20 Selective Production of Ethyl Acetate by Candida utilis David W. Armstrong Division of Biological Sciences, National Research Council of Canada, Ottawa, Canada, K1A 0R6 Ethyl acetate produced via bioconversion processing has potential use as a 'natural' flavor and fragrance compound; for this reason the physiological manipulation of the yeast C. utilis to produce significant yields of this ester from glucose or ethanol has been studied. Production of the ester was dependent on the stage of growth. By use of iron-limited conditions, under adequate aeration, ethyl acetate accumulated from ethanol. Studies using specific metabolic inhibitors implicated acetyl-CoA as a key intermediate in formation of the ester. Chelators could be used to increase yield and specific production of ethyl acetate from glucose and ethanol. EDTA appeared to function at the level of cell permeability whereas EGTA or NTA may have encouraged larger acetyl-CoA pools to accumulate. Currently the bulk of flavor and fragrance compounds is provided through traditional methods which include chemical synthesis or extraction of desired components from natural sources such as plants. Accordingly, with the great current interest in "natural" products more pressure has been placed on expensive and labor intensive extraction processes since the FDA specifies that only products derived from living sources can be termed "natural". Bowing to consumer demand for these products, the flavor industry is beginning to enlist the help of biotechnology to produce natural flavor and aroma compounds via fermentative routes. At this time, the production of these fermentation compounds is a largely untapped area of bioconversion research in which bioesterification has great potential as esters play a key role in flavors. Simple organic acid esters, compounds of an alcohol with a monobasic acid, are produced in small amounts by some microorganisms as by-products in their utilization of organic compounds. However, comparatively few microorganisms are able to form significant amounts of esters (1). Various yeasts of the genera Saccharomyces (2) and Hansenula (3) produce ethyl acetate from glucose and/or This chapter not subject to U.S. copyright. Published 1986, American Chemical Society
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ethanol. Saccharomyces sp. produce very low concentrations of ethyl acetate ( i . e . levels found in alcoholic beverages) whereas Hansenula anomala produces s i g n i f i c a n t amounts of the ester 0 0 . Hansenula anomala 00 produced maximum amounts of the ester after about 8 d in a n u t r i t i o n a l l y complex medium while J. took up to 50 d to achieve the same levels (2.6 g/L ethyl acetate from 20 g/L glucose) in a simple s a l t s medium. More recently Thomas and Dawson (5), while studying the e f f e c t of iron l i m i t a t i o n on energy metabolism in the yeast Candida u t i l i s , found s i g n i f i c a n t rates of ethyl acetate formation from glucose in cultures synchronized by repeated d i l u t i o n with iron-limited minimal medium. Physiological studies on the formation of esters such as ethyl acetate have been targeted towards the alcoholic beverage industry (2,6) and production of single c e l l protein (SCP) (7). The studies have looked at regulation of the l e v e l s of these esters by environ mental factors in order to prevent o f f - f l a v o r s due to their organo l e p t i c properties in beverages or to maximize SCP y i e l d s from ethanol. More recently, interest has grown in the b i o l o g i c a l pro duction of certain esters s p e c i f i c a l l y for use in flavors and fragrances. E s t e r i f i c a t i o n of racemic alcohols with fatty acids and other organic acids by Candida cylindracea lipase (8) and in work by Paterson and B e l l using Rhizopus arrhizus esterase (9) have indicated potential for microbiological flavor and fragrance processing. The present work examines the physiological control of ethyl acetate production by C. u t i l i s . Materials and Methods Culture and Medium Formulation. Candida u t i l i s NRC 2721 (NRRL Y-900; ATCC 9950) was grown at 28°C in the minimal-salts medium of Thomas and Dawson (5). Glucose, as indicated, was added before adjusting the f i n a l volume, while 95% ethanol was added postautoclaving where indicated. The medium was adjusted to pH 5.8 and s t e r i l i z e d at 121°C for 15 minutes. A l l chemicals were of a n a l y t i c a l grade. A n a l y t i c a l . Viable c e l l counts: Samples, suitably diluted, were spread on the surface of agar medium (minimal s a l t s medium described above containing 20 g/L glucose and agar at 18 g/L). Colonies were counted after incubation at 28°C for 48 h. C e l l mass density (A ) C e l l mass density was determined by absorbance at 620 nm in c y l i n d r i c a l cuvettes having a l i g h t path of 1 cm. Dry c e l l weight: DCW was determined gravimetrically. Analysis of fermentation products: Products were i d e n t i f i e d by gas chromatography/mass spectrometry with routine analysis by gas chromatography. Glucose analysis: The d i n i t r o s a l i c y l a t e method of M i l l e r (H)) was used to "measure glucose content of the medium. Determination of iron concentration of medium: The α,α-dipyridyl method of Herbert et a l . (11) was used. :
6 2 0
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Ethanol Uptake Studies. Uptake of ethanol was studied using [ 1 - C ] ethanol suitably diluted in deionized d i s t i l l e d water to make a stock solution of 100 yCi/mL (= stock solution A). Where indicated 50 yL of stock solution A and 450 mL of 10" M non-radioactive ethanol were mixed (= stock solution B). C e l l s isolated at 3
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d i f f e r e n t points in the growth curve ( A readings of 0.35, 0.61 and 1.05, Figure 3), were collected on membrane f i l t e r s (0.45 μ) and washed three times with carbon-free medium (10 mL volumes) with f i n a l c e l l suspension ( A - 0.4) in medium to which glucose was or was not added (10 g/L f i n a l concentration). Aliquots (450 pL) of the l a t t e r suspensions were placed in 100 mL Pyrex screw-cap tubes to allow for adequate aeration. Stock ethanol solution Β (50 \ih) was then added (1 yCi/mL). Samples (100 uL) were spotted on Whatman 3 mm discs at 1.5 h and subsequently rinsed with three portions of 150 mL of ice-cold 5% t r i c h l o r o a c e t i c acid (TCA) and then with three portions of 100 mL ether/ethanol (1 :1) with suction on a large f i l t e r paper supported on a Biichner funnel. F i l t e r discs were a i r dried (on aluminum f o i l ) then oven dried at 110°C. Radioactivity was counted in 0.4J Omnifluor in toluene (5 mL) in a Beckman LS 7000 s c i n t i l l a t i o n counter. Results are averages of duplicate samples. 6 2 0
6 2 0
Fermentation Studies. Butyl rubber-stoppered v i a l s (Wheaton 160 mL) were used. The r a t i o of headspace t o culture volume (H/C) indicated the degree of aeration; an H/C of ca. 4 represented a 'high l e v e l of aeration while an H/C of ca. 1.5 represented a 'low' l e v e l of aeration. 1
Results and Discussion Kinetics of Ethyl Acetate Accumulation and E f f e c t of Iron. Candida u t i l i s accumulated s i g n i f i c a n t l e v e l s of ethyl acetate when grown on glucose in medium l i m i t e d f o r iron [Figure 1] whereas the addition of iron (FeCl at 100 μΜ) severely i n h i b i t e d t h i s c a p a b i l i t y (12). In both cultures, grown under adequate aeration (H/C - 1.5), ethanol and c e l l mass accumulated u n t i l glucose was depleted and the y i e l d of ethanol was similar (about 90$ of theoretical y i e l d ) . In the culture without iron supplementation, the c e l l s began to u t i l i z e ethanol after a lag period and accumulated a c e t i c acid and c e l l mass. Following t h i s , ethyl acetate accumulation began and con tinued while a c e t i c acid declined. I t was demonstrated that both acetic acid and ethyl acetate resulted from ethanol u t i l i z a t i o n . Ethanol is also known to be an intermediate for ethyl acetate synthesis by H. anomala (3,4). Apart from a severe i n h i b i t i o n of ethyl acetate accumulation, the addition of iron (100 μΜ) to C. u t i l i s c e l l s resulted in a s i g n i f i c a n t prolongation of the l a g before the s t a r t of ethanol u t i l i z a t i o n . A more rapid u t i l i z a t i o n of ethanol has been also observed previously in cultures of C. u t i l i s (13) and S. cerevisiae (14) grown under conditions of iron limitation. In order to study the e f f e c t of iron on the u t i l i z a t i o n of ethanol more closely, F e C l was added at various l e v e l s to glucosecontaining medium. The presence of iron did not affect the l e v e l s to which ethanol accumulated from glucose (24 h) [Figure 2]; however the subsequent u t i l i z a t i o n of ethanol was i n h i b i t e d in proportion to the l e v e l of iron present. I t is possible that iron may i n t e r f e r e with some early stage of ethanol u t i l i z a t i o n thereby leading to delayed ethyl acetate accumulation. 3
