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Asymmetric bioreduction of a β-ketoester to (R)-β-hydroxyester by the fungus Mortierella alpina MF 5534

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Asymmetric bioreduction of a β-ketoester to (R)-β-hydroxyester by the fungus Mortierella alpina MF 5534
  JOURNAL OF FERMENTATION ND BIOENGINEERING Vol. 80 No. 2 176-179. 1995 Asymmetric Bioreduction of a ,kKetoester to (R)-p-Hydroxyester by the Fungus zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA ortierella alpina MF 5534 MICHEL CHARTRAIN,* JOSEPH ARMSTRONG, LORRAINE KATZ, JENNIFER KELLER, DAVID MATHRE, AND RANDOLPH GREASHAM zyxwvutsrqponmlkjihgfedcbaZYXWVU Merck Research Laboratories R Y 8OY-105 PO Box 2000 Rahway NJ07065 USA Received 1 March 1995/Accepted 20 May 1995 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPO Two hundred and sixty strains of microorganisms, (60 strains of yeasts, 60 strains of bacteria and 140 strains of fungi), were evaluated for their ability to stereoselectively bioreduce a ,8-ketoester {[2s-(2a,4B)]-l-[(l,l- dimetbylethoxy)carbonyl]-4-hydroxy-B-oxo-2-pyrrolidinepropanoic acid l,l-dimethylethyl ester] to the cor- responding (R)-,9-hydroxyester ([2~-(2a(~*),4~)]-~,4-dihydroxy-l-[(l,l-dimethylethoxy)carbonyl]-2-pyr- rolidinepropanoic acid l,l-dimethylethyl ester], a precursor to the p-methyl carbapenem antibiotic BO 2727. Among all the microbes evaluated, only one fungal strain, Mortierella alpina MF 5534 (ATCC 8979) was found to catalyze the desired reaction. The scaled-up bioconversion process in laboratory bioreactor (23-1 scale) supported (R)-p-hydroxyester titers of 550 mg/Z during a 250-h cultivation cycle and allowed the timely production of gram quantities of diastereomerically pure materials (diastereomeric excess >98 ). [Key zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA ords: bioreduction fermentation process development bioconversion] The past few years have seen an increase in the use of chiral precursors in the synthesis of new pharmaceutical drugs (1). Biocatalysis employing either whole cells or isolated enzymes has emerged as an attractive alternative to chemical chiral catalysts and to difficult chemistry (2- 5). Because of the importance of prochiral carbonyls as synthons, asymmetric bioreduction of ketones and keto- esters catalyzed by yeasts and to a lesser extent by fungi and bacteria is one of the fields of biocatalysis that has recently received a great deal of attention (6-9). Because of their broad spectrum, ;3-la&am antibiotics such as Imipemem have been widely used in the past decade but their instability requires that they be ad- ministered in conjunction with cilastatin, a dihydropepti- dase 1 inhibitor (10). This instability can be overcome when a f-methyl group is introduced at the one position of the nucleus, and several new ,3-methyl carbapenem have been recently developed (11). BO 2727 is a new very promising broad spectrum f-methyl carbapenem that contains 7 chiral centers (ll), one of which is locat- ed in the side chain of the molecule and derived from the chiral precursor (R)-;3-hydroxyester. As an attractive alternative to a difficult and expensive chiral chemical synthesis of this (R)- -hydroxyester, we investigated a chiral biocatalytic process as a valuable and cost effective step. This report presents the identification of the fungus Mortierella alpina MF 5534 (ATCC 8979) as a suitable biocatalyst for the asymmetric bioreduction of the ,?