ C for 3 hours, warmed to room temperature, and allowed to stir overnight. The reaction was quenched with 9mL of 5% ammonium hydroxide solution, and extracted three times with 10 mL ethyl acetate at pH 10. The aqueous layer was collected, acidified to pH 3 with 6M hydrochloride solution and extracted three times with 15mL ethyl acetate. The combined organics were washed with brine, dried with sodium sulfate, and concentrated by rotary evaporation to yield 3 g (86% yield) of the desired product.

Synthesis of 4S-hydroxy-E-L-isoleucine

[0032] In a 10 mL round bottom flask, 4S-hydroxy-L-isoleucine (200mg, 1.5mmol), sodium bicarbonate (435 mg, 3.1 mmol) and 1.6 mL of water were combined, and the solution cooled

to 0°C. Ethyl chloroformate (281 mg, 1.65 mmol ) was slowly added and the reaction was allowed to stir at 0 ⁰ C for 3 hours, warmed to room temperature and allowed to stir overnight. The reaction was quenched with 5% ammonium hydroxide solution, and extracted three times with 1 ml. of ethyl acetate at pH 10. The aqueous layer was collected, acidified to pH 3 with 6M hydrochloride solution and extracted three times with 2mL of ethyl acetate. The combined organics were washed with brine, dried with sodium sulfate, and concentrated by rotary evaporation to yield 240 mg (80%) of the desired product.

Synthesis of 4S-hydroxy-Z-L-isoleucine

[0033] In a 10 ml_ round bottom flask, 4S-hydroxy-L-isoleucine (200mg, 1.5mmol), sodium bicarbonate (435 mg, 3.1 mmol) and 1.6 ml. of water were combined and the solution cooled to 0°C. Benzyl chloroformate (282 mg, 1.65 mmol ) was slowly added, and the reaction was allowed to stir at O ⁰ C for 3 hours, warmed to room temperature and allowed to stir overnight. The reaction was quenched with 5% ammonium hydroxide solution, and extracted three times with 1 ml. of ethyl acetate at pH 10. The aqueous layer was collected, acidified to pH 3 with 6M hydrochloride solution and extracted three times with 2ml_ of ethyl acetate. The combined organics were washed with brine, dried with sodium sulfate, and concentrated by rotary evaporation to yield 240 mg (80%) of the desired product.

Example 2: Microbial conversion of isoleυcine to 4-HIL

[0034] It was surprisingly discovered that certain microorganisms were capable of converting isoleucine to 4-hydroxyisoleucine.

[0035] Glycerol stocks of all microbial strains were prepared from liquid cultures of the organisms. After five days growth at 26 ⁰ C, cultures were diluted with one volume of a sterile solution containing 100 g/L glycerol and 50 g/L lactose, dispensed into 1.5 ml_ sterile screw- cap microtubes and stored at -80 ⁰ C.

[0036] A screening culture was prepared in a 25 mL glass culture tube filled with 3 mL of growth media, topped with an autoclavable tube cap and autoclaved. Once the tube returned to room temperature, the tube was inoculated with 30 μl_ of glycerol stock of choice, and allowed to incubate with 200 RPM agitation at a 45 degree slant at 26°C. After three days, L-

isoleucine was added to the culture (1 g/L, from a 100 g/L ethanol stock), and incubated for five additional days. The culture was then quenched with one volume of ethanol, allowed to incubate for an additional hour to extract compounds from the cell mass, and centrifuged. The resulting supernatant was analyzed for the production (presence) of 4-hydroxyisoleucine by HPLC. Alternatively, growth medium for Streptomyces albus and Gluconobacter oxydans was composed of 10 g/L malt extract, 4 g/L yeast extract, 4 g/L glucose, all dissolved in deionized water, pH-adjusted to 6.0 with 1 M sodium hydroxide or 1 M hydrochloric acid, and sterilized by autoclaving. Growth medium for Streptomyces rimosus was composed of 5 g/L yeast extract, 5 g/L soy peptone, 20 g/L glucose, 5 g/L sodium chloride, 5 g/L monobasic potassium phosphate, all dissolved in deionized water, pH-adjusted to 7.0 with 1 M sodium hydroxide or 1 M hydrochloric acid, and sterilized by autoclaving. Growth medium for Aspergillus ochraceus, Phanerochaete chrysosporium, Beauveria bassiana, Syncephalastrum racemosus, Mortierella isabellina, Umbelopsis vinacea, Neurospora crassa, and Rhizopus arrhizus was composed of 10 g/L corn steep liquor, 30 g/L glucose, 1 g/L monobasic potassium phosphate, 2 g/L dibasic potassium phosphate, 2 g/L sodium nitrate, 0.5 g/L potassium chloride, 0.5 g magnesium sulfate heptahydrate, 0.02 g ferrous sulfate heptahydrate, all dissolved in deionized water, pH-adjusted to 6.5 with 1 M sodium hydroxide or 1 M hydrochloric acid, and sterilized by autoclaving.

