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2530469 792.798Appl Microbiol Biotechnol (2000) 54: 792±798 M. Saayman á H. J. J. van Vuuren á W. H. van Zyl M. Viljoen-BloomDifferential uptake of fumarate by Candida utilis Received: 15 March 2000 / Received revision: 4 July2000 / Accepted: 9 July2000 Abstract The dicarboxylic acid fumarate is an impor- of the TCA cycle (Barnett and Kornberg 1960). Previous tant intermediate in cellular processes and also serves as studies have shown that L-malate can be utilised by a precursor for the commercial production of ®ne Candida utilis (CaÂssio and LeaÄo 1993), Candida sphaer- chemicals such as L-malate. Yeast species dier re- ica (CoÃrte-Real et al. 1989), Hansenula anomala (CoÃrte- markablyin their abilityto degrade extracellular di- Real and LeaÄo 1990) and Kluyveromyces marxianus carboxylic acids and to utilise them as their only source (Queiros et al. 1998) as their onlysource of carbon and of carbon. In this studywe have shown that the yeast energy. In these species, the dissociated form of L-malate Candida utilis eectivelydegraded extracellular fumarate is transported across the plasma membrane bya and L-malate, but glucose or other assimilable carbon H+-symport system that is substrate-inducible and sources repressed the transport and degradation of these subject to glucose repression. In contrast, Schizosac- dicarboxylic acids. The transport of both dicarboxylic charomyces pombe and Zygosaccharomyces bailii can acids was shown to be stronglyinducible byeither degrade L-malate onlyin the presence of an assimilable fumarate or L-malate while kinetic studies suggest that carbon source (Rodriguez and Thornton 1990; Osothsilp the two dicarboxylic acids are transported by the same and Subden 1986a). Other yeasts such as Saccharomyces transporter protein. In contrast, Schizosaccharomyces cerevisiae can import L-malate and other dicarboxylic pombe eectivelydegraded extracellular L-malate, but acids onlyvia simple diusion (Salmon 1987) and is not fumarate, in the presence of glucose or other as- therefore unable to eectivelydegrade or utilise extra- similable carbon sources. The Sch. pombe malate cellular L-malate.
transporter was unable to transport fumarate, although In Sch. pombe, the dissociated form of L-malate is fumarate inhibited the uptake of L-malate.
activelytransported via a H+-symport system that op- erates constitutively, whereas the undissociated acid enters the cell via simple diusion (Baranowski and Radler 1984; Osothsilp and Subden 1986b; Sousa et al.
1992). The dicarboxylic acids fumarate, D-malate, succinate, oxaloacetate, maleate, malonate and a-keto- The C4-dicarboxylic acid fumarate serves as an inter- glutarate acted as competitive inhibitors for the uptake mediate of the tricarboxylic acid (TCA) cycle that allows of L-malate (Sousa et al. 1992), suggesting a common for the metabolic ¯ow of carbon between various met- transporter for the uptake of dicarboxylic acids in ®ssion abolic pathways. Yeast species dier remarkably in their yeast. However, Grobler et al. (1995) showed that abilityto transport and utilise one or more intermediates L-malate, succinate and malonate, but not a-ketogluta- rate, were activelytransported bySch. pombe cells.
In addition to its role in metabolic processes, fuma- rate is also an important precursor for the commercial M. Saayman á W. H. van Zyl á M. Viljoen-Bloom (&) Department of Microbiology, University of Stellenbosch, production of ®ne chemicals such as L-malate. The D,L- Private Bag X1, Matieland 7602, South Africa malate racemic mixture is routinelyused in a varietyof foods and beverages whereas the L-isomer is used for the treatment of conditions such as hyperammonaemia (Rosenberg et al. 1999). The racemic mixture is com- merciallyproduced via chemical hydratation of maleate B.C. Wine Research Centre, Facultyof Agricultural Sciences, Universityof British Columbia, Vancouver BC, or fumarate, and the L-isomer through the enzymatic conversion of fumarate using fumarase-containing microbial cells. The bioconversion of fumarate to L- malate has been obtained bystrains of Brevibacterium (Takata et al. 1980), Candida rugosa (Yang et al. 1992), Pichia (Keruchen'ko et al. 1995) and Dipodascus (Ro- senberg et al. 1999) that exhibit high fumarase activities.