- ketoester to diastereomerically pure (R)+hydroxyester (“de”>98%). Scale up of the bioreduction process in laboratory bioreactors achieved the production of (R)-,3- hydroxyester (“de” >98%) in gram quantities. MATERIALS AND METHODS Chemicals and cultivation media All chemicals used were of reagent grade and purchased from either the Sigma Chemical Co. (St. Louis, MO, USA) or from Fisher Scientific Products (Springfield, NJ, USA). {[2S- * Corresponding author. (2a,4b)]- 1- [(l,l -dimethylethoxy)carbonyl] -4-hydroxy-b- oxo-2-pyrrolidinepropanoic acid zyxwvutsrqponmlkjihgfedcba   1-dimethylethyl ester} [,%ketoester] was obtained from Merck and Co (Rah- way, NJ, USA). The composition of the various cultivation media is as follows: YME: yeast extract (Difco, Detroit, MI, USA) 4 g/l, malt extract (Difco) 20 g/l and glucose 4 g/l. KF: Corn steep liquor (CPC Corp, Engelwood, NJ, USA) 5 g/l, tomato paste (Heinz) 40 g/l oatmeal flour (Quaker oats) 10 g/l, glucose 10 g/l, 10ml KF trace element solution, and sodium hydroxide to bring the pH to 6.8. Trace element solution contains: FeS04.7H20, 1 g/l; MnS04. 4H20, 1 g/l; CuClz. 2H20, 0.025 g/l; CaCl*, 0.1 g/l; H3B03, 0.056 g/l; (NH&Mo,O~~. 4H20, 0.019 g/l; ZnS04.7H20, 0.2 g/l. Sabouraud Dextrose (SD) and Tryptic Soy (TS) were purchased from Difco and were used at the concentra- tion of 30 g/l. Microorganisms All microorganisms employed in our screen were obtained from the Merck Microbial Resources Culture Collection (Rahway). Screening for bioreduction activity Cells preserved on agar slants at 4°C (SD for yeast, YME for fungi and TS for bacteria) were used to inoculate 250-ml flasks con- taining 50ml of the appropriate seed medium (YME for fungi, SD for yeasts, TS for bacteria). The flasks were in- cubated aerobically at 28°C on an orbital shaker operat- ed at 220rpm. Flasks (250-ml) containing 50ml of the appropriate growth medium were inoculated with 2.5 ml of a 48-h old seed culture and aerobically incubated at 28°C on an orbital shaker operated at 220rpm. ;3- Ketoester dissolved in ethanol was added to each flask after 48 h of incubation to give an ethanol concentration of 20 ml/l and a +ketoester concentration of 1 g/l. Three days following the addition of ,%ketoester, the flasks were harvested, and the broths were extracted and analyzed by HPLC for the presence of hydroxyester. Bioconversion methods Shake flask scale A frozen glycerol suspension of M. alpina MF 5534 was used to inoculate a 250-ml flask 176  VOL. 80, 1995 containing 50 ml of KF medium. The first stage seed cul- ture was incubated aerobically at 28°C on a rotary shak- er operated at 220 rpm. A 2-1 Erlenmeyer flask contain- ing 500 ml of YME was inoculated with 25 ml of a 72-h old first stage seed culture and incubated at 28°C on an orbital shaker operated at 180rpm (2 in. throw). Two days following inoculation, 10ml of an ethanol solution containing 0.5 g of crude f-ketoester were added to the 2-1 flask. Visual observations of the flasks revealed that good growth had been achieved at the time of fi- ketoester addition. The flasks were returned to the same environmental conditions, and the progress of the bio- conversion was periodically monitored. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Bioreactor scale 23-l) A frozen glycerol suspen- sion of M. alpina MF 5534 was used to inoculate a 250- ml flask containing 50ml of KF medium. The first stage seed culture was incubated aerobically at 28°C on a ro- tary shaker operated at 220rpm. The second stage seed culture was prepared by inoculating a 2-1 Erlenmeyer flask containing 500 ml of KF medium with 25 ml of the 48-h old first stage seed culture. The 2-l flask was incu- bated aerobically at 28°C on a rotary shaker operated at 180 rpm. A 23-1 bioreactor containing 15 I of YME was inoculated with 500ml of the 72-h old second stage seed. The bioreactor was operated with a 220 rpm agita- tion (minimum set point), a 0.6 PSI back pressure and was sparged with air at a rate of 6 f/min. The dissolved oxygen tension was maintained above 30% of initial saturation by computer controlled ramping of the agita- tion. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Analytical techniques Chemical extraction Cultures were harvested by centrifugation in a Beckman table top centrifuge model TJ 6 (Beckman Instruments, Fullerton, CA, USA) oper- ated at 2,500rpm for 10 min. The supernate was mixed with an equal volume of ethyl acetate, employing a reciprocal shaker for 10 min. The resulting emulsion was broken by centrifugation (10 min at 2,500 rpm) in a Beck- man bench top centrifuge. The ethyl acetate layer was separated and taken to dryness. The dryed materials were resuspended in 2 ml of methanol and stored at 4°C prior to analysis by HPLC. Chromatography The harvested supernate was loaded on a 120-ml chromatography column containing 80ml of Amberchrome 161 resin (Supelco, Bellfonte, PA, USA). The materials were eluted with a step gradient of methanol in water, (0% to 100% methanol by increments of 10%). Collected fractions were ana- lyzed by HPLC and NMR for the presence of hydrox- yester . Reverse phase HPLC The separation of hydrox- yester and ketoester was achieved, employing a Rainin HPLC system (Rainin, Woburn, MA, USA) comprised of two analytical pumps, a UV detector, an autoinjec- tor, and a Macintosh computer system. The column was a Zorbax RX C8 (4.6 x 250 mm) (Mac-mod Analytical, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB Mortierella alpina FIG. 1. Asymmetric bio-reduction of the +ketoester to its cor- responding (R)+hydroxyester, a precursor for the novel wide spec- trum antibiotic BO 2727. ASYMMETRIC BIOREDUCTION OF ,?-KETOESTER 177 Chadds Ford, PA, USA). Separation was obtained by us- ing a gradient of acetonitrile (0.1% H3P04) and water (0.1% H,P04) at a flow rate of 1.5 ml/min. The gradient was 20/80 (v/v) acetonitrile/water to 70/30 (v/v) over lOmin, and held for 20min. The system was re- equilibrated back to 20/80 (v/v) for 7 min before per- forming the next injection. Detection was done at 200nm, and hydroxyester and -ketoester eluted after 10.7 and 12.9 min, respectively. Supercritical @id chromatography SFC) An EM diol silica (EM science, Gibbstown, NJ, USA) and a Chiralcel OD(H) column (Chiral Technologies) were used in tandem, employing a Hewlett Packard model 1205A SFC (Hewlett Packard). The system was operated at 35°C with a CO* pressure of 100 Bar and methanol as the modifier co-solvent (8%, v/v) at a flow rate of 1 ml/min. Detection was performed at 200 nm, using a diode array detector. (R)-p-Hydroxyester and (s)-p- hydroxyester diastereomers eluted after 18.78 min and 19.70 min respectively. Calculation of the diastereomeric excess (de) was performed as follows: de (%)=[(R)-_(S)I/[(R)+(S)l*lOO AJMR The dried materials were resuspended in deu- terated methanol prior to analysis. i3C NMR spectra were recorded from CD30D solutions at 75.5 MHz with CDjOD (49.1 ppm) used as the as internal standard, em- ploying a Brucker spectrometer model AM 300 (Brucker instruments). RESULTS Screening for microorganisms with , ketoester reduc- ing activity Two hundred and sixty microbial strains (60 strains of yeasts, 60 strains of bacteria, 140 strains of fungi) were screened for the presence of the desired bio-reducing activity. Among all the microorganisms test- ed, only one fungal culture, M. aIpina MF 5534 was found to bioreduce the ,3-ketoester to the desired /3- hydroxyester (Fig. 1). After isolation of the molecule was performed as described in the Materials and Methods section, the structure of the (R)-,3-hydroxyester was confirmed by NMR studies (Fig. 2). (R)- -Hydrox- yester 13C NMR resonances are listed below: d 172.6, 172.3, 157.2, 156.7, 81.8, 81.3, 81.0, 70.8, 70.4, 69.2, 69.0, 61.8, 56.8, 56.2, 41.7, 41.1, 34.8, 34.4, 28.9, 28.5. zyxwvutsrq I I L I I I I I 1 I I 7 65 6 55 5 45 4 35 3 twm FIG. 2. NMR traces of the (@-,%hydroxyester.  