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Biosynthesis of p-Hydroxylated Aromatics Joseph J. Salvo,* David P. Mobley, Daren W. Brown, Lisa A. Caruso, Andrew P. Yake, Jay L. Spivack, and David K. Dietrich Biological Sciences Laboratory, GE Research and Development Center, P.O. Box 8, Schenectady, New York 12301
A bioconversion process with Aspergillus parasiticus has been developed to enhance p-hydroxylation activities with respect to aromatic substrates such as m-terphenyl. Through modified growth conditions, we are improving product concentration, while streamlining fermentation and downstream processing requirements. We routinely carry out biosynthetic conversions with m-terphenyl at the 300-L scale and produce over 200 g of 4,4"-dihydroxy-m-terphenyla t a concentration of 0.7 g / L in the fermentation vessel. ~
Introduction Hydroxylated aromatic molecules have commanded considerable interest in industry due to their many uses in the manufacture of plastics, liquid crystals, and dyes. However, some large-scale selective hydroxylations are difficult or expensive to carry out by any means (Doddema, 1988). One potential route leading to the production of these and other specialty compounds is biotransformation. Often relatively inexpensive starting materials can be biologically converted to higher value products. One organism capable of performing an interesting bioconversion is Aspergillus parasiticus (A. parasiticus) (Smith et al., 1980; Golbeck and Cox, 1984; Cox and Golbeck, 1985). It has been reported that this fungus can transform biphenyl to 4,4'-dihydroxybiphenyl in batch and continuous cultures, but the reported rates and concentrations were judged to be too low to be economically attractive. In addition, A. parasiticus produces carcinogenic secondary metabolites, aflatoxins, which make largescale fermentations less desirable from a processing standpoint. We report progress in developing a fermentation system for the routine production of 100-g quantities of hydroxylated compounds suitable for research purposes.
Materials and Methods Strains and Small-Scale-Culture Conditions. A. parasiticus strain ATCC 11517 was chosen for the initial hydroxylation studies. Sabouraud dextrose (SD) medium (Difco) was used for routine shake flask cultures (37 "C, 300 rpm), but corn steep liquor/corn syrup medium (CSS), 22 g/L Karo light corn syrup and 43 g/L Argo Steepwater E801, was also used where indicated. The solid medium for colony purification, and for the propagation of mutagenized spores, was SD with Bacto agar (Difco) added to 1.5%. Aflatoxin production plates were as described by Lennox and Davis (1983), except that the final concentration of Bacto agar was 3 5%. Spore Germination and Large-Scale Cultures. Starter cultures were prepared in five 2-L baffle flasks that contained 400 mL each of SD broth inoculated with 2 x los spores and incubated at 37 "C for 24 h at 300 rpm. The cultures were added to a 400-L-capacity fermentation vessel charged with 6.6 kg of Karo light corn syrup, 12.9 kg of corn steep liquor (ArgoSteepwater ESOl), 8756-7938/90/3006-0193$02.50/0