Over-expression of the Sac. cerevisiae fumarase gene, FUM1, also resulted in an increased conversion rate of fumarate to L-malate (Peleg et al. 1990). Since Sac. ce- revisiae can onlyimport fumarate through diusion, the introduction of a fumarate transporter gene into Sac.
cerevisiae could enable this yeast to actively transport fumarate and consequentlyimprove the bioconversion Heterologous expression of the Sch. pombe malate transporter gene, mae1, in a strain of Sac. cerevisiae resulted in the active transport and ecient degradation L-malate (Volschenk et al. 1997a, b). Our ®rst ap- proach was therefore to determine whether expression of the mae1 gene in Sac. cerevisiae would also enable the recombinant strain to transport fumarate. We found that neither the recombinant Sac. cerevisiae strain nor the wild type Sch. pombe strain could transport fuma- rate. In search of an alternative fumarate transporter, several yeast species were evaluated for their ability to degrade extracellular fumarate. Since C. utilis proved to be able to degrade both fumarate and L-malate, the transport of these dicarboxylic acids was further inves- The yeast strains used in the transport studies included C. utilis ATCC 9950 T, Sch. pombe 972 h) (Osothsilp 1987), Sch. pombe 972 h) leu1-32 TR) mae1) (Osothsilp and Subden 1986b) and Sac.
cerevisiae YPH259 (MATa ura3-52, lys2-801a, ade2-101o, his3D200, leu2-D1) (Sikorski and Hieter 1989). The strains used for the screen on fumarate/malate indicator plates are listed in Table 1. Unless otherwise stated, the growth media contained 0.17% YNB (yeast nitrogen base without amino acids and ammonium sulphate [Difco with amino acids and buered at pH 3.5. Dierent concentrations of fumarate, L-malate and/or a carbon source were added as indi- cated for the dierent experiments.
Degradation of extracellular fumarate and Indicator agar plates were used to screen dierent yeast species for the degradation of fumarate and L-malate in the presence of dif- ferent carbon sources. The yeast strains were streaked onto YNB agar plates containing 0.05% bromocresol-green, 0.3% fumarate or L-malate, together with 2% glucose, fructose, galactose, glyc- erol, maltose, ranose or sucrose as carbon source. The plates were incubated at 30 °C for 2 days and evaluated for a colour change from yellow at pH 3.3 to blue at pH 5.2.
The utilisation of extracellular fumarate and C. utilis and Sch. pombe was determined after growth in liquid YNB media containing 2% glucose, ranose or glycerol/ethanol as carbon source, supplemented with either 0.5% fumarate or malate. Cells were harvested at dierent time intervals and high performance liquid chromatography(HPLC) was used to deter- mine the residual levels of fumarate and L-malate. Glucose con- centrations were measured with the glucose oxidase method (Glucose [Trinder], Sigma, St Louis, Mo.) and cell growth was determined spectrophotometricallyat OD600. All assays were done Results Cells of Sac. cerevisiae YPH259 transformed with plasmid pHV3 Strains of Sac. cerevisiae cannot transport extracellular containing the Sch. pombe mae1 gene (Volschenk et al. 1997b), and wild type Sch. pombe 972 cells were grown in YNB media con- dicarboxylic acids such as L-malate or fumarate (Salmon taining 2% glucose. For C. utilis, cells were cultured in 0.5% 1987). However, transport studies with a recombinant fumarate, 0.5% L-malate, 2% glucose, 2% ranose or 2% glyc- Sac. cerevisiae strain expressing the mae1 gene of Sch.
erol/ethanol as the onlysource of carbon. To further investigate the pombe, showed that active transport of 14C-malate was eect of dierent carbon sources on the transport of fumarate and obtained, whereas fumarate was not transported L-malate, C. utilis cells were cultured to OD600 of 0.6 in media (Fig. 1a). The active transport of 14C-malate bythe wild glycerol/ethanol and divided into two batches. One batch of cul- type Sch. pombe 972 h) strain was con®rmed (Fig. 1b), tures was assayed immediately while the other was transferred to but not in the Sch. pombe mae1) mutant strain that has fresh medium containing either 0.5% L-malate or 2% glucose as a defective malate transporter. No transport of 14C- carbon source and incubated for another 6 h.
