Vol. 59, No. 12APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1993, p. 4230-42350099-2240/93/124230-06$02.00/0Copyright © 1993, American Society for Microbiology

Fermentation of Lactose by Yeast Cells SecretingRecombinant Fungal Lactase

SUNDARAM RAMAKRISHNANt* AND BRIAN S. HARTLEY

Centerfor Biotechnology, Imperial College of Science Technology and Medicine,South Kensington, London SW7 2AZ, United Kingdom

Received 24 May 1993/Accepted 1 October 1993

Strains ofSaccharomyces cerevisiae transformed with a yeast multicopy expression vector carrying the cDNAfor Aspergilus niger secretory 13-galactosidase under the control of ADH1 promoter and terminator werestudied for their fermentation properties on lactose (V. Kumar, S. Ramakrishnan, T. T. Teeri, J. K. C.Knowles, and B. S. Hartley, Biotechnology 10:82-85, 1992). Lactose was hydrolyzed extracellularly intoglucose and galactose, and both sugars were utilized simultaneously. Diauxic growth patterns were notobserved. However, a typical biphasic growth was observed on a mixture of glucose and galactose underaerobic and anaerobic conditions with transformants of a haploid S. cerevisiae strain, GRF167. Polyploiddistiller's yeast (Mauri) transformants were selected simply on the basis of the cloned gene expression on X-Gal(5-bromo-4-chloro-3-indolyl-13-D-galactopyranoside) plates. Rapid and complete lactose hydrolysis and higherethanol (0.31 g/g of sugar) and biomass (0.24 g/g of sugar) production were observed with distiller's yeastgrown under aerobic conditions. A constant proportion (101%) of the population retained the plasmidthroughout the fermentation period (48 h). Nearly theoretical yields of ethanol were obtained under anaerobicconditions on lactose, glucose, galactose, and whey permeate media. However, the rate and the amount oflactose hydrolysis were lower under anaerobic than aerobic conditions. All lactose-grown cells expressed partialgalactokinase activity.

The uses of whey, a lactose-rich effluent from cheeseindustries, have been reviewed by Kosaric and Asher (7).Although much of the nutritious protein of whey is recov-ered, the lactose portion is largely unutilized and its disposalrepresents a serious environmental concern (11). A varietyof microorganisms can ferment lactose to ethanol and bio-mass, but traditional brewing yeasts with high ethanol andsugar tolerance cannot utilize lactose because they lacklactose permease and ,-galactosidase. Moreover, the use ofprehydrolyzed lactose results in glucose repression of galac-tose utilization, leading to a prolonged fermentation periodand lower ethanol yields (14). Even though this problem wasovercome with the selection of catabolite-resistant mutantsthat can utilize glucose and galactose simultaneously, theprocess still involves two steps and is economically unfeas-ible (22). Hence, genetic engineering has been used toconstruct recombinant strains of Saccharomyces cerevisiaethat express the 3-galactosidase and permease genes from alactose-fermenting yeast, Kluyveromyces lactis, allowinguptake by the permease and intracellular breakdown by the,B-galactosidase. However, slow growth, instability, andcatabolite repression of expression of these genes have beenreported (20, 25).

In an alternative approach, we reported the constructionof a genetically engineered strain of S. cerevisiae thatexpresses and secretes a lactase from Aspergillus niger (5,8). A cDNA for the secreted 3-galactosidase was cloned inthe yeast multicopy vector yEP24 and expressed under thecontrol of alcohol dehydrogenase (ADHI) promoter andterminator. Transformants of this recombinant plasmid

* Corresponding author. Electronic mail address: ramakris@rwja.edu, ramakrishnan@biovax.rutgers.edu.

t Present address: Department of Neuroscience and Cell Biology,UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane,Piscataway, NJ 08854.

(pVK1.1) secreted recombinant fungal lactase by using itsown signal sequence, and therefore were able to grow onlactose as a sole carbon source and on whey.

