Levels of pentose phosphate pathway enzymes from Candida shehatae grown in continuous culture

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1 Appl Microbiol Biotechnol (1988) 29: Springer-Verlag 1988 Levels of pentose phosphate pathway enzymes from Candida shehatae grown in continuous culture M. A. Alexander 1, V. W. Yang 2, and T. W. Jeffries 2 1 Department of Chemical Engineering, University of Wisconsin, Madison 53706, USA Institute for Microbial and Biochemical Technology, Forest Products Laboratory, Madison, Wisconsin 53705, USA 2 Summary. Candida shehatae exhibits different fermentative capacities when grown under different aeration conditions. These studies investigated the titers of xylose reductase, xylitol dehydrogenase, glucose-6-phosphate dehydrogenase and alcohol dehydrogenase in crude extracts of Candida shehatae grown in continuous culture with various specific aeration rates. Carbon source, aeration rate, dilution rate and temperature were examined as variables. Xylose reductase and xylitol dehydrogenase were induced by xylose and were largely absent in glucose-grown cells. Alcohol dehydrogenase levels were higher in glucose-grown cells than in xylose-grown cells. The levels of this enzyme also correlated with the fermentative character of metabolism, having a low value under fully aerobic conditions, a high value under anaerobic conditions, and intermediate levels under various semi-aerobic conditions. Temperature had no effect on any enzyme level over the range of C. Introduction Candida shehatae is one of a handful of yeasts that is capable of rapidly converting xylose into ethanol (du Preez and van der Walt 1983). It exhibits three different types of metabolic behavior. (1) Under fully aerobic conditions, in which oxygen is available in excess, respirative growth occurs without fermentation (Alexander et al. 1987). (2) Fermentation and respirative growth occur simultaneously under semiaerobic conditions * Maintained in cooperation with the University of Wisconsin-Madison Offprint requests to: T. W. Jeffries wherein growth is limited by the oxygen supply (Alexander et al. 1987, 1988a). (3) Under anaerobic conditions fermentation occurs without growth (du Preez et al. 1984; Wayman and Tsuyuki 1985; Sreenath et al. 1986; van Dijken and Scheffers 1986; Alexander et al. 1988a). Induction appears to be required for fermentation because C. shehatae grown under fullyaerobic conditions does not ferment when transferred to anaerobiosis, whereas cells grown under semi-aerobic conditions do (Alexander et al b). In this respect, C. shehatae is similar to P. tannophilus. Cells of the latter grown under fullyaerobic conditions ferment only poorly when shifted suddenly to anoxia (Bruinenberg et al. 1984). In contrast, cells grown under oxygen limitation carry out a vigorous fermentation following such a shift (Jeffries 1982). In batch culture, a gradual adaptation seems to occur. When started from a small inoculum, P. tannophilus undergoes a transition from a non-fermentative to a fermentative state (du Preez et al. 1984; Mahmourides et al. 1985). P. tannophilus requires oxyen for growth and enzyme induction (Maleszka et al. 1982; Nierinck et al. 1984), and a similar requirement seems to exist for C. shehatae. Aerobic and anaerobic xylose metabolism may be limited by different factors. Aerobic xylose uptake in continuous culture appears to be transport-limited (Alexander et al. 1988a). Alternatively, aerobic xylose consumption could be affected by the levels of xylose reductase (XOR) or glucose 6-phosphate dehydrogenase (GPD) (this enzyme provides the reductant necessary for NADPH-linked xylose reduction). Anaerobic metabolism proceeds at only a third the aerobic rate and may be limited by low levels of key enzymes such as NADH-linked XOR activity, xylitol dehydrogenase (XID) or alcohol dehydrogenase