[0037] Eight fungal or bacterial species (Streptomyces albus, Neurospora crassa, Rhizopus arrhizus, Streptomyces rimosus, Beauveria bassiana, Syncephalastrum racemosus, Mortierella isabellina, Umbelopsis vinacea) grown in medium containing Z-L-isoleucine resulted in the formation of a chromatographic peak with the same retention time as the (2S,3R,4S)-hydroxy-Z-isoleucine standard, with a m/z of 264. Peaks corresponding to the lactone form as well as other potential hydroxyisomers of 4-hydroxy-Z-L-isoleucine were also detected. Six of the bacterial strains exceeded 5% product formation, with strain Syncephalastrum racemosus producing approximately 45% desired product with 90% specificity.

[0038] Twelve fungal or bacterial species/strains (Streptomyces albus, Neurospora crassa- BCC061, Neurospora crassa-BCC068, Streptomyces-BCC070, Streptomyces rimosus, Gluconobacter oxydans, Beauveria bassiana, Mortierella isabellina, Cunninghamella echinulata, Umbelopsis vinacea, Aspergillus ochraceus and Phanerochaete chrysosporium) grown in medium containing E-L-isoleucine resulted in the formation of a chromatographic peak with the same retention time as the (2S,3/?,4S)-hydroxy-E -isoleucine standard, with a

m/z of 218. Peaks corresponding to the lactone form as well as other potential hydroxyisomers of 4-hydroxy-E-L-isoleucine were also detected. Among these strains, six exhibited product formation exceeding 5%.

[0039] TABLE 1 summarizes the product distribution of six strains for isoleucine conversion as well as the observed m/z for the analytes. The BCC# correspond to the strain number from Biocatalytics (Biocatalytics Inc. 129 N. Hill Avenue, Suite 103, Pasadena, CA, 91106 USA). BCC3: Streptomyces Albus, BCC61 : Neurospora crassa, BCC68: Neurospora crassa, BCC73: Streptomyces rimosus, BCC78: Beauveria bassiana, BCC82: Syncephalastrum racemosus, BCC109: Mortieralla isabellina, BCC114: Umbelopsis vinacea, BCC141 : Aspergillus ochraceυs.

TABLE 1 CONVERSION OF E-L-ISOLEUCINE AND Z-L-ISOLEUCINE

E-lsoleuciπe Microbial Screen Preliminary Results

Conversion Scale-Up Cultures

[0040] Conversion experiments were run in 50 mL scale-up cultures. A baffled 250 mL glass Erlenmeyer flask was filled with 50 mL of growth media, topped with a polyester foam stopper followed by aluminum foil and autoclaved. Once the flask returned to room temperature, the flask was inoculated with 0.5 mL of glycerol stock, and allowed to incubate with 200 RPM agitation at 26 °C. After three days, L-isoleucine was added to the culture (1 g/L, from a 100 g/L ethanol stock), and incubated for five additional days. The culture was

quenched by pH adjustment to 3.0 with hydrochloric acid and immediately extracted with three volumes of ethyl acetate. The extracts were evaporated to dryness and reconstituted with 5 ml_ methanol containing 0.2% triethylamine.