Cells were harvested in the exponential growth phase (OD fumarate was observed in either strain (Fig. 1b). How- 0.6), washed twice with ice-cold distilled water and resuspended in ever, increasing concentrations of fumarate progres- 0.1 M KH2PO4 (pH 3.5) to a ®nal concentration of approximately sivelyinhibited L-malate uptake bythe recombinant Sch.
20 mg dryweight ml)1 (adapted from Grobler et al. 1995). Cell pombe malate transporter (Fig. 1c). The HPLC analyses suspensions were pre-incubated for 5 min at 30 °C in a shaker waterbath at 100 rpm. Assays were initiated by adding 10 ll of con®rmed that Sch. pombe cells removed a signi®cant an aqueous solution of [1-14C]-fumarate (6.62 lCi/lmol; ICN portion (approximately65%) of the L-malate from the Pharmaceuticals, CA) or L-[1,4(2,3)-14C]-malate (55 lCi/lmol; glucose-containing growth media within 28 h (Fig. 2a), Amersham, Bucks, UK). Non-speci®c binding of 14C-fumarate or whereas less than 15% of the fumarate was removed 14C-malate to the yeast cells was determined by pre-boiling the cells (Fig. 2b). Similar results were obtained for cells grown for 5 min at 100 °C. Samples of 0.5 ml were withdrawn at dierent time intervals and the reactions were stopped bydilution with 5 ml in media containing ranose or glycerol/ethanol as ice-cold distilled water. The cells were rapidly®ltered through 0.45 lm membranes (Millipore Corporation, Bedford, Mass.) and Since fumarate inhibited the transport of L-malate in immediatelywashed with 5 ml ice-cold distilled water. The ®lters the recombinant Sac. cerevisiae strain without being were air dried for 10 min and placed in scintillation vials with 5 ml scintillation reaction mixture (EcoLite, ICN Pharmaceuticals, transported itself, the uptake and subsequent cellular Calif.). Levels of radioactivitywere measured with a Beckman localisation of 14C-malate and fumarate was further in- LS 3801 scintillation counter (Beckman Instruments, Calif.).
vestigated in wild type Sch. pombe cells (Table 2). An hour after the addition of 14C-malate to glucose-grown Cellular fractionation for localisation of dicarboxylic acids cells, approximately30% of the 14C-malate was re- moved from the extracellular fraction. The majorityof Cultures of Sch. pombe in 10 ml YNB medium containing 2% this was alreadyfurther metabolised to pyruvate and glucose were harvested at OD600 of 0.6 and resuspended in 1 ml of CO 2 with only0.36% and 0.32% retained in the cell 2PO4 (pH 3.5). Cultures were incubated for another hour with 1 ll of either 14C-fumarate (6.62 lCi/lmol) or 14C-malate debris and intracellular fractions, respectively. The ad- (55 lCi/lmol). A ®nal concentration of 0.5% non-labelled fuma- dition of unlabelled fumarate decreased the uptake of L-malate was added to the 14C-malate or 14C-fumarate C-malate by20% and reduced the localisation of 14C- cultures, respectively. Cells were harvested and the supernatant was malate in the cell debris and intracellular fractions by transferred to scintillation vials containing 5 ml scintillation reac- tion mixture. The cells were resuspended in 300 ll of 0.1 M When 14C-fumarate was added to the Sch. pombe 2PO4 (pH 3.5) together with 0.3 g glass beads (106 lm diame- ter). Cells were broken with 10 pulses of 15 s with 1 min on ice cells, only1.9% was removed from the extracellular between pulses. The supernatant and cell debris were separated fraction after 1 h, but almost 10% of this was retained in through centrifugation and transferred to scintillation vials con- the cell debris (Table 2). Although the addition of un- taining 5 ml scintillation reaction mixture. The levels of radioac- tivitywere determined as described above.