3
E f f e c t of Glucose on Ethanol Uptake by C. u t i l i s . Glucose can repress metabolism of other carbon sources by yeasts (]5_). In
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Escherichia c o l i the primary e f f e c t of glucose is the i n h i b i t i o n of transport of other carbon sources (]_6). As indicated above [Figure 1] sequential production of various products (ethanol, acetic acid and ethyl acetate) from glucose could be seen under an appropriate degree of aeration. Also c e l l mass accumulation ( A ) showed a diauxic increase [Figure 1] which r e f l e c t e d u t i l i z a t i o n of d i f f e r e n t carbon sources by C. u t i l i s . Ethanol u t i l i z a t i o n began only after glucose was depleted. This suggests a p o s s i b i l i t y that glucose i n h i b i t s ethanol uptake and thus conversion of ethanol to ethyl acetate. I t is of interest, especially from a biotechnolog i c a l standpoint, to know whether ethyl acetate production is possible in the presence of glucose. Therefore, the e f f e c t of glucose on the uptake of ethanol by C. u t i l i s was studied [Figure 3 ] . Candida u t i l i s was isolated at the three d i f f e r e n t growth phases indicated [Figure 3] and resuspended in fresh medium with or without glucose (10 g/L). Small volume (450 \ih) aliquots were placed in tubes to which l C] ethanol along with non-labelled ethanol c a r r i e r was added. The amount of the ethanol taken up by c e l l s without glucose was divided by the uptake by c e l l s with glucose. A r a t i o greater than 1.0 indicated i n h i b i t i o n of ethanol uptake by glucose. I t would appear that the results are consistent with the general phenomenon of cataboli te repression (15,16). Thus, without additional environmental or genetic manipulation, production of ethyl acetate may not occur e f f i c i e n t l y in the presence of g l u cose. A production phase for the ester should be separated from the fermentative conversion of glucose to ethanol. 6 2 0
1H
E f f e c t of I n h i b i t i o n of Acetyl-CoA Formation on Ethyl Acetate Accumulation. E t h y l acetate has been shown to be formed from acetic acid and ethanol without any cofactors in S. cerevisiae suggesting an esterase mechanism of biosynthesis (j_7). Other studies (18) found c e l l - f r e e synthesis of ethyl acetate with ethanol and a c e t y l CoA but not with acetic acid as a substrate. From the l a t t e r study J. was proposed that ester formation in yeasts was primarily v i a alcoholysis of acyl-CoA compounds. Others have also suggested acetyl-CoA as an intermediate for ethyl acetate accumulation by S. cerevisiae (19). Yeasts generally catabolize ethanol as follows: ethanol — acetaldehyde — a c e t i c acid — a c e t y l - C o A — o t h e r oxidative pathways [Figure 4]. I t has been speculated that C. u t i l i s forms ethyl acetate by the reaction of acetyl-CoA with ethanol (5). Thus i f the flow of metabolites into the TCA cycle is inhibited or limited, as might occur under iron-limited conditions, acetyl-CoA would be expected to accumulate thus providing a precursor pool for e s t e r i f i c a t i o n of ethanol. Acetyl-CoA synthesis is known to be inhibited by arsenite (20). Therefore, to investigate the s i g n i f i c a n c e of acetyl-CoA in the formation of ethyl acetate, arsenite was added to ethanol-adapted C. u t i l i s c e l l s suspended in fresh ethanol-containing medium [Figure 5]. Even at low concentrations of arsenite (0.1 and 0.2 mM) ethyl acetate accumulation was severely i n h i b i t e d . At an elevated l e v e l of arsenite (1.0 mM) no ethyl acetate was detected whereas the l e v e l s of acetaldehyde and acetic acid increased s i g n i f i c a n t l y . Many metabolic i n h i b i t o r s exert secondary e f f e c t s (20).
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2 Time (d)
4
6
8
10
Figure 1. Kinetics of ethyl acetate accumulation by Candida u t i l i s . Growth on glucose medium without (a) or with iron [100 uM F e C l ] (b). Glucose , ethanol ( A ) , a c e t i c acid (?) shown 10 X actual l e v e l , ethyl acetate ( + ), A (·). (Reproduced with permission from Ref. 12. Copyright 1984, John Wiley & Sons, Inc.) 3
6 2 0
8h
0
25 FeCI
50 3
75
100
(μΜ)
Figure 2. Effect of iron l e v e l s on ethanol u t i l i z a t i o n by Candida u t i l i s . Ethanol accumulated in cultures from glucose at 24 h ( Π ) and 120 h ( » ) .