178 CHARTRAIN ET AL. J. zyxwvutsrqponmlkjihgfedcb ERMENT. BIOENG., Min. FIG. 3. Chiral super critical fluid chromatography separation of the hydroxyester (R) and (s) enantiomers. Super critical fluid chromatography analyses con- firmed that the orientation of the hydroxy group was in the desired (R) position, and that the optical purity (diastereomeric excess) of this (R)-,3-hydroxyester was greater than 98% (Fig. 3). zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Process development studies Bioreduction kinetics of the ,l-ketoester to (R)-,9-hydroxyester were first investi- gated at the 2-1 shake flask scale. Figure 4 shows that a maximum (R)-j-hydroxyester titer of 5.50 mg/l was achieved after 170 h of cultivation and that the rate of (R)-,3-hydroxyester production was fairly linear during the entire course of the cultivation. Carbon sources, especially glucose, have in some in- stances been reported to negatively regulate bioreduction activity and/or to adversely affect the optical purity of the final reduced product (12-14). Consequently, several candidates for replacing glucose as a carbon source were evaluated in 2-1 shake flasks. Table 1 shows that all the carbon sources tested supported the production of highly optically pure (R)-;3-hydroxyester at comparable level. Scale-up studies were performed in 23-1 bioreactors, employing sucrose as the carbon source. Growth of the microorganisms measured by on-line (oxygen uptake rate) and off-line (packed cell volume) occured rapidly and reached stationary phase about 24 to 36 h post in- oculation for the 1X and 3X medium strength respective- ly. ,%Ketoester (1 g/l) was added in ethanol (0.18,0d v/v, final concentration) to the bioreactors when the cells reached late log phase as indicated by on-line respiratory activity. Figure 5 shows that a slower rate of bioconver- sion was observed in the fermentor when employing the same production medium that was used in the shake flask studies. However, increasing the medium strength by 2 and 3 fold alleviated this problem, and a per- formance similar to that achieved in shake flasks was achieved in the laboratory bioreactors (Fig. 5). It is postulated that in a bioreactor setting, the nutrients used to provide energy for the bioconversion process may be depleted too quickly in favor of biomass synthesis. This scaled up process was able to produce 550 mg// of (R)-,3- hydroxyester after 250 h of cultivation. The diastereom- eric excess of the (R)-,%hydroxyester produced by this process was found to be greater than 989:. DISCUSSION We identified a fungal strain, M. zyxwvutsrqponmlkjihgfedcbaZY lpina MF 5534, capable of bioconverting the ;3-ketoester to diastereomeri- tally pure (R)- hydroxyester, and successfully scaled up the bioconversion process in laboratory bioreactors to achieve the production of gram quantities of materials. The observed lack of bioreduction activity by all the yeasts, bacteria and most of the fungi evaluated in this screen was surprising since ;i-ketoesters are usually con- sidered as easy and sure targets for enantioselective bioreductions. Yeasts (especially Baker’s yeast) are often recommended as obvious biocatalyst for this type of 600 500 - c h 400 - 2 j 300 - ?? g ” 200 - zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 1 200 300 400 Cultivation time h) FIG. 5. Time course for the bio-reduction of the ,?-ketoester to the (R)-,3-hydroxyester in 23-l bioreactors under various conditions. Symbols: C: , shake flask lx; A, bioreactor 3x; 0, bioreactor 2x; 0, bioreactor 1 x. Bioconversion time h) FIG. 4. Time course for the bio-reduction of the +ketoester to the (R)-,?