[0041] The samples were treated to deprotect and convert any lactone form into the corresponding open form as follows:

CBZ Removal

[0042] To a 2 mL of liquid content from mother solution in a 5 mL round bottom flask was added 30 mg of Palladium, 10 wt % (dry) on carbon powder, wet (Aldrich Batch # 04115MB) and a filled balloon of hydrogen. Reaction was monitored on thin layer chromatography (TLC) and stopped when starting material (CBz Lactone) was consumed (16h) and then the reaction mixtures were filtered through a pipet with cotton and celite (2mm) in order to filter off palladium, rinced 2x with methanol and recovered filtrate in a 4 mL DRAM vial. In the DRAM vial charged with LiOH (250 uL of 1M solution) was added the content of the previous step with stirring at room temperature (1-2h, based on TLC monitoring of disappearance of lactone). Then the mixtures were lyophilized overnight and re-dissolved with methanol to their original concentration after which they were analyzed for their 4-HIL content.

Ethyl Carbamate Removal

[0043] 2 mL of sample were placed in the culture tube and was evaporated under nitrogen. The samples were then subjected to E group hydrolysis. To the above samples (0.2 mL) HBr-AcOH (33%) were added at O ⁰ C. The samples were then allowed to stir at room temperature for 1 hour. The reaction mixture was diluted with 2 mL of ethyl acetate and was evaporated under nitrogen for 2 hours. To each of the sample, 2 mL of MeOH was added, followed by 0.2 mL of 1 N-LiOH. The reaction mixtures were allowed to stir overnight (pH=8- 10). All the samples were evaporated under nitrogen and were diluted with 2mL of MeOH. Samples were filtered through 0.2μm filter and were submitted to chiral HPLC. The deprotected samples solutions were diluted in methanol and 10 μL of internal standard working solution of l-leucine-d3 was added to the solution to monitor any possible drift in retention

Chromatographic Conditions

[0044] Analytical Column : Chirobiotic T 250*4.6 mm 5μm

Column Temperature : 2O ⁰ C

Injection volume : 5μL

Mobile Phase A : 80% Ethanol

B : 20% 20 mM Ammonium Acetate pH 4.0

Flow rate : 0.5 mL/min

Retention time

SRR : 13.7 min

SRS : 15.5 min

SSR : 19.3 min

SSS : 23.5 min

RSS &RSR : 26.2 min

RRS : 31.6 min

RRR : 39.8 min l-leucine-d3 (IS) : 14.1 min

Split : 1 in 2

Mass Spectrometric Conditions

Source : Electrospray

Ionization mode : Positive

Tune file : atune.tun

Drying gas : 12.0L/min

Drying gas Temperature : 350 ⁰ C

Nebulizer Pressure : 35 psig

Capillary Voltage : 3000V

Fragmentor : 70V

SIM (m+H)+ isomers : 148.1 amu

IS : 135.1 amu

Z-L-lsoleucine conversion results in scale-up cultures are shown in TABLE 2 and E-L- lsoleucine conversion results in scale-up cultures are shown in TABLE 3.

Table 2 Z-L-lsoleucine Conversion

TABLE 3 E-L-ISOLEUCINE CONVERSION

E derivative

[0045] The rate of conversion to the (2S,3R,4S)-HIL isomer expressed as a function of starting isoleucine material varied between 0.5 to 16%. The two best strains were

Neurospora crassa with 8% conversion and Syncephalastrum racemosus with 16% conversion.

[0046] The rate of conversion to the isomer (2S,3R,4S)-HIL expressed as a function of starting isoleucine material varied between about 1 and 20%. The best strain for E-L- isoleucine conversion was Beauveria bassiana with about 20% conversion. Also, unprotected isoleucine was converted to 4-HIL by Cunninghamella echinulata.

Example 3: Further Fungal Conversion

[0047] Further fungal strains were tested for the conversion of isoleucine to 4-HIL. Standard conditions were developed for detection of L-isoleucine and (2S,3fi,4S)-hydroxy-isoleucine at minimal concentrations on a LC/MS-Bruker MicroTOF instrument equipped with an Agilent 1100 series LC instrument (Agilent Technologies, Inc.) with electrospray ionization in positive mode using the following conditions: 10μl injection volume on a Phenomenex-Luna-3μ-C8- 150X4.6cm, elution with a gradient system of 95:05; WaterAcetonitrile at 0.4ml_/min over 10 minutes.

