Strain 1403-7A is a MAL constitutive strain that lacks invertase activity, but it still grows and ferments efficiently sucrose due to its active transport into the cell, and intracellular hydrolysis by a cytoplasmic α-glucosidase 424445. Since several different permeases (with differing kinetic properties) have been described as capable of active sucrose-H⁺ symport 40, we decided to investigate the role that sugar transport could have on sucrose utilization by this yeast cells. Sucrose transport kinetics by strain 1403-7A (Figure 1) indeed indicated the presence of both a high-affinity (K_m~7 mM) and low-affinity (K_m~120 mM) transport activities, as has already been described for other wild-type yeast strains 40. The AGT1 permease is responsible for the high-affinity component, while the low-affinity transport activity is mediated by any of the MALx1 maltose permeases. Indeed, this strain contains in its genome (Figure 2) at least 3 different MALx1 permeases, MAL21 (chromosome III), MAL31 (chromosome II), and MAL41 (chromosome XI), while the AGT1 gene is located in chromosome VII. It should be stressed out that both genetic and molecular studies 4647 have already shown that in strain 1403-7A the MAL31, MAL41 and AGT1 permeases are functional, and we do not know if the MAL21 gene encodes for a functional permease. Our PFGE and blotting analysis also showed the presence of a unique SUC gene (SUC2 in chromosome IX, data not shown) in the genome of strain 1403-7A, probably a mutant suc2 allele of the gene since this strain lacks invertase activity 424445.

In order to develop a new yeast strain that would take up sucrose from the medium by just the low-affinity transport activity, we deleted from strain's 1403-7A genome the high-affinity sucrose-H⁺ symporter encoded by the AGT1 gene (see strain LCM001 in Fig. 2). As expected, the kinetics of active sucrose transport indicated that in the agt1Δ strain LCM001 sucrose-H⁺ symport was mediated by a low-affinity and low-capacity transport activity (Fig. 1). Since the AGT1 permease is a low-affinity (K_m 20–30 mM) maltose transporter 41, in strain LCM001 maltose uptake from the medium was normally mediated by the above indicated high-affinity MALx1 (MAL21, MAL31 and MAL41) maltose transport activities (data not shown).

The kinetics of sucrose consumption and ethanol production during batch fermentations (Figure 3) indicated that the agt1Δ strain LCM001 had a significant slower capacity to consume sucrose from the medium (at a concentration of 20 g/L), producing less ethanol than the parental wild-type strain 1403-7A. This phenotype was more pronounced when fermentations were performed in synthetic medium, compared with rich YP medium (Fig. 3). Strain 1403-7A showed rates of maximum sucrose consumption of 0.25 and 0.78 g sucrose (g cell dry weight)^-1 h^-1 in synthetic and rich medium, respectively, while the maximum sucrose consumption rate was reduced fourfold in the agt1Δ LCM001 strain, to 0.06 and 0.17 g sucrose (g cell dry weight)^-1 h^-1 in synthetic and rich medium, respectively. The beneficial effect of complex nitrogen sources (peptides or amino acids) compared with ammonium sulfate present in the synthetic medium has been described previously 484950. Furthermore, no major difference in the rates of sucrose consumption or ethanol production were observed among both strains when high sucrose concentrations (> 200 g/L) were used (Figure 4). The maximum sucrose consumption rates for the wild-type and the agt1Δ strains were 0.99 and 0.78 g sucrose (g cell dry weight)^-1 h^-1, respectively, which is in accordance with the kinetics of active sucrose transport presented by each strain (Fig. 1). At 20 g/L sucrose (~58 mM) the low affinity transport system present in strain LCM001 is far from saturated (Fig. 1), while at this concentration the wild type strain's high-affinity transporter is operating with high capacity. At concentrations of ~0.73 M sucrose (250 g/L), probably all transport activities present in both strains are operating with high capacity allowing efficient sucrose fermentation. An intriguing observation was that even when the yeast cells were consuming sucrose more slowly, the increases in biomass during these batch fermentations were the same for both strains (Fig. 3 and 4). This result indicated that strain LCM001 is preferring to respire more of the carbon source (sucrose), and not fermenting it into ethanol.