2 M. A. Alexander et al.: Levels of pentose phosphate pathway enzymes 283 (ADH). Previous studies in our laboratory (Lachke and Jeffries 1986) have shown mutants of P. tannophilus capable of more rapid anaerobic fermentation possess a higher ratio of NADH to NADPH-linked xylose reductase activity and higher ADH activities than the parent cells. NADH-linked XOR can relieve reductant imbalance that arises in cells under anoxic conditions (Bruinenberg et al. 1984). Alcohol dehydrogenase is responsible for ethanol production and may be implicated in the lack of anaerobic fermentation by fully-aerobic cells. Xylitol dehydrogenase may catalyze a rate-limiting step because xylitol occurs as a major byproduct of xylose metabolism, especially under anaerobic conditions (du Preez et al. 1984). The objective of this research was to examine the titers of key enzymes in cells grown under well-defined culture conditions that would affect their physiology. Four important variables which may affect enzyme titers were examined in continuous culture: dilution rate, temperature, aeration condition (fully aerobic, semi-aerobic or anaerobic) and carbon source (xylose vs glucose). Methods and materials Cultivation Candida shehatae ATCC 22984, was maintained on slants of yeast base-malt extract-peptone-glucose agar (YMPG, Difco). Inocula were grown on 0.17% yeast nitrogen base (without amino acids or ammonium sulfate, Difco, Detroit, USA) using 0.66% peptone as a nitrogen source and 2.0% xylose as a carbon source. All fermentation studies were performed using a defined medium that was not nutrient limiting in continuous culture (Alexander et al. 1987). To obtain cells grown under fully-aerobic conditions, excess oxygen was provided to the reactor and the carbon source was constrained so that no residual sugar was observed in the effluent. Such cells were therefore carbon-limited. To obtain cells grown under semiaerobic conditions, the carbon source was provided in excess and the oxygen source was constrained. Such cells were therefore oxygen-limited. An effort was made to maintain a nearconstant cell density of about 10 g dry wt/1 (values ranged from 6 to 13 g/l) by decreasing the aeration rate at the same time as the dilution rate. By these methods, different specific aeration rates could be obtained. The details of the methodologies and the resulting transient and steady-state fermentation kinetics have been previously reported (Alexander et al. 1988a). For the data reported here, only cells from steady-state conditions were employed. Preparation of homogenates Effluent from the continuous culture apparatus was collected by a refrigerated fraction collector and held at 3 C. Periodically, about 3-4 g (wet wt) of cells were pooled for enzyme analysis. The cells were centrifuged, washed twice and suspended in 0.1 M (ph 6.8) MOPS (3-[N-morpholino] propanesulfonic acid) buffer (=2 g wet wt cells/3 ml of slurry), quickfrozen in a dry ice-acetone bath, and stored at 78 C. After thawing, cell slurries (1.0 ml) were placed in a 13 mm i.d. glass tube containing 1 g of 0.5 mm acid-washed glass beds. The cell-bead mixture was chilled in ice and blended in a highspeed vortex mixer for two one-minute bursts with in-between cooling, after which approximately 60% cell disruption was attained (Ciriacy 1975). Cell homogenates were centrifuged at g for 15 min. The supernatant solution was used for enzymatic assays. Enzymatic assays All assays were performed within four hours of cell breakage. D-Xylose reductase (EC ) activity was measured by following the oxidation of NADPH (and NADH) according to the method of Chiang and Knight (1966). Xylitol dehydrogenase (EC ) activity was measured by following reduction of NAD according to the method of Chakravorty et al. (1962). D-Glucose-6-phosphate dehydrogenase (EC ) activity was determined by the method of Koby and Noltmann (1966). Alcohol dehydrogenase (EC ) activity was determined by following the reduction of NAD according to the method of Vallee and Hoch (1955). Analytical Protein determinations were made by the method of Bradford (1976) using bovine serum albumin as standard. All specific activities are expressed in international units per mg protein (IU mg -1 ) where an international unit is defined as µmoles substrate consumed per min. The results for each cell sample are the average of 2-5 assays of each enzyme. Except where noted, the standard deviations reported represent the variance between different samples which had been obtained under comparable conditions. Results No effect of temperature on any enzyme level was evident (data not shown), so for statistical purposes, cells grown at different temperatures (20-30 C) were treated as though they were identical. Neither was an effect of dilution rate apparent on any enzyme except ADH, and then only under semi-aerobic conditions (Table 1). Under these circumstances, in which the growth rate was limited by the oxygen supply, a decreasing dilution rate correlated with an increasing ADH titer (see below). Under fully aerobic growth conditions, no significant changes in any of the enzyme titers were observed over a range of dilution rates (Table 2). However, fewer runs were performed under fully-aerobic conditions (n= 4 for fully-aerobic, versus 13 for semi-aerobic grown cells). In addition, two batches of cells grown semi-aerobically were held for 18 or 30 h under anaerobic conditions prior to breakage and assay. Average values for each enzyme titer (other than ADH) in aerobic, semi-aerobic, and anae-

3 284 M. A. Alexander et al.: Levels of pentose phosphate pathway enzymes robic xylose-grown cells were used to compare the effects of aeration. Aeration did not have a discernible effect on the activity of either NADHor NADPH-linked XOR activity (Table 3). Xylitol dehydrogenase levels were somewhat higher in cells held under anaerobic conditions, but no significant difference was seen between fully-aerobic and semi-aerobic XID levels. Aeration had a strong negative effect on ADH levels. In the case of ADH, the enzyme titer under semi-aerobic conditions varied with the dilution rate (Table 1). Reducing the dilution rate (D) was equivalent to reducing the specific aeration rate because cell density in the reactor was maintained at a near constant value (see cultivation method). As D (= µ) decreased, ADH activity increased (Table 4). Composite values for enzyme titers in xylosegrown cells versus glucose-grown cells were calculated from all of the available data, and the standard deviations for enzyme titers are shown in Table 5. For XID and XOR activities, n = 19.