and water to a volume of 300 L. The corn steep liquor was autoclaved, prior to addition to the vessel, as a 50% solution with water at 120 "C for 20 min, while all other liquids were filter sterilized. In some large-scale runs, corn syrup was added continuously at a rate of 0.1 (g/L)/h. The fermentation tank was outfitted with four baffles and two impellers that stirred the contents at 300 rpm, while the temperature was maintained at 37 "C and filter-sterilized air was sparged in at a rate of 80 standard L/min. Silicone-based antifoam (Sigma A5757) was used as needed. Mutagenesis and Selection. Spores were plated on SD agar and irradiated with a germicidal UV light source for 60 (pW.h)/cm2 measured at the culture plate surface. Approximately 99% of the irradiated spores failed to germinate. Spores that did germinate were transferred to aflatoxin production plates. Four colonies were inoculated per plate with a wild-type control at the tenter. Decreased aflatoxin producers were screened by illuminating the plates with a long-wave UV lamp (366 nm) and by looking for the absence of a blue halo, which normally surrounds a wild-type colony (Lennox and Davis, 1983). Aflatoxin Detection. Thin-layer chromatography (Dugan, 1989) and high-pressure liquid chromatography (HPLC; see text below) were used to detect aflatoxins after extraction from spent media, mycelia, or agar plates. Aflatoxin standards (Sigma) were always run in parallel. Substrate Addition. After 24 h had passed following the inoculation of the 300-L fermentor, 1.5 kg of Triton X-100, 300 g of m-terphenyl, and 15 g of 4,4'-dihydroxybiphenyl, an inducer, were added (Golbeck and Cox, 1984). Both the m-terphenyl and 4,4'-dihydroxybiphenyl were added as fine dispersions in water that were stabilized with gelatin (Grayslake). Product Recovery. For large-scale product isolation from mycelia and broth, the fermentor contents were adjusted to pH 12 with NaOH once the biotransformation activity had diminished (usually 120 h after substrate addition). This dissolved the m-terphenyldiol in the aqueous medium as the sodium salt. The mycelia were separated from the broth with use of a basket centrifuge, resuspended in 70 L of water, adjusted to pH 12 with NaOH, and washed to recover any residual product associated with the biomass. The spent broth and wash
0 1990 American Chemical Society and American Institute of Chemical Engineers
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Figure 1. Effect of Triton X-100 on the peak 4,4”-dihydroxym-terphenyl concentration during fermentation. 250-mL shake flasks with 50 mL of fungal culture (SDmedium) were jncubated at 37 O C with a variable amount of Triton X-100. After 48 h, m-terphenyl was added a t the 1.0 g/L level as a dispersion (see Materials and Methods). The maximum amount of 4,4”-dihydroxy-m-terphenylproduced during each run is plotted with respect to surfactant concentration. All fermentations were continued until diol production plateaued.
water were combined and the surfactant removed at pH 14 by extraction with methylene chloride. The aqueous phase was brought to pH 7 with HC1 and extracted with ethyl acetate, which recovers the product in the bispheno1 form. The final product was obtained after purification on a silica gel column and recrystallization from a concentrated solution in ethanol/water. High-Performance Liquid Chromatography. The course of the bioconversion process was followed by analyzing methanol extracts of mycelia and broth by highperformance liquid chromatography (HPLC; Waters 840). Samples were loaded onto a (2-18 reverse-phase column (Perkin-Elmer) and eluted with a 5-100% (v/v) gradient of acidified (0.06% acetic acid) acetonitrile/HzO. Integrated peak areas from UV absorbance scans were used in conjunction with standards to estimate substrate, product, and aflatoxin levels. Polar products (believed to be further oxidation products of 4,4”-dihydroxy-m-terphenyl) were estimated to have a response factor identical with that of 4,4”-dihydroxy-m-terphenyl.Due to errors in sampling and analysis, reported concentrations may differ as much as 20% from actual values.