-hydroxyester at the 2-1 shake flask scale.  VOL. 80, 1995 ASYMMETRIC BIOREDUCTION OF ,%KETOESTER 179 TABLE 1. Effect of carbon source on bioconversion activity and (R)-hydroxyester optical purity Carbon source Hydroxyester titer (mg/n Diastereomeric excess (%) Glucose 700 >98 Fructose 730 >98 Galactose 760 >98 Glycerol 660 >98 Glutamate 760 >98 Sucrose 750 >98 Experiments were performed in 2-l shake flasks employing YME medium. Carbon source replacements were employed at the same concentration as glucose (4 g/f). The DE was calculated according to the following formula: de (%)=[(R)-(S)]/[(R)+(S)]*lOO asymmetric reduction (2, 6-9) however, none of the 60 diverse yeast strains evaluated here was able to catalyzed the desired bioreduction. One could speculate that the presence of the BOC protected nitrogen atom positioned near the site of reduction may create a steric hindrance to most oxydoreductases. Another explanation may be that the membrane of most of the microbes evaluated in this screen may not be permeable to the 1%ketoester. Initial process development studies indicated the need for some source of nutrient for the bioreduction process to occur at a reasonable rate. Beside medium develop- ment and the evaluation of physico-chemical parameters, additional work should focus at developing a fed-batch process capable of achieving a high rate of bioconver- sion and elevated (R)-,&hydroxyester volumetric produc- tion. REFERENCES 1. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA tinson, S.: Chiral drugs. C & EN, Sept., 38-72 (1994). 2. Jones, B.: Enzymes in organic synthesis. Tetrahedron., 42, 3351-3403 (1986). 3. Kieslich, K.: Biotransformations of industrial use. Acta Bio- technol., 11, 559-570 (1991). 4. Lilly, M.: Advances in biotransformation processes. Chemical Engineering Science, 49, 151-159 (1994). 5. Margolin, A.: Enzymes in the synthesis of chiral drugs. En- zyme Microb. Technol., 15, 266-280 (1993). 6. Csuk, R. and Glanzer, B.: Baker’s yeast mediated transforma- tions in organic chemistry. Chem. Rev., 91, 49-97 (1991). 7. Nakamura, K., Kawai, Y., Kitayama, T., Miyai, T., Ogawa, M., Mikata, Y., Higaki, M., and Ohno, A.: Asymmetric reduc- tion of ketones with microbes. Bull. Inst. Chem. Res., Kyoto Univ., 67, 157-168 (1989). 8. Servi, S.: Baker’s yeast as a reagent in organic synthesis. Syn- thesis., 1, l-25 (1990). 9. Ward, 0. and Young, C.: Reductive biotransformations of organic compounds by cells or enzymes of yeast. Enzyme Microb. Technol., 12, 482-492 (1990). 10. Maki, E.: General pharmacology of imipenem, a new beta- lactam antibiotic, and cilastatin sodium, a specific competitive inhibitor of dehydropetidase-1 and the combination of these agents. Chemotherapy, 33, 329-365 (1985). 11. Nakagawa, S., Hashizume, T., Matsuda, K., Sanada, M., Okamoto, O., Fukatsu, H., and Tanaka, N.: In zyxwvutsrqponmlkjih itro activity of a new carbapenem antibiotic, BO 2727, with potent anti- pseudomonal activity. Antimicrob. Agents Chemoterapy, 37, 2756-2759 (1993). 12. Christen, M., Crout, D., Holt, R., Morris, J., and Simon, H.: Biotransformations using clostridia: stereospecific reductions of a iii-ketoester. J. Chem. Perkin Trans., 1, 491-493 (1992). 13. Ehrler, J., Giovannini, F., Lamatsch, B., and Seebach, D.: Stereoselectivity of yeast reductions-an improved procedure for the preparation of ethyl (S)-3-hydroxybutanoate and (S’)-2- hydroxymethylbutanoate. Forschung Chimia, 40, 172-173 (1986). 14. Ushio, K., Inouye, K., Nakamura, K., Oka, S., and Ohno, A.: Stereochemical control in microbial reduction. IV. Effect of cultivation conditions on the reduction of ,3-keto esters by methylotrophic yeasts. Tetrahedron Lett., 27, 2657-2660 (1986).
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