4 M. A. Alexander et al.: Levels of pentose phosphate pathway enzymes 285 Average values and standard deviations for GPD and ADH under fully aerobic (n= 4) and semiaerobic (n= 13) conditions were also calculated. Only two continuous culture runs (one fully aerobic, one semi-aerobic) were made using glucose as a carbon source, and only a single run was made using a mixture of the two carbon sources, so for these values, the standard deviation represents the variance attributable to the assay rather than to differences between runs or cell samples. For comparison purposes, enzyme titers from cells grown on xylose under the same conditions used for the glucose experiments are also shown. The carbon source had a clear effect on the enzyme levels observed (Table 5). Xylose reductase and XID levels were 15- and 6-fold higher when cells were grown on xylose rather than glucose. Alcohol dehydrogenase levels were 3-fold higher on glucose than on xylose under both aeration conditions. Where data were collected, ADH

5 286 M. A. Alexander et al.: Levels of pentose phosphate pathway enzymes levels intermediate between those obtained from xylose-grown and glucose-grown cells were seen with cells grown on xylose-glucose mixtures. Discussion With some xylose-metabolizing yeasts, their inability to produce ethanol anaerobically has been accounted for by an imbalance between NADH production and NADH consumption (Bruinenberg et al. 1983a, 1984). The imbalance arises because xylose is first reduced to xylitol by an NADPH-dependent aldehyde reductase (= xylose reductase, EC ), and the resulting xylitol is oxidized to D-xylulose by an NAD + -dependent xylitol dehydrogenase (Fig. 1). Xylose reductases are generally specific for NADPH, but show wide specificity for aldehyde substrates (Scher and Horacker 1966; Suzuki and Onishi 1975; Sheys et al. 1971). Yeasts known to convert xylose to ethanol under anoxic conditions (i. e., ferment) also possess an XOR that is active with NADH as well as NADPH (Bolen et al. 1985, Verduyn et al. 1985a, b). In one instance, an XOR from P. tannophilus has been reported as specific for NADH (Ditzelmüller et al. 1985). In P. tannophilus, the ratio of NADH- to NADPH-linked XOR activity varies with aeration conditions (Verduyn et al. 1985a). This finding suggested that P. tannophilus has multiple forms of XOR, which is indeed the case (Ditzelmüller et Fig. 1. Initial steps in the metabolism of D-Xylose by yeasts. Xylose is first reduced to xylitol by NAD(P)H and subsequently oxidized to xylulose by NADH. After phosphorylation, xylulose-5-phosphate is rearranged by non-oxidative reactions to yield hexose phosphate and triose phosphate. A portion of the hexose phosphate can be oxidized via glucose- 6-phosphate dehydrogenase to yield NADPH for assimilation. Otherwise, metabolism continues through the glycolytic pathway to yield ethanol in a balanced fermentation al. 1985; Verduyn et al. 1985a). The invariant ratio of NADH- to NADPH-linked XOR activity observed with C. shehatae under all conditions examined here suggests that there might be only a single form of XOR. In this respect, C. shehatae appears to be similar to Pichia stipitis, which possesses only a single XOR (Verduyn et al b). However, other explanations are possible. Multiple enzymes might be constitutively expressed or coordinately regulated or they might be differently regulated under conditions not yet examined. The effect of carbon source was not surprising: xylose induces the xylose-processing enzymes XOR, XID and possibly, GPD. Glucose- 6-phosphate dehydrogenase levels in xylosegrown cells tended to be 60% greater than in glucose-grown cells, but it is not clear whether this difference is significant (Table 5). In aerobicallygrown Candida utilis, GPD activities in xylosegrown cells are twice those in cells grown on glucose (Bruinenberg 1986). Higher levels of this enzyme in xylose-grown cells are to be expected because growth on xylose requires twice the NADPH as that on glucose (Bruinenberg et al b), and hence needs more GPD activity to produce it. Correlation of ADH with the fermentative character of metabolism The compounded effects of aeration and dilution rate on ADH titers can be interpreted as different aspects of the same phenomenon. In Table 4, measured ADH activities are tabulated along with fermentation rates and cell yield data (µ/q S ) obtained with these same cells (Alexander et al. 1988a). Using the fermentation and cell yield data from cells grown under the various aeration and dilution rate conditions, we constructed an index which represents the fraction of metabolism that is fermentative. This was accomplished by subtracting the respirative fraction of metabolism from the whole. The cell yield, Y X/S (=µ/qs), is approximately equal to 0.5 for purely respirative (i.e. fully-aerobic) conditions and no fermentation occurs (Table 2). The cell yield is zero under purely fermentative (i.e. anaerobic) conditions because no growth occurs. Under semi-aerobic conditions µ/ Q S is proportional to the dilution rate and approaches 0.5 as µ approaches µ MAX, equal to 0.28 h -1 at 30 C (Table 4). Under semi-aerobic conditions, the specific fermentation rate (Q E ) increases as the growth rate decreases (Alexander et