Results and Discussion Effect of Triton X-100 Surfactant on Hydroxylation Activity. The ability to p-hydroxylate biphenyl biologically has been well documented in the literature, but yields and solution concentrations were deemed too low to justify large-scale syntheses. Recently, conditions were found that increased product concentrations more than 10-fold from previous reports (Smith et al., 1980; Cox and Golbeck, 1985). Greater aeration during the bioconversion phase and the addition of the substrate as a protein-stabilized dispersion instead of a solution with dimethyl formamide led to the major increase (Abramowicz et al., 1990). In this study we report that the addition of the nonionic surfactant, Triton X-100, has a dramatic effect on improving the maximum concentration of 4,4”-dihydroxy-mterphenyl produced during fermentation (Abramowicz and Keese, 1989). Figure 1 shows the results from typical bioconversions of m-terphenyl to 4,4”-dihydroxy-m-terphenyl. In the absence of surfactant, the conversion is
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Figure 2. Time course of p-hydroxylation of m-terphenyl in SD medium. 50-mL aliquots of a stationary-phase fungal culture were incubated with 1.0 g/L m-terphenyl (nominal), 50 mg/L 4,4’-dihydroxybiphenyl (inducer), and 0.5 % Triton X-100. Substrate and product levels are shown with respect to time. Polar products are believed to derive from further oxidation of the 4,4”-dihydroxy-m-terphenyl and not the intermediate product, 4-hydroxy-m-terphenyl. The data shown is from a single flask sampled repeatedly throughout the time course of the experiment. The standard deviation of the concentration measurements between replicate flasks in other similar runs was 510%. I
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Figure 3. Time course of p-hydroxylation of m-terphenyl in CSS medium. Fermentation conditions were identical with those described in Figure 2 except that CSS media was substituted for SD. Note that the overall bioconversion of m-terphenyl appears accelerated compared to that in Figure 2. The data shown are the average of two identical flasks in the same run. The standard deviation of the concentration measurements between replicate flasks was 7 % .
barely detectable, while sample cultures that include 0.5% Triton X-100 produce over 0.2 g/L. In general, one of the difficulties in performing bioconversions with biphenyl analogues is that they have a very low solubility in the aqueous fermentation medium. We have found that Triton X-100 makes the substrate much more soluble. For example, the solubility of m-terphenyl in SD broth is only about 1ppm, but the apparent solubility in 0.5% Triton X-lOO/SD is nearly 90 ppm. While this observation may explain the enhanced hydroxylation activity, we cannot rule out the possibility that Triton may effect cell-wall permeability or induce specific enzymatic pathways. The dramatic loss of activity at 0.7%Triton X-100 is complete in that no conversion to intermediates or polar products occurs. Current investigations with different surfactants are aimed at elucidating the mechanism of Triton-stimulated hydroxylations.
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Figure 4. (A) Identification of a low-level aflatoxin mutant. A potential low-level aflatoxin producer was transferred to a CSS plate and incubated a t 30 “C for 48 h and then a t room temperature until a fluorescent halo could be detected from the wild-type control colony (see Materials and Methods). The exposure on the left was taken with room lighting, while that on the right was taken with illumination from a long-UV source. The colony with a halo is A. parasiticus ATTC 11517; the mutant is JS 1-89. (B) Thin-layer chromatography of extracts from mutant JS 1-89. Chloroform extracts from spent culture media and mycelia were separated by thin-layer chromatography and compared to standards of aflatoxins R1, R2, G1, and G2 (Sigma): (lane 1) mixture of B1, B2, G1, and G2,O.l pg each; (lane 2) 0.1-pg R1; (lane 3) 0.1-pg R2; (lane 4) 0.1-pg GI; (lane S) 0.1-pg 62; (lane 6) 1p L of a 50X concentrated chloroform extract from a typical 300-1,fermentation broth and mycelia mixture; (lane 7) same as lane 6 except 0.1 pg of each aflatoxin (BI,B2, G1, G2) was added to the sample. As little as 0.001 pg of aflatoxin could easily be detected by this method.