labelled L-malate did not signi®cantlyin¯uence the up- take of 14C-fumarate, it decreased its localisation in the cell debris bymore than 50%. These results suggested Kinetic parameters for protein-mediated transport that fumarate competes for the uptake of L-malate by Cells of C. utilis were cultured in YNB medium containing 0.5% inhibiting its binding to the malate transporter, although fumarate as the onlycarbon source. Cells were harvested in the only L-malate is activelytransported bythe protein.
exponential growth phase (OD600 of 0.6), washed twice with ice- cold distilled water and resuspended in 0.1 M KH2PO4 (pH 3.5) to a ®nal concentration of 7 mg dryweight ml)1. Transport assays were initiated byadding increasing concentrations of 14C-fumarate Screening of yeast species for degradation (0.015±2 mM) in the presence or absence of 2 mM non-labelled L-malate. Estimates of kinetic parameters were obtained from Lineweaver-Burk plots of the initial uptake rates of 14C-fumarate.
In a screen for yeasts capable of transporting fumarate m for total dicarboxylic acids was based on the concentra- tions of both anionic and undissociated dicarboxylic acids.
and L-malate, several yeast species were screened for Fig. 2 HPLC analyses of extracellular concentrations of a L-malate and b fumarate during growth of Sch. pombe 972 h) on medium their abilityto degrade extracellular fumarate or L- malate incorporated into fumarate/malate indicator agar plates (Table 1). The yeasts Sch. pombe and Sac. cere- visiae are not able to utilise intermediates of the TCA cycle as their only source of carbon (Barnett and Kornberg 1960), whereas the other species that were investigated are known for their abilityto utilise TCA cycle intermediates. No degradation of either fumarate or L-malate was found for Sac. cerevisiae, since the yeast is unable to transport either of the dicarboxylic acids. In Sch. pombe, L-malate was eectivelydegraded in the presence of all the carbon sources that sustained growth, but no degradation of fumarate was observed. For C. utilis, degradation of both fumarate and L-malate Fig. 1 Transport studies to determine the uptake of L-malate and were found in all the carbon sources investigated, except fumarate bystrains of Schizosaccharomyces pombe and Saccharomy- for glucose (Table 1). The other yeast species showed ces cerevisiae. a Uptake of 14C-malate and 14C-fumarate in Sac.
varying abilities to utilise fumarate or L-malate that cerevisiae cel s transformed with the Sch. pombe mae1 gene. b Uptake of 14C-malate and 14C-fumarate by Sch. pombe 972 h) (wt) and Sch.
seemed to be dependent on the available carbon source.
pombe mae1) (mae1)) grown in 2% glucose. c Competition by Since the indicator plates onlyprovided limited infor- fumarate for the transport of 4 mM 14C-malate at pH 3.5 bythe Sac.
mation, further investigation was required to better cerevisiae YPH259 host strain (control) or transformed with the Sch.
understand the regulatorymechanisms involved in the pombe mae1 gene. The yeast strains were grown in 2% glucose without fumarate, or with 10 mM, 20 mM or 30 mM non-labelled degradation and transport of fumarate and L-malate by fumarate added simultaneouslywith the 14C-malate Cellular distribution (% of total 14C added) Grown in 2% glucose, add fumarate and 14C-malate Grown in 2% glucose, add L-malate and 14C-fumarate both fumarate and L-malate is subject to catabolite When C. utilis cells were pre-cultured in either fum- Cells of C. utilis eectivelydegraded extracellular fum- arate or L-malate as the onlycarbon source, most of the arate when grown in YNB medium containing fumarate 14C-fumarate was taken up within 10 s of addition as the onlycarbon source (Fig. 3a). However, the de- (Fig. 4a). However, the uptake of 14C-fumarate bycells gradation of fumarate was less ecient when grown in grown on either glucose or ranose as the onlycarbon the presence of either ranose or glycerol/ethanol as source was almost non-detectable, with onlya small carbon source, suggesting that other assimilable carbon amount transported bycells grown on glycerol/ethanol.
sources mayresult in catabolite repression of fumarate Similar results were obtained for the transport of L- transport. In support of this, the degradation of fuma- malate in C. utilis (data not shown), indicating that ac- rate bycells grown in glucose/fumarate media only tive transport of both fumarate and L-malate was subject commenced once the glucose had been depleted to substrate induction byeither dicarboxylic acid.