6 M. A. Alexander et al.: Levels of pentose phosphate pathway enzymes 287 Fig. 2. Alcohol dehydrogenase activity versus degree of fermentative character. Alcohol dehydrogenase specific activity is given as IU/mg protein. The fraction of metabolism considered fermentative is given by equation (1). Data from aerobically-grown (Table 2), semi-aerobically grown and anaerobically-held cells (Table 4) are included al. 1988a). If we multiply µ/q S by two, we obtain a quantity which varies from zero for purely fermentative metabolism to unity for completely non-fermentative (i.e. respirative) metabolism. In order to obtain an index with a positive correlation with ADH activity, 2 (µ/q S ) is subtracted from one: Fraction fermentative = 1 2 (µ/q S ) (1) Alcohol dehydrogenase levels were plotted against the cell yield as expressed in the index given by Eq. (l). Fully aerobic cells, which were not fermentative, showed constant ADH levels independent of the dilution rate. Cells grown under semi-aerobic conditions showed varying ADH levels, depending on the dilution rate at which they were maintained. As shown in Fig. 2, up to about 30% of the xylose taken up could be metabolized to ethanol before ADH activity was induced to a higher level. Implication of enzymatic differences in different fermentation capacities The inability of fully-aerobic cells to metabolize xylose anaerobically was not due to a lack of NADH-linked XOR activity because this activity was present in xylose-grown cells, regardless of the degree of aeration. Alcohol dehydrogenase levels were lower under fully aerobic than under semi-aerobic conditions suggesting that this enzyme was involved in the lack of anaerobic fermentation by aerobically-grown cells. However, fully aerobic cells did exhibit significant ADH activities. Thus, the absence of this enzyme by itself can not explain the lack of anaerobic fermentation by aerobic cells. Several types of ADH exist in Saccharomyces cerevisiae (Wills and Phelps 1975), one of which is located in the mitochondria (Ciracy 1975). Cytoplasmic ADH is necessary for anaerobic fermentation (Ciriacy 1975). Net ethanol production by mitochondrial ADH is prevented by a redox imbalance caused by metabolic compartmentation: NAD produced by ADH in the mitochondria cannot penetrate the mitochondrial membrane, and so cannot enter the cytoplasm where it is required for glycolysis (Wills and Phelps 1975). Other studies in our laboratory have shown that when C. shehatae is grown under various aerobic conditions, it possesses a single band of ADH. The total ADH activity increases dramatically and the number of isozyme bands increases from one to three following a shift to semi-aerobic, fermentative conditions (Prior et al. 1988). These data suggest that the increased ADH activity that we observe is attributable to an increase in the cytoplasmic ADH component. Implication of enzymatic factors in anaerobic rate limitations The specific xylose uptake rate by C. shehatae in semi-aerobic continuous culture is a constant independent of the growth rate (Table 4) and is equal to the maximum rate of xylose uptake in xylose-limited culture (=µ MAX /Y X/S =0.55 g g -1 h -1 at 30 C), suggesting a transport limitation. Xylose uptake rates under anaerobic (anoxic) conditions are only a third of those observed under semi-aerobic conditions (Table 5), suggesting that some factor other than transport limits anaerobic xylose metabolism. Potential candidates for a rate-limiting enzyme should show anaerobic activities not greater than one third of their semi-aerobic values. Of the four enzymes examined, only XOR meets this criterion: NADH-linked XOR activity (the relevant XOR activity in anaerobiosis) is only 27% of NADPHlinked XOR activity (the relevant XOR activity under aerobic conditions). Under aerobic or semiaerobic conditions, both the NADPH- and the NADH-linked XOR activities can function because respiration can recycle the excess NADH formed as a consequence of xylose assimilation back to NAD (Fig. 1). Under anaerobic conditions, the NADH-linked XOR activity functions as an electron acceptor permitting xylose

7 288 M. A. Alexander et al.: Levels of pentose phosphate pathway enzymes metabolism to proceed. The NADPH-linked activity cannot recycle NADH to NAD and so is inactive under anoxic conditions. In summary, NADH-linked XOR is probably rate-limiting under anaerobic conditions by fermentative cells. The amount of ADH present in cells grown under various specific aeration rates increases with fermentative activity and decreases with aeration, suggesting that larger amounts of ADH are synthesized in proportional response to the metabolic demand. The absence of cytoplasmic ADH is postulated to account for the inability of respiratively-grown cells to ferment when they are placed under anaerobic conditions without a chance to adapt. Received February 25, 1988/Accepted April 7, 1988