Fermentation Media. Sabouraud dextrose medium (SD) is often used to culture fungi; however it is far too expensive to be considered as a medium for routine largescale synthesis of specialty chemicals. Figures 2 and 3 compare the hydroxylation activity in SD medium with that of an inexpensive corn syrup/corn steep liquor medium (CSS). We find that the hydroxylation rate is faster in CSS than in SD. For example, the reaction profile a t 25 h in CSS medium is nearly identical with that in SD after 140 h of fermentation. Peak concentrations of 4,4”-dihydroxy-m-terphenylare higher in CSS, but it appeared that longer incubations in SD may continue to produce hydroxylated compounds. Subsequent experiments have shown that incubations longer than 140 h in
SD produce less than 25% additional 4,4”-dihydroxy-mterphenyl (data not shown). Aflatoxins. A considerable drawback of A. parasiticus is its tendency to produce aflatoxins, potent carcinogens, and mutagens (Busby and Wogan, 1984) during the bioconversion reactions of interest. Corn steep liquor stimulates aflatoxin production, and a t the 300-L scale, significantquantities of aflatoxins could be produced (Lennox and Davis, 1983; Henderberg et al., 1988). Therefore, we screened a collection of mutants for a strain that retained hydroxylation activity but had greatly reduced aflatoxin production capacity. Aflatoxin minus strains of A. parasiticus have been generated in the past by standard UV or chemical mutagenesis techniques (Bennett
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or the additional carbon source. In any event, with use of CSS media and the addition of supplemental corn syrup, peak concentrations of 0.7 g/L 4,4"-dihydroxy-mterphenyl can be routinely produced at the 300-L scale versus 0.4 g/L in shake flasks with no supplemental feeding (for a typical large-scale conversion, see Figure 5B). With this inexpensive CSS media, we can isolate 200-g quantities of specific p-hydroxylated compounds such as 4,4"-dihydroxy-m-terphenylfrom a 300-L batch culture at a more reasonable cost than before. Larger scale syntheses can now be envisioned. Product Recovery. Previously (Abramowicz et al., 1990) the fermentor contents were extracted with an organic solvent such as methanol in order to recover the aromatic products, which partition in both the cell mass and broth. This procedure releases many unwanted compounds such as pigments and complicates further purification steps. We find that after raising the pH of the culture to 12 with NaOH, nearly all of the hydroxylated products can be recovered in the aqueous phase as the corresponding sodium salts. The surfactant can be selectively removed at pH 14 with methylene chloride. Extraction of the aqueous phase (adjusted to pH 7) with ethyl acetate allows facile concentration of the products before final purification via standard silica gel chromatography and recrystallization from a concentrated ethanol/water solution.
Conclusions and Future Prospects
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Figure 5. (A) Time course of p-hydroxylation of m-terphenyl in a mutant with reduced aflatoxicity. A. parasiticus strain JS 1-89was incubated under conditions identical with thoee described in Figure 3. The p-hydroxylated product profile was similar to that observed with the wild type. The data shown are the average of two identical flasks in the same run. The standard deviations of concentration measurements between replicate flasks were 10%. (B) p-Hydroxylation of m-terphenyl at the 300-L scale. A. parasiticus strain JS 1-89 was cultured in CSS media as described in Materials and Methods, with a feed rate of 0.1 (g/L)/h of corn syrup.
and Papa, 1988). We isolated several low-level aflatoxin producers after screening several thousand colonies that developed from UV-irradiated spores (Figure 4A). Mutants were initially identified by the absence of a blue aflatoxin halo on corn steep liquor supplemented plates when illuminated with a long-wavelength UV source (peak at 366 nm). Subsequent thin-layer chromatography (Figure 4B) and HPLC analysis (not shown) of extracts from mycelia and broth showed that strain JS 1-89 produced no detectable aflatoxins (less than 20 ppb), i.e. at least 100-fold lower than wild-type A. parasiticus grown under identical (unpublished observations) or similar conditions (Lennox and Davis, 1983). In addition, the hydroxylation activity of this strain in shake flasks was comparable to wildtype isolates, i.e. 0.4 g/L 4,4"-dihydroxy-m-terphenyl (Figure 5A). Scaleup. A t the 300-L scale, corn syrup was fed at a rate of 0.1 (g/L)/h for maintenance energy during the bioconversion stage. Under these conditions there is a longer induction period than was seen in the shake flasks after which the rate of conversion is comparable. We have not determined what factor(s) are responsible for this lag, but possibilities include poor mixing, aeration,
We have developed a straightforward bioconversion process for the synthesis of laboratory-scale quantities of a p-hydroxylated aromatic molecule. The addition of the nonionic surfactant, Triton X-100, greatly improved substrate and product solubilities without any deleterious effects on reaction rates. An inexpensive corn syrup/ corn steep liquor based medium accelerated both the initial onset and rate of bioconversion and proved to be more than adequate for moderate-scale syntheses. Further improvements in the process likely can be made through strain manipulations. m-Terphenyl undergoes two separate oxidations, presumably by the same enzyme, to yield 4,4"-dihydroxy-m-terphenylvia a monohydroxylated intermediate, 4-hydroxy-m-terphenyl. Further oxidation steps leading to polar product formation probably involve other enzymes. Current efforts have focused on isolating the enzymets) and gene(s) responsible for these activities and identifying the structure of the polar products. If it is possible to eliminate the conversion of the starting material to polar products while maintaining hydroxylation activity, we may realize higher concentrations of dihydroxylated product in the culture and consequently a more economical process.