2
4 6 Time (d)
8
Figure 3. E f f e c t of glucose on ethanol uptake by Candida u t i l i s . See text for d e t a i l s . A r a t i o greater than 1.0 indicates i n h i b i t i o n of ethanol uptake by glucose. Ethanol uptake r a t i o (O), A of o r i g i n a l culture (·-·). 6 20
Production of Ethyl Acetate by Candida utilis
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Environment
Cell
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- > Ethanol
It;
Γ alcohol I dehydrogenase Acetaldehyde Ethyl Acetate
259
aldehyde 4^— J.:dehydrogenase Acetic A c i d [ * - CoA acetyl-Co A synthetase Acetyl - CoA
TCA cycle
—
y i » Acetaldehyde / - /
'Ethyl Acetate > Acetic A c i d — \ $
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Figure 4. Pathway of ethanol u t i l i z a t i o n and ethyl acetate formation (model). Control
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E f f e c t of s e l e c t i v e i n h i b i t o r s on product accumulation Figure 5 (24 h) by Candida u t i l i s Ethyl acetate ( © ) , acetaldehyde (H)t a c e t i c acid ( Q ) ·
260
BIOGENERATION OF AROMAS
Arsenite is known to have some effect on oxidative phosphorylation. However, as can be seen [Figure 5c] the use of an uncoupler of oxidative phosphorylation (2,4-dinitrophenol) did not affect accumul a t i o n of ethyl acetate. From t h i s J. would appear that the main i n h i b i t o r y effect of arsenite on ethyl acetate accumulation by C. u t i l i s is at the l e v e l of acetyl-CoA formation. Thus acetyl-CoA is implicated as a key precursor f o r synthesis of ethyl acetate supporting a model presented e a r l i e r [Figure 4], It has been found that in cultures of C. u t i l i s , ethanol concentrations exceeding about 35 g/L causes a progressive s h i f t in product d i s t r i b u t i o n from ethyl acetate to acetaldehyde (21_) [Figure 6]. Acetaldehyde is known to i n h i b i t acetyl-CoA synthetase which catalyses the formation of acetyl-CoA from a c e t i c acid (7). The r e s u l t s [Figure 6] suggest that higher ethanol concentrations cause higher l e v e l s of acetaldehyde which in turn i n h i b i t s acetyl-CoA formation and thus ethyl acetate accumulation is reduced. These l a t t e r r e s u l t s along with those involving s e l e c t i v e i n h i b i t o r s implicate acetyl-CoA as a key intermediate in ethyl acetate accumulation in C. u t i l i s . Ethyl Acetate Accumulation by C. u t i l i s C e l l s Isolated at Different Phases of Growth. The accumulation of ethyl acetate was previously shown [Figure 1 ] to be concomitant with the onset of u t i l i z a t i o n of ethanol. The question to be asked then was whether the accumulation of ethyl acetate is merely dependent upon the a v a i l a b i l i t y of the ethanol or is there also a requirement for a modification of the metabolic capacity of the c e l l ? To explore t h i s question, C. u t i l i s was grown under a low degree of aeration (H/C = 1.5) in order to delineate d i s t i n c t phases of diauxie [Figure 7]. Aliquots of the culture were i s o l a t e d at d i f f e r e n t points (indicated by arrows), c e l l s were collected and resuspended in fresh ethanol-containing medium under a high l e v e l of aeration (H/C = 4). The capacity of these c e l l s to accumulate ethyl acetate was shown to depend upon the phase of growth during which they were i s o l a t e d . The r e s u l t s of Table I indicate that c e l l s isolated from the second r i s e in the diauxie c e l l mass density curve (stage III) exhibited the greatest e f f i c i e n c y of ethanol conversion to ethyl acetate. I t is postulated that the presence of ethanol alone was not s u f f i c i e n t for e f f i c i e n t conversion to the ester, but the c e l l s needed to undergo some adaptation to allow e f f i c i e n t ester formation. However ethanol u t i l i z a t i o n by C. u t i l i s i s o l a t e d at the different points were s i m i l a r . Thus the main difference between the c e l l s isolated at the d i f f e r e n t stages may be in TCA cycle a c t i v i t y leading to different l e v e l s of acetyl-CoA pools rather than the induction of enzymes for ester synthesis. E f f e c t of Chelating Agents on Product Distribution. In e a r l i e r studies, J. was shown that the addition of EDTA to a culture of C. u t i l i s , growing on glucose, encouraged a rapid production of ethyl acetate (22). The ester accumulated even though glucose was present. In addition, when EDTA was added to a glucose medium supplemented with a high l e v e l of iron, C. u t i l i s produced amounts of the ester comparable to iron-limited cultures. Thus use of EDTA could be b e n e f i c i a l in the r a p i d fermentation of glucose to ethyl acetate even in the presence of r e l a t i v e l y high l e v e l s of iron. The
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