(Fig. 3b). Similar results were obtained for L-malate The transport of 14C-malate by C. utilis was further (data not shown), indicating that the degradation of investigated byshifting cultures grown on dierent carbon sources to fresh medium containing either 0.5%L-malate or 2% glucose (Fig. 4b, c). 14C-malate was quicklytransported bycells grown on either fumarate orL-malate, but transport ceased when cells were trans- ferred to glucose-containing medium (Fig. 4b). Cells were unable to transport 14C-malate when grown on glucose or glycerol/ethanol medium, not even when 0.5%L-malate was included in the glucose medium (Fig. 4c).
However, cells grown on glucose medium regained their abilityto transport 14C-malate when transferred to medium containing L-malate as the onlycarbon source Preliminarykinetic studies were done to determine whether C. utilis uses the same transporter protein for the uptake of fumarate and L-malate. Lineweaver-Burk plots of the initial rates of uptake of 14C-labelled fum- arate at pH 3.5 were linear over the concentration range of 0.08±2 mM (Fig. 5). The following kinetic parameters were calculated: Vmax(fumarate)(pH 3.5) 1.058 nmol s)1 mg (dryweight) cells)1; Km(pH 3.5) = 0.11 mM. These results indicated that fumarate and L-malate were mu- tuallycompetitive inhibitors, suggesting that theymight share the same carrier protein in C. utilis.
The dicarboxylic acid L-malate is widelyemployed in both the pharmaceutical and food industries. Due to its industrial importance, several groups have investigated the bioconversion of fumarate to L-malate using mi- crobial cells (Takata et al. 1980; Yang et al. 1992; Fig. 3 HPLC analyses showing the residual levels of fumarate after Keruchen'ko et al. 1995; Rosenberg et al. 1999).
growth of C. utilis on a 0.5% fumarate, 2% ranose or 2% glycerol/ ethanol or b 2% glucose as carbon source (residual concentration of Increased bioconversion of fumarate to L-malate (80.4 mmol fumaric acid/h per g of cell wet weight) was Fig. 5 Lineweaver-Burk plots of the initial uptake rates of 4 mM 14C- fumarate byfumarate-grown cells as a function of the fumarate concentration in the media. Assays were done in the presence or obtained byover-expression of the Sac. cerevisiae fu- marase gene, FUM1 (Peleg et al. 1990). This eciency maybe further improved if the Sac. cerevisiae cells were able to activelytransport fumarate and not have to rely onlyon diusion of the substrate. This could be realised through heterologous expression of a suitable fumarate transporter from another yeast in Sac. cerevisiae.
A screen for yeast strains that could degrade extracel- lular fumarate showed signi®cant dierences in the regu- lation and speci®cityfor the uptake of fumarate andL-malate between yeast species. A common dicarboxylic acid transporter was suggested for Sch. pombe strain ICV'M (Sousa et al. 1992), but results presented here showed that neither the wild type Sch. pombe 972 h) strain nor a recombinant strain of Sac. cerevisiae containing the Sch. pombe malate transporter gene was able to transport fumarate (Fig. 1). However, increasing concentrations of fumarate were able to progressivelyinhibit the uptake ofL-malate bythe recombinant strain. Cellular fractiona- tion of glucose-grown cells (Table 2) showed that the addition of unlabelled fumarate decreased both the up- take and membrane localisation of 14C-malate. The data suggested that fumarate can also bind to the malate transporter and therefore inhibit the uptake of L-malate.