Acknowledgment Thanks to Dr. Herman Finkbeiner for helpful discussions, advice, and support.
Literature Cited Abramowin, D. A.; Keese, C. R. Process for the Microbial Hydroxylation of Biphenyl Compounds. Patent 01/364,218, 1989. Abramowicz, D. A.; Keese, C. R.; Lockwood, S. H. Regiospecific Hydroxylation of Biphenyl and Analogs by Aspergillus parasiticus. In Biocatalysis; Abramowicz, D. A., Ed.; Van Nostrum-Rheinhold, New York, 1990, in press. Bennett, J. W.; Papa, K. E. Genetics of Aflatoxigenic Aspergillus Species. In Advances in Plant Pathology; Sidhu, G. S., Ed.; Genetics of Pathogenic Fungi, Vol. 6 ; Academic Press: London, 1988; pp 263-280.
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Busby, W. F.; Wogan, G. N. Aflatoxins. In Chemical Carcinogens; Searle, C. E., Ed.; ACS Monograph Series 182; American Chemical Society: Washington, DC, 1984; pp 945-1136. Cox, J. C.; Golbeck, J. H. Hydroxylation of Biphenyl by Aspergillus parasiticus: Approaches to Yield Improvement in Fermenter Cultures. Biotechnol. Bioeng. 1985,27, 13951402. Doddema, H. J. Site-specific Hydroxylation of Aromatics by Polyphenol Oxidase in Organic Solvents and in Water. Biotechnol. Bioeng. 1988, 32, 716-718. Dugan, E. A. The Detection of Aflatoxins by TLC. Am. Biotech. Lab. 1989, 7, 46-48. Golbeck, J. H.; Cox, J. C. The Hydroxylation of Biphenyl by Aspergillus toxicarius: Conditions for a Bench Scale Fermentation Process. Biotechnol. Bioeng. 1984, 26, 434-441. Golbeck, J. H.; Albaugh, S. A.; Radmer, R. J. Metabolism of Biphenyl by Aspergillus toxicarius: Induction of Hydroxy-
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lating Activity and Accumulation of Water-Soluble Conjugates. J. Bacteriol. 1983, 156, 49-57. Henderberg, A.; Bennett, J. W.; Lee, L. S. Biosynthetic Origin of Aflatoxin G1: Confirmation of Sterigmatocystin and Lack of Confirmation of Aflatoxin B1 as Precursors. J. Gen. Microbiol. 1988, 134, 661-667. Lennox, J. E.; Davis, C. K. Selection of and Complementation Analysis Among Aflatoxin Deficient Mutants of Aspergillus parasiticus. Exp. Mycol. 1983, 7, 192-195. Smith, R. V.; Davis, P. J.; Clark, A. M.; Glover-Milton, S. Hydroxylations of Biphenyl by Fungi. J. Appl. Bacteriol. 1980,49, 65-74. Accepted April 27, 1990. Registry No. m-Terphenyl, 92-06-8; 4,4'-dihydroxy-m-terphenyl, 124526-56-3.