In this article, we present the fermentation properties of S.cerevisiae cells transformed by pVK1.1. Since these cellshydrolyze lactose extracellularly, one could expect glucoserepression of galactose utilization that results in diauxicgrowth, causing reduced ethanol yield and an extendedfermentation period (22). However, we describe the absenceof catabolite repression and simultaneous utilization of glu-cose and galactose resulting from lactose hydrolysis. Wealso present an effective and simple method for transforma-tion and selection of polyploid yeast cells based on theexpression and secretion of the cloned ,B-galactosidase.

MATERLILS AND METHODS

Yeast strains and media. The haploid S. cerevisiae strainGRF167 (a ura3-167his-A206 Gal') was from the laboratorystrain collection, and Mauri distiller's yeast strain wasobtained from Grand Metropolitan Plc, Ruislip, UnitedKingdom.For strain maintenance, yeast minimal medium containing

0.67% yeast nitrogen base (Difco) without amino acids,supplemented with lactose, glucose, or galactose (2% [wt/vol]) and uracil or histidine (20 mg/liter) as appropriate, wasused. Fermentations were performed either in YP medium(1% yeast extract and 2% peptone with appropriate sugarsupplement) or modified D, medium. Modified D, mediumcontained [per liter] 20 g of NH4SO4, 11 g of KH2PO4, 1.2 gof MgSO4. 7H20, 0 to 100 g of a carbon source (lactose orglucose), 1 ml of vitamin solution, and 10 ml of traceelements. Trace elements consisted of a 10Ox concentratecontaining (per liter) 9 g of NaEDTA, 2.7 g ofZnSO4 7H20, 0.6 g of MnCl2 4H20, 0.6 g ofCoCl2- 6H20, 0.18 g of CuSO4- 5H20, 0.24 g of

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Na2MoO4. 2H20, 2.7 g of CaCl2. 2H20, 1.0 g ofFeSO4- 7H20, 0.6 g of H3BO4, and 0.06 g of KI. Thevitamin solution consisted of a 1,OOOx concentrate contain-ing (per liter) 0.3 g of D-biotin, 6.0 g of calcium pantothenate,6.0 g of nicotinic acid, 10 g of myoinositol, 6.0 g of thiamine-HCI, and 6.0 g of pyridoxine-HCl (4).

Fermentations. Batch culture experiments were performedin a pH-controlled 2-liter bioreactor (Life Science Laborato-ries Ltd., Luton, England) with a working volume of 1.5 literand air sparging at 300 ml min-1. The dissolved oxygen waskept above 20% by regulating the stirrer speed. For anaero-bic conditions, oxygen-free nitrogen was used (100 mlmin-') to sparge the bioreactor.Shake flask anaerobic fermentations were carried out in

500-ml Erlenmeyer flasks with 250 ml of medium per flask.The flasks were fitted with a fermentation lock (Boots plc,Nottingham, England) containing sterile glycerol. All fer-mentations were carried out at 30°C. The inoculum (10%[vol/vol]) was routinely taken from an aerobically grownculture after 16 to 18 h of growth in modified D, mediumcontaining 2% (vol/vol) glycerol. Samples were removedperiodically for the analysis of growth (optical density at 600nm) and glucose, galactose, lactose, and ethanol content.

Analytical procedures. Glucose, lactose, and galactosewere estimated with enzyme kits from Boehringer Mann-heim. Protein content was estimated by the method ofBradford (2a) by using Bio-Rad dye-reagent with bovineserum albumin standards.For the determination of biomass (dry weight), 10 ml of

culture liquid was filtered onto preweighed 0.45-,um-pore-size cellulose acetate paper (Sartorius), washed with distilledwater, dried in a 70°C hot-air oven for 12 h, and then weighedafter cooling the paper to room temperature under vacuum.The amount of dry weight per liter of culture was calculated.

Ethanol was estimated by gas chromatography with aPhilips PU4500 gas chromatogram equipped with a dual-flame ionization detector and a Poropak QS column (WatersAssociates, Inc.). The injector, column, and detector tem-peratures were 200, 180, and 250°C, respectively.