The binding of both fumarate and L-malate to the Sch.
pombe malate transporter can be ascribed to the struc- Fig. 4 Transport studies to determine the uptake of 14C-labelled L- tural relatedness of the two dicarboxylic acids. Similarly, malate and fumarate by C. utilis. a Uptake of 14C-fumarate after Grobler et al. (1995) reported that a-ketoglutarate was growth on 2% glucose, 2% ranose, 2% glycerol/ethanol, 0.5% fumarate or 0.5% L-malate as onlycarbon source. b Uptake of 14C- not transported by Sch. pombe, although it competed for malate after growth on 0.5% fumarate or 0.5% L-malate as only the uptake of L-malate (Sousa et al. 1992).
carbon source, and shifted to fresh medium containing 2% glucose. c The results presented here indicate a signi®cant dif- Uptake of 14C-malate after growth on 2% glucose or 2% glycerol/ ference in the transport of fumarate and L-malate by ethanol with or without 0.5% L-malate. Glucose-grown cells were also C. utilis and Sch. pombe. Cells of Sch. pombe 972 eec- shifted to fresh medium containing 0.5% L-malate tivelytransported L-malate, but not fumarate, and no evidence for substrate induction or glucose repression for the uptake of L-malate was found. In contrast, the C. utilis ATCC 9950 T strain eectivelytransported both fumarate and L-malate and the uptake of both References dicarboxylic acids was induced by either of the sub- strates. The kinetic data suggest that fumarate and Baranowski K, Radler F (1984) The glucose-dependent transport malate are transported bythe same carrier protein in C.
of L-malate in Zygosaccharomyces bailii. Antonie Van Leeu- utilis, which explains the similar regulatorymechanisms Barnett JA, Kornberg HL (1960) The utilisation byyeast of acids observed for the transport of fumarate and L-malate.
of the tricarboxylic acid cycle. J Gen Microbiol 23: 65±82 The degradation of either fumarate or L-malate by CaÂssio F, LeaÄo C (1993) A comparative studyon the transport of C. utilis was sensitive to the presence of glucose (Fig. 3).
l(-)malic acid and other short-chain carboxylic acids in the yeast Candida utilis: evidence for a general organic acid permease.
This supports previous reports that the utilisation of L-malate in C. utilis strain IGC 3092 was subject to CoÃrte-Real M, LeaÄo C (1990) Transport of malic acid and other glucose repression (CaÂssio and LeaÄo 1993). In addition, dicarboxylic acids in the yeast Hansenula anomala. Appl Envi- we observed that the transport of either fumarate or CoÃrte-Real M, LeaÄo C, Van Uden N (1989) Transport of L-malic L-malate was also insigni®cant in the presence of other acid and other dicarboxylic acids in the yeast Candida sphaerica.
carbon sources such as ranose and glycerol/ethanol (Fig. 4). This con®rmed that C. utilis employs a double Grobler J, Bauer F, Subden RE, Van Vuuren HJJ (1995) The mae1 gene of Schizosaccharomyces pombe encodes a permease for fumarate with the dicarboxylic acids only being trans- malate and other C4 dicarboxylic acids. Yeast 11: 1485±1491 Keruchen'ko YS, Kerucheno'ko ID, Gladilin KL (1995) Forma- ported in the presence of either of the inducers and when tion of malate byyeast of the genus Pichia. Prikl Biochim no alternative carbon source is available.