Indeed, when we analyzed the kinetics of growth on sucrose by both strains (Figure 5), strain 1407-7A clearly presented a typical diauxic growth curve 51, where initially cell growth is coupled to efficient sugar consumption and ethanol production, when the carbon source is exhausted cells stop growing and the diauxic shift takes place, allowing the further consumption of the ethanol produced during sugar fermentation. However, strain LCM001 had a quite different pattern of sucrose utilization (Fig. 5). This agt1Δ yeast grow on sucrose efficiently, with the same specific growth rate (μ = 0.24 ± 0.02 h^-1) as strain 1403-7A (μ = 0.24 ± 0.01 h^-1) or other SUC⁺ strains 44, although sugar consumption was slightly slower than with the wild-type strain (0.26 and 0.51 g sucrose [g cell dry weight]^-1 h^-1 for strains LCM001 and 1403-7A, respectively). These LCM001 yeast cells did not enter into the diauxic shift (Fig. 5), but continued to grow efficiently through time, reaching higher cell densities than the parental strain 1403-7A. Although it is expected that these batch cultures become oxygen limited during exponential cell growth, favoring sugar fermentation, strain LCM001 not only produced less ethanol from sucrose, but also started to consume the ethanol produced during growth even when significant amounts of sugar (~40% of initial value) still remained in the medium (Fig. 5). These results indicated that strain LCM001 had shifted its metabolism into a more aerobic sucrose utilization pattern than the parental strain.

We thus analyzed the biomass yield of these two strains during growth using three different carbon sources (glucose, maltose and sucrose) and different amounts (and quality) of nitrogen sources (Figure 6). It is evident that strain LCM001 produces from 1.5- to 2-fold more biomass during batch growth on sucrose (compared to the parental strain), while the biomass yields on glucose or maltose were unaffected. Conversely, while the ethanol yields on glucose by both the wild-type and the agt1Δ strain were similar and varied from Y_e/s values between 0.16 ± 0.02 and 0.31 ± 0.03 g ethanol (g glucose)^-1 when ammonium sulfate or peptone were used as nitrogen source, respectively, the agt1Δ strain LCM001 produced only 10 to 40% of the ethanol produced by the wild-type strain 1403-7A when sucrose was used as carbon source. In order to confirm that this increase in biomass production by the LCM001 strain is due to respiration of the sugar, we added amtimycin A to the medium. Under this condition (respiration blocked), the biomass yield on sucrose of strain LCM001 was exactly the same as for the parental strain 1403-7A (Fig. 6), and both strains fermented sucrose efficiently (Y_e/s = 0.50 ± 0.01 g ethanol [g sucrose]^-1).

Cells were routinely grown on rich YP medium (10 g/L yeast extract and 20 g/L peptone), or synthetic medium (2 g/L yeast nitrogen base without aminoacids containing 75 mg/L L-tryptophane and 150 mg/L uracil) supplemented with different quantities of ammonium sulfate or peptone as nitrogen source, and 20 g/L glucose, sucrose or maltose as carbon source. The pH of each medium was adjusted to pH 5.0 with HCl, and media was either sterilized by filtration (synthetic medium), or autoclaved at 120°C for 20 min (rich YP medium). When required, 20 g/L agar, 3 mg/L antimycin A, or 200 mg/L geneticin (G-418) sulfate were added to the medium. Cells were grown aerobically at 28°C with shaking (160 rpm) in cotton plugged Erlenmeyer flasks filled to 1/5 of the volume with medium. The inoculum for growth assays was prepared by transferring aseptically a single colony from a plate into 5–10 mL of the selected medium containing 20 g/L glucose, and allowed to growth into stationary phase for 2 to 3 days before they were used to inoculate (by a 100 × dilution) new media containing the indicated carbon sources. Culture samples were harvested regularly, centrifuged (5,000 g, 1 min), and their supernatants used for the determination of sugars and ethanol. For batch fermentations yeasts were pregrown on YP-20 g/L sucrose until the exponential phase (~1 g of dry yeast/L), centrifuged (3,500 g, 3 min) and washed twice with cold water, and inoculated at a high cell density (10 g of dry yeast/L) into synthetic medium (4 g/L yeast nitrogen base without aminoacids and 10 g/L ammonium sulfate) or rich YP medium containing the indicated amounts of sucrose. Batch fermentations were incubated as described above for growth assays, and samples were collected regularly, centrifuged, and their supernatants analyzed as described below.

The S. cerevisiae strains and oligonucleotides used in the present study are described in Table 1. The yeast strains were kept at -80°C in 25% sterile glycerol, and from these frozen stocks yeast cells were streaked onto solid YP-2% glucose plates, incubated for 2 days at 28°C, and stored at 4°C (for a maximum of 1 month) until use. Standard methods for yeast transformation, DNA manipulation and analysis were employed 74. The AGT1 gene was deleted according to the polymerase chain reaction (PCR)-based gene replacement procedure as described previously 4350. Briefly, the kanMX cassette from plasmid pFA6a-kanMX6 75 was amplify with primers AGT1-pFA6-F1 and AGT1-pFA6-R1, and the resulting PCR product of 1,579-bp was used to transform competent yeast cells. After 2-hour cultivation on YP-20 g/L glucose, the transformed cells were plated on the same medium containing G-418 and incubated at 28°C. G-418-resistant isolates were tested for proper genomic integration of the kanMX cassette at the AGT1 locus by Southern analysis (see below) and analytical colony PCR using 3 primers (V-AGT1-F, V-AGT1-R and V-kanr-R; Table 1). These set of 3 primers amplified a 1,938-bp fragment from a normal AGT1 locus, or yielded an 813-bp fragment if the kanMX cassette was correctly integrated at this locus and replaced the AGT1 gene.