Exit gases from the bioreactor were first passed through avertical stainless-steel condenser and then dried over ananhydrous calcium sulfate column (Hammond Drierite, Xe-nia, Ohio) before being analyzed on-line by use of a quadru-ple mass spectrometer (model 200F; VG Gas, Surry, UnitedKingdom). The respiratory quotient (RQ) was determined bythe equation RQ = rate of CO2 production/rate Of 02consumption. The rates of CO2 production and 02 consump-tion were determined as described by Postma et al. (16).Yeast cell extracts for enzyme analyses were prepared by

centrifuging, washing, and resuspending the cells in anappropriate buffer to give a heavy paste. To obtain lysis, 3 gof 0.45-mm-diameter glass beads was added per ml of cellpaste, and the suspension was subjected to three or more30-s periods of disruption with a vortex mixer. Cells werechilled in an ice bath for 1 min between each cycle. Theresulting homogenate was centrifuged at 10,000 x g for 10min to remove cellular debris and unbroken cells, and thesupernatant was used for enzyme assays. Cell fractionationfor ,B-galactosidase localization was carried out as describedby Pentilla et al. (15).Western blot (immunoblot) analysis for the detection of

recombinant ,-galactosidase in various cell fractions wascarried out by using previously described methods (8). Thepurified antisera againstA. niger P-galactosidase used in thisanalysis were a gift from I. Salovuori (VTT BiotechnicalLaboratory, Espoo, Finland).

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Time (h )FIG. 1. Effect of pH on the aerobic growth of S. cerevisiae

GRF167 transformants on modified Dw medium containing 4%lactose. Symbols: 0, biomass, pH 5.5; 0, biomass, pH 4.5; *,residual lactose, pH 5.5; El, residual lactose, pH 4.5. Levels ofglucose and galactose were negligible.

Measurement of enzyme activity. 3-Galactosidase activitywas determined by the method of Nevalainen (13) by using 5mM o-nitrophenyl-13-D-galactopyranoside (ONPG) in 0.075M sodium actetate buffer. The reaction was initiated by theaddition of 100 ,ul of suitably diluted enzyme to give a totalvolume of 1 ml. After incubation at 30°C for 10 min, thereaction was stopped by adding 1 ml of 0.1 M sodiumbicarbonate. The A420 was measured. Enzyme activity wasexpressed in nanomoles ofONPG hydrolyzed per minute permilliliter.

Galactokinase activity was determined by the method ofAdams (1). The reaction cocktail mixture consisted of 3 mMATP, 5 mM MgCl2, 0.1 mM dithiothreitol, and 3 mM[I4C]galactose (Amersham, Buckinghamshire, England) in0.1 M Tris-HCl buffer (pH 8.0). The reaction was initiated byadding 25 ,ul of cell homogenate to a total volume of 250 ,lj.Phosphorylated sugar was separated on Dowex Ag 1-8columns (1). The labelled [14C]galactose-l-phosphate wasdetermined by scintillation counting. Enzyme activity wasexpressed in nanomoles of galactose phosphorylated perminute per milligram of protein.Yeast transformation. Transformation of yeast cells was

carried out by electroporation (with the Bio-Rad GenePulser) with modifications (2). Yeast cells were grown aero-bically in YPD (YP medium plus 2% glucose) broth at 30°Cto a cell density of 2 x 108 cells per ml. The cells were thenharvested, washed with sterile water five to six times,resuspended in 30% glycerol in 1 M sorbitol, and frozen in40-,u aliquots. The yeast cells were electroporated with 100ng of plasmid DNA at 1.5 kV, 25 mF, and 200 fl in 0.2-cmcuvettes. To the electroporated cells, 1 ml of cold 1 Msorbitol was added and 100 ,ul of cell suspension was platedonto a selective agar medium (yeast minimal agar containing2% glucose and X-Gal). Transformants of pVK1.1 wereidentified by their blue colony morphology.