The carbon sensitivityand substrate induction ob- Osothsilp C (1987) Genetic and biochemical studies of malic acid metabolism in Schizosaccharomyces pombe. PhD dissertation, C. utilis could be interpreted in the context of its ability Osothsilp C, Subden RE (1986a) Isolation and characterisation of to utilise intermediates of the TCA cycle as the only Schizosaccharomyces pombe mutants with defective NAD- source of carbon and energy. The yeast C. utilis is dependent malic enzyme. Can J Microbiol 32: 481±486 Crabtree-negative and can therefore ferment sugars only Osothsilp C, Subden RE (1986b) Malate transport in Schizosac- charomyces pombe. J Bacteriol 168: 1439±1443 under oxygen-limited conditions (Van Dijken et al. Peleg Y, Rokem JS, Goldberg I, Pines O (1990) Inducible overex- 1993). Under aerobic growth conditions, the yeast pression of the FUM1 gene in Saccharomyces cerevisiae: Lo- tended to channel most of its pyruvate into the TCA calization of fumarase and ecient fumaric acid bioconversion cycle, resulting in an adequate supply of intracellular to L-malic acid. Appl Environ Microbiol 56: 2777±2783 TCA cycle intermediates such as fumarate and Queiros O, Casal M, Altho S, Moradas-Ferreira P, LeaÄo C (1998) Isolation and characterization of Kluyveromyces marxianus Since the degradation of glucose, ranose or glycerol/ mutants de®cient in malate transport. Yeast 14: 401±407 ethanol can provide pyruvate for the TCA cycle, the Rodriguez SB, Thornton RJ (1990) Factors in¯uencing the utili- dicarboxylic acids will most likely only be utilised if a sation of L-malate byyeasts. FEMS Microbiol Lett 72: 17±22 more ecient carbon source is not available. The results Rosenberg M, MikovaÂ H, KrisÏtofõÂkovaÂ L (1999) Formation of presented here support the notion that C. utilis cells al- low the transport of fumarate and L-malate onlyin the Salmon JM (1987) L-malic acid permeation in resting cells of an- presence of the inducers and when an alternative carbon aerobicallygrown Saccharomyces cerevisiae. Biochim Biophys source is not available. Furthermore, the results pre- Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast sented in Fig. 3 indicate that the catabolite repression is host strains designed for ecient manipulation of DNA in stronger when cells are grown on glucose than on the Saccharomyces cerevisiae. Genetics 122: 19±27 less favourable carbon source glycerol/ethanol.
Sousa MJ, Mota M, LeaÃo C (1992) Transport of malic acid in the Although the transport of dicarboxylic acids has been yeast Schizosaccharomyces pombe: evidence for a proton± described for a number of yeast species, the Sch. pombe dicarboxylate symport. Yeast 8: 1025±1031 Takata I, Yamamoto K, Tosa T, Chibata I (1980) Immobilization mae1 gene is the onlymalate transporter gene cloned of Brevibacterium ¯avum with carrageenan and its application and sequenced thus far (Grobler et al. 1995). In this for continuous production of L-malic acid. Microb Technol 2: study, we demonstrated signi®cant dierences between Sch. pombe and C. utilis concerning the uptake of fum- Van Dijken JP, Weusthuis RA, Pronk JT (1993) Kinetics of growth and sugar consumption in yeasts. Antonie Van Leeu- arate and L-malate and the regulation thereof. However, a proper investigation into the molecular basis for the Volschenk H, Viljoen M, Grobler J, Bauer F, Lonvaud-Funel A, transport of fumarate and L-malate by C. utilis can only Denayrolles M, Subden RE, Van Vuuren HJJ (1997a) Malo- be done once the fumarate/malate transporter gene from lactic fermentation in grape musts bya geneticallyengineered strain of Saccharomyces cerevisiae. Am J Enol Vitic 48: 193± Volschenk H, Viljoen M, Grobler J, Petzold B, Bauer F, Subden Acknowledgements We thank Q. Willemse and M. Blom for RE, Young RA, Lonvaud-Funel A, Denayrolles M, Van technical assistance with HPLC analyses. This work was funded by Vuuren HJJ (1997b) Engineering pathways for malate degra- WINETECH, FRD grant 2016111 and THRIP grant 2038508 to dation in Saccharomyces cerevisiae. Nat Biotechnol 15: 253±257 H.J.J.v.V., and THRIP grant 2040900 to M.V. Experiments were Yang LW, Wang XY, Wei S (1992) Immobilization of Candida conducted in accordance with South African law on the handling of rugosa having high fumarase activity with polyvinyl alcohol.
Evolution and Human Behavior 22 (2001) 61±69Second to fourth digit ratio and male ability in sport:implications for sexual selection in humansaPopulation and Evolutionary Biology Research Unit, School of Biological Sciences, University of Liverpool,bFootball Research Unit, Department of History, University of Liverpool, Liverpool L69 3BX, UKReceived 14 July 2000; accepted 16 October 2000