Liquid Chromatography (LC)-Conditions

[0048] Mobile Phase : water (0.1% HCOOH) and Acetonitrile (0.1% HCOOH)

[0049] Column : Phenomenex-Luna-3u-C8-150 x 4.6cm

[0050] Run Time : 10 minutes

[0051] Elution : lsocratic 95:5; Water: Acetonitrile

[0052] Flow rate ; 0.4mL/min

[0053] Injection Volume : 10μl

Mass spectrometry Conditions

[0054] Ionization technique : Electrospray [0055] Ionization mode : Positive

Fungal Strains Preparation

[0056] Fungal isolates were initiated from frozen stock cultures and plated onto appropriate agar slants solid medium (either potato dextrose agar -PDA-, yeast extract agar -YEA- or malt extract agar -MEA-) and grown for an average of 7 days.

Fermentation Initiation

[0057] Fermentation was initiated by inoculating liquid cultures from previously prepared agar slants. All remaining agar slants were either sealed with parafilm wax and stored at 4 ⁰ C or were used to prepare glycerol stock cultures to be stored at -80 ⁰ C.

[0058] Liquid culture for all fungal isolates consisted in a complex medium comprising the following: 2Og dextrose, 5g NaCI, 5g K ₂ HPO4, 5g Difco bacto peptone, 5g Difco yeast extract, 1 L distilled water.

[0059] All fermentations were conducted on a 25ml_ scale using 125ml_ flasks and a previously described two-stage fermentation procedure (Herath et al. 2003):

Stage 1 : growth at room temperature for 3 days

Stage 2: Inoculation of fresh medium with first stage growth culture at day 3 and incubation for an additional period of time (e.g. up to 14 days).

lsoleucine Addition

[0060] L-isoleucine was added to stage 2 cultures following the first three days of stage 1 culture and incubated for a further period of 14 days.

Conversion Monitoring

[0061] Conversion was monitored at day 3, 7 and 14 following L-isoleucine addition. 5mL of supernatant was sampled at each interval with the entire culture (including mycelia) harvested at day 14. All samples were stored frozen at -20 ⁰ C until analysis.

[0062] All frozen culture samples were freeze-dried to remove all water. Lyophilized samples were extracted at 3 days and 7 days with 5.0 ml. and at 14 days with 15ml_ of distilled water. The water soluble extract was sonicated at room temperature for 15 minutes and filtered using a Millipore syringe driven filter unit (0.2pM). All samples were analyzed by LC/MS for 4-hydroxyisoleucine production.

4S-Hvdroxy-L-isoleucine Conversion Test

[0063] To assure that the fungal isolates were not metabolizing the product after conversion, cultures were initiated using R. stolonifer (ATCC 24795) and M. rammanianus (ATCC 9628) in which the fungi were fed L-isoleucine as well as (2S,3R,4S)-hydroxy-L- isoleucine on day 4. The amount of 4-hydroxyisoleucine recovered was in the range of 90- 93% showing that the strains did not metabolize (2S,3R,4S)-hydroxy-L-isoleucine following production by fungus. The results are presented in FIGURE 2 in which the % yield correspond to the rate of conversion from isoleucine to 4-HIL

[0064] It was discovered that the following fungi were able to convert isoleucine into 4- hydroxyisoleucine: (the number in parenthesis correspond to the American Type Culture Collection-ATCC number) Rhodoturla mucilaginous (20129), Aspergillus ochraceus (22947), Rhizopus stolonifer (24795) and Mucor ramannianus (9628).

[0065] Although the present invention has been described by way of exemplary embodiments, it should be understood by those skilled in the art that the foregoing and various other changes, omission and additions may be made therein and thereto, without departing from the spirit and scope of the present invention.

References

Broca et al., Am. J. Physiol. 277:E617-E623, 1999

Broca et al., Eur. J. Pharmacol. 390:339-345, 2000

Broca et al., Endocrinol. Metab. 287:E463-E471 , 2004

Greene's Protective Groups in Organic Synthesis, 4th Edition, 2006

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