Yeast chromosomes were prepared as previously described 76 from 1 ml of yeast cells pre-grown in YP-2% glucose medium and collected at the stationary phase of growth. Cells were washed with 10 mM Tris-HCl, pH 7.5, containing 50 mM EDTA, and resuspended in the 0.4 ml of 4 mM Tris-HCl, pH 7.5, containing 95 mM EDTA, 130 mg/L of Zymolyase 20T and 7 g/L of molten (42°C) low-melting-point agarose. After solidification in a mold (Pharmacia Biotech), the agarose blocks were immersed in 10 mM Tris-HCl, pH 7.5, containing 0.5 M EDTA, and incubated at 37°C for 8 hours. Following a subsequent incubation in 10 mM Tris-HCl, pH 9.5, containing 0.5 M EDTA, 1% N-lauroylsarcosine, and 2 g/L proteinase K at 50°C overnight, the blocks were washed in 10 mM Tris-HCl, pH 7.5, containing 50 mM EDTA, and stored at 4°C in the same buffer. Each low-melting-point agarose block was transferred to a 10 g/L agarose gel in 50 mM Tris-HCl, pH 8.3, containing 50 mM boric acid and 1 mM EDTA. Pulsed field gel electrophoresis (PFGE) was performed at 10°C using a Gene Navigator pulsed-field system (Pharmacia Biotech) for a total of 27 hours at 200 V. The pulse time was stepped from 70 seconds after 15 hours to 120 seconds for 12 hours. Following electrophoresis, the gel was stained with ethidium bromide and photographed. The chromosomes separated by PFGE were transferred to a nylon filter (Biodyne A, Gibco BRL) by capillary blotting 74, and labeling of DNA probes (see below), including the pre-hybridization, hybridization, stringency washes and chemiluminescent signal generation and detection was performed by using a AlkPhos kit (GE Healthcare/Amersham Biosciences) as recommended by the manufacturer. After hybridization, an autoradiography film (Hiperfilm™ ECL – Kodak) was exposed to the membrane for 2 to 3 h before it was developed. Images were obtained by scanning with an ImageScanner™ (Amersham Biosciences) and annotated with Microsoft PowerPoint. Probes corresponding to nucleotides +1 through +1848 on the AGT1 ORF, -73 through +1845 of the MAL31 gene, or +77 through + 1333 of the SUC2 locus were generated by PCR using primers AGT1-F and AGT1-R, MAL31-F and MAL31-R, and SUC2-F and SUC2-R (Table 1), respectively.

Sucrose was quantified using 50 U/mL of yeast β-D-fructosidase (Sigma) in 50 mM citrate-phosphate buffer, pH 4.5, followed by glucose determination. Glucose was measured by the glucose oxidase and peroxidase method using a commercial kit (BioDiagnostica-Laborclin). Maltose was determined spectrophotometrically at 540 nm with methylamine in 0.25 M NaOH as described previously 50. Ethanol was determined with alcohol oxidase and peroxidase as described previously 4450. Cellular growth was followed by turbidity measurements at 570 nm after appropriate dilution, and yeast cell dry weight was determined as described elsewhere 77. Briefly, from 1 to 5 mL of fermentation broth was filtered through pre-weighed filters (0.45 μm mixed nitrocellulose and cellulose acetate filters), washed twice with 5 mL of distilled water, and after placing in a small (5 cm diameter) covered Petri dish, dried for 3 to 5 min in a microwave oven at maximum power (900 W) until constant weight. The sugar consumption rates were calculated using samples harvested from the logarithmic growth phase and/or in intervals during which maximal rates were attained. Mean values of dry weight in the specified time intervals were used in the rate calculations. Specific growth rate (μ, h^-1) was determined as slope of a straight line between ln OD_{570 nm} and time (h) during the initial (~12 h) exponential phase of growth. Biomass and ethanol yield coefficients (Y_x/s and Y_e/s, respectively) were obtained at the end of cell growth or ethanol production, taking into account the amount of sugar utilized. The kinetics of active H⁺-sucrose or H⁺-maltose symport were determined as previously described 4041 using a PHM84 research pH meter attached to a TT1 Servograph (Radiometer, Copenhagen). Initial rates of sugar-induced proton uptake were calculated from the slope of the initial part (< 10 s) of the curve obtained in the recorder, subtracting the basal rate of proton uptake observed before addition of 0.1–100 mM of the sugar. All determinations were done at least in duplicate, and assays were monitored so that no more than 5% of the substrate was depleted. All activities were expressed as nmol of substrate transported per mg dry cell weight per min.