RESULTS

Growth of haploid yeast transformants on modified D,medium containing 4% lactose. Growth of S. cerevisiaeGRF167 transformants on 4% lactose at pH 5.5 is shown inFig. 1. About 63% of the lactose was utilized in 55 h, with no

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4232 RAMAKRISHNAN AND HARTLEY

accumulation of glucose or galactose, but the growth ratewas relatively low (p. = 0.1 h-1). The specific growth rate isdetermined by the rate of glucose and galactose formation,which depends on the secreted P-galactosidase activity, andtherefore is considerably influenced by the temperature andpH of the medium. The A. niger 0-galactosidase is thermo-stable (optimum temperature, 65°C), with a pH optimum ofabout 3.5 (24). By increasing the temperature or decreasingthe pH of the fermentation, an increase in lactase activityand consequent improved lactose hydrolysis might be ex-pected. As shown in Fig. 1, a dramatic increase in lactosehydrolysis (72%) was achieved by dropping the pH from 5.5to 4.5 (50% hydrolysis of total lactose was achieved within38 h compared with 54 h at pH 5.5). An increase in thegrowth rate (p. = 0.14 h-1) was also observed, but there wasno accumulation of galactose or glucose at any stage of thefermentation (48 h).Growth of haploid yeast transformants on modified Dw

medium containing 2% glucose and 2% galactose. To estab-lish whether transformants of S. cerevisiae GRF167 arenormally subject to glucose repression, their fermentationpattern on a mixture of glucose and galactose was studied.Yeast strains resistant to catabolite repression utilize bothglucose and galactose simultaneously (22), but most yeaststrains can metabolize galactose only aerobically (18).Hence, anaerobic growth studies were also undertaken toreveal the possible oxygen requirement for galactose metab-olism.

Figure 2 shows the aerobic growth of yeast transformantsof plasmid pVKl.l on a mixture of 2% glucose and galactoseat pH 4.5 and 30°C. The pattern is of classical diauxic growthin which glucose is used first (0 to 22 h) with a high growthrate (p. = 0.206 h-1) and then galactose is used (22 to 40 h)at a slower growth rate (p. = 0.060 h-1). Note also that theethanol yield from the glucose (9.5 g/liter) is higher than thatfor the galactose utilization (5.5 g/liter). The RQ values ofaerobically grown cells were particularly revealing. Thesevalues rise during the period 0 to 17 h, indicating a switch tofermentative metabolism. From 22 to 24 h, when the glucoseis almost exhausted and galactose utilization has not yetbegun, ethanol production ceases and there is a rapid fall inthe RQ. This shows that metabolism has switched fromfermentative to respiratory (Crabtree Effect) at the diauxicswitch point. When galactose consumption begins, ethanolproduction resumes and the RQ rises to a maximum (2.0),which is lower than the maximum on glucose (3.8), and fallsrapidly at 39 h when galactose is exhausted. This is indica-tive of mixed respiratory and fermentative metabolism.These results confirm that the yeast transformants ofpVK1.1 were not resistant to catabolite repression.

Figure 2 also shows the anaerobic growth on a similarmedium. Diauxy is again observed, with glucose being usedfirst (0 to 22 h), and a slightly longer lag phase precedesgalactose utilization. The anaerobic growth rates on glucose(p. = 0.165 h-1) and galactose (p. = 0.050 h-') were onlyslightly lower than the corresponding aerobic growth rates,but in this case ethanol yields on glucose (10.2 g/liter) andgalactose (9.8 g/liter) were comparable, since respiratorymetabolism is impossible, and final biomass yields wereslightly lower.Table 1 summarizes the final biomass and ethanol yields in

these fermentations. Although the aerobic fermentations on4% lactose were not complete in 50 h, ethanol yields weresimilar to the aerobic fermentation on 2% glucose-2% galac-tose. Moreover, more biomass was produced in the lactosefermentations, so the molar ethanol yields are close to the

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FIG. 2. Growth of S. cerevisiae GRF167 transformants at 30°C,pH 4.5, on modified Dw medium containing 2% glucose and 2%galactose. (Upper panel) Biomass during aerobic growth (0) oranaerobic growth (A) and respiratory quotient (RQ = rate of CO2evolution/rate of 02 uptake) during aerobic growth (O). (Lowerpanel) Residual sugars and ethanol production during aerobicgrowth (filled symbols) or anaerobic growth (open symbols). Sym-bols: *, 0, ethanol; *, O, galactose; A, A, glucose.

theoretical maximum of 2.0 mol of ethanol per mol of hexosecatabolized. This shows that the aerobic lactose metabolismwas strictly fermentative, unlike that of glucose-galactose,which was about 80% fermentative and 20% respiratory. Thelactose fermentation rate appears to be limited by theextracellular activity of the secreted 0-galactosidase andtherefore could probably be increased by the use of astronger promoter or, more simply, a larger initial cellinoculum. Although diauxy is observed even in the anaero-bic fermentations on glucose-galactose, the ethanol yieldsare now the theoretical maximum, corresponding to 100%fermentative metabolism. Hence, it is probable that thiswould apply to anaerobic fermentations on lactose, althoughthis was not tested.

Effect of carbon source on galactokinase activity. The effectof glucose on galactokinase activity is twofold (inhibition ofenzyme activity and repression of synthesis), and thereforeits activity in yeast transformants of plasmid pVK1.1 grownon various carbon sources was measured to determine thelevel of repression of the GAL pathway genes under variousconditions (1, 10, 22). The results are presented in Table 2and include experiments with a pVK1.1-transformed indus-trial yeast strain (see below). The extent of repression ofenzyme activity was calculated by assuming 100% derepres-

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TABLE 1. Ethanol and biomass yield from different carbon substrates by S. cerevisiae GRF167 transformantsa

EthanolCarbon substrate Growth Carbon Biomass yield Amt of CO2 producedCarbonWsubstrate conditions utilized (g/g of sugar Concn Yield Yield (mol/mol of sugar

M utilized (g/liter)(gig of sugar (mol/mol of sugar catabolized)(wt/vol)conditions (%) utilized) (glliter) ~utilized) catabolized)

2% Glucose-2% galactose Aerobic, pH 4.5 99 0.13 15.7 0.38 1.72 2.29Anaerobic, pH 4.5 99 0.09 19.6 0.48 2.08 1.97

4% Lactose Aerobic, pH 4.5 72 0.22 1.18 0.38 1.98 1.98Aerobic, pH 5.5 62 0.21 0.97 0.37 1.91 2.14

a Fermentations were conducted in modified D, medium supplemented with carbon substrates under aerobic and anaerobic conditions. The analyses for sugar,biomass, and ethanol content were performed at the end of the fermentation (48 h). The amount of sugar catabolized was calculated by subtracting the biomassequivalent from the sugar uptake. The amount of CO2 was calculated assuming that biomass and ethanol were the only other products.

sion in cells grown on 2% galactose. Complete repressionwas seen in cells grown on 2% glucose or 5% glucose-2%galactose, but around 50% repression was seen with 2%glucose-2% galactose or 4% lactose. This indicates that theGAL operon is partially repressed in lactose-utilizing cellseven though glucose and galactose do not accumulate in themedium (Fig. 1). However, there was much less GAL operonrepression in the Mauri yeast strain grown on 10% lactose.

Transformation of polyploid distiller's yeast with plasmidpVKl.l. Transformation of polyploid industrial yeasts ishampered by the absence of readily selectable auxotrophicmarkers in such strains, but pVK1.1 transformants are easyto select. After transformation of Mauri distiller's yeast withplasmid pVK1.1, blue colonies among white confluent colo-nies were easily detected on a 2% glucose minimal mediumcontaining X-Gal. Further purification and maintenance oftransformants were carried out on lactose minimal medium.The presence of plasmid pVK1.1 was established by prepa-ration of the plasmid from transformants, and then restric-tion analysis followed (17). In addition, Western blot analy-sis of various cell fractions, culture supernatant, and endoH-treated fractions confirmed the presence of heteroge-neously glycosylated recombinant protein as reported earlier(8). When the cell fractions were assayed for ,3-galactosidaseactivity, most of the activity was present in the culturesupernatant (61%), the rest being distributed betweenperiplasm (16%) and the insoluble and cytoplasmic fractions(23%).

TABLE 2. Galactokinase activity in yeast transformants grownon different carbon substrates

S. cerevisiae (GRF167) S. cerevisiae (MauriCarbon source distiller's yeast)

(wt/vol) Galactokinase % Galactokinase %activity' inductionb activity' inductionb

2% Galactose 41 100 31 1002% Glucose 0 0 0 02% Glucose- 20 502% galactose

5% Glucose- 0 02% galactose

4% Lactose 25 624% Lactosec 22 5310% Lactose 26 83

a The enzyme activity (nanomoles of galactose phosphorylated per minuteper milligram of protein) was estimated from cells grown on modified D,medium supplemented with the appropriate carbon source for 48 h at pH 4.5.

b Assuming 100% induction on 2% galactose.c Fermentation at pH 5.5.

Moreover, the total amount of 3-galactosidase synthe-sized (assuming that the enzyme made in yeasts has aspecific activity similar to that of the purified Aspergillusenzyme) corresponds to 0.38% of total cell protein comparedwith 0.16% for the transformed laboratory yeast.

Aerobic growth of distiller's yeast transformants on modi-fied D, medium containing 10%S lactose. Growth of pVK1.1transformants of Mauri distiller's strain on medium with ahigh lactose concentration is shown in Fig. 3. Comparedwith the laboratory strain of S. cerevisiae GRF167 (Fig. 1), ahigher growth rate (p, = 0.28 h-1) and lactose hydrolysiswith simultaneous uptake of glucose and galactose wereobserved in Mauri distiller's strain. Galactose utilization wasalso indicated by a higher galactokinase activity (Table 2).Complete hydrolysis was achieved within 48 h with higherbiomass (0.25 g/g of lactose) but lower ethanol (0.31 g/g oflactose) yields, indicating relatively more respiratory metab-olism.

Stability studies of the plasmid indicated that only about10% of the population retained the plasmid, and this fractionremained fairly constant throughout the fermentation period,representing 19 generations (Table 3). This presumablyreflects a balance between direct selection for strains retain-ing the plasmid and cross-feeding of cured cells by secreted,-galactosidase activity in the culture supernatant (Fig. 3).Anaerobic fermentations with transformed distiller's yeast.

The results of anaerobic fermentations with this transfor-mant are summarized in Table 4. Ethanol and biomass

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FIG. 3. Aerobic growth of Mauri distiller's yeast transformantsat 30'C, pH 4.5, on modified D, medium containing 10% lactose.Symbols: El, optical density at 600 nm; 0, ethanol (in grams perliter); 0, residual lactose. Glucose and galactose concentrationswere negligible.

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TABLE 3. Stability of distiller's yeast transformants grown onmodified DW medium containing 10% lactosea

No. of cells/mlGrowth stage of yeast (104) % of cells secreting

transformant (h) 3-galactosidaseBlue White

0 25 218 11.56 38 272 1212 130 1,060 1124 400 4,100 948 600 6,000 9

a Fermentation was carried out aerobically under controlled growth condi-tions in a bioreactor. The colonies containing plasmid were identified by theirblue morphology on 2% glucose minimal agar plates containing X-Gal.

production from whey permeate (obtained from Grand Met-ropolitan Plc) medium containing 10% lactose was comparedwith ethanol produced in YP medium supplemented withequivalent concentrations of glucose, galactose, or lactose.As in the case of aerobic fermentation of lactose, no catab-olite repression of galactose utilization was observed. Etha-nol yields (0.46 to 0.47 g/g of sugar) were higher in anaerobicthan in aerobic fermentation (0.31 g/g of sugar) and close tothe theoretical maximum (0.51 g/g of sugar), but only 35% ofthe lactose in YP medium and 21% in whey permeatemedium was fermented in 72 h. Since the fermentations onglucose or galactose were complete in this period, the slowgrowth on lactose can be attributed to lower ,-galactosidaseproduction under the anaerobic conditions, since the bio-mass produced was less than half that produced aerobically.In the case of whey permeate, there also seems to be someinhibition of growth by other components in the crudefeedstock (14).

DISCUSSION

These results show that plasmid pVK1.1 can be usedreadily to select transformants of industrial yeasts that givehigh ethanol yields directly on concentrated whey permeate.This simple approach would be better than the more expen-sive alternatives of prehydrolysis of lactose or coimmobili-zation of S. cerevisiae cells with ,-galactosidase. Moreover,since almost any yeast strain could be easily transformed inthis way, the approach appears superior to cloning andexpressing a lactose permease gene and an intracellularI-galactosidase gene in the chosen host, as explored byothers (20, 25). In addition, our yeast transformant does notshow diauxic growth on lactose, and both the glucose and

galactose are utilized simultaneously without accumulationat any stage of the fermentation.

This shows that the rate of lactose hydrolysis is lower thanthe rates of uptake of glucose and galactose by the yeast cellsand explains why the fermentation is slower on lactose thanon glucose. An increase in rates of ,-galactosidase secretionprobably could be achieved if the A. niger gene were to bestably integrated into the yeast chromosome (21), since therewas considerable plasmid loss during the fermentation.However, the rate of ethanol production by current strainscould be increased simply by using a larger inoculum.

S. cerevisiae exhibits diauxic growth on a mixture ofglucose and galactose because glucose represses the GALoperon, which encodes regulator proteins and enzymesresponsible for galactose utilization. This glucose repressionpersists even in the presence of galactose, the inducer for theGAL operon: hence, the glucose is used up first. Growth isslower on galactose, indicating that either its uptake, phos-phorylation, or epimerization to glucose-i-phosphate arerate limiting to the common pathway of glycolysis thereafter.This slow growth reduces intracellular pyruvate levels,thereby reducing the flux through pyruvate decarboxylaserelative to that through pyruvate dehydrogenase. Hence,ethanol yields are lower in the diauxic phase.These problems are bypassed by using lactose with our

strain because extracellular levels of glucose are very low,and therefore glucose repression of the GAL operon isremoved. Hence, both sugars are utilized simultaneouslyand the total glycolytic flux is high, giving high ethanolyields.The mechanism of glucose repression and galactose acti-

vation of the GAL operon is complex (6, 9, 12), but ourexperiments help clarify it. There are three or more regula-tory genes: GAL4, which encodes a positive regulatoryprotein; GAL80, which encodes a negative regulator thatinterferes with the GAL4 product in the absence of inducer(galactose); and GAL3, which may function in the synthesisof a coinducer (3). On the basis of galactokinase levels invarious genetical constructs or mutants grown on 5% glu-cose-2% galactose or 1% glucose-5% galactose, Matsumotoet al. (10) proposed that glucose affects GAL4-GAL80 regu-lation by inhibiting galactose uptake (inducer exclusion).Our experiments support this, showing that there was nogalactokinase activity at high concentrations of glucose evenin the presence of inducer, while galactokinase activity waspartially induced at lower concentrations of glucose. Adams(1) also observed partial galactokinase activity with 2%glucose plus either 0.5 or 2% galactose, and Torchia et al.(23) observed partial GAL expression on 2% glucose but not

TABLE 4. Anaerobic ethanol and biomass yield on different carbon substrates by distiller's yeast transformantsa

Ethanol CO2Carbon EhnlC2Carbonsubstrate Medium % Carbon Biomass yield Yield Yield Yield recovery(wt/vol) utilized (g/g of sugar) Concn (g/g of sugar (mol/mol of sugar Cotncn (mol/mol of sugar (%)

catabolized) catabolized) (g/lier) catabolized)

Glucose YP 99 0.035 49 0.49 2.00 44 1.89 97Galactose YP 97 0.050 46 0.47 1.80 42 1.87 94Lactose YP 35 0.103 16.5 0.47 2.00 14 1.82 95

Whey 21 0.086 9.7 0.46 1.99 10 2.14 102Lactoseb Modified Dw 97 0.247 30 0.31 1.63

a Fermentations were carried at 30'C in anaerobic 500-ml Erlenmeyer flasks. Analyses for sugar, biomass, and ethanol content were performed after 72 h. Theamount of sugar catabolized was calculated by subtracting the biomass equivalent from the sugar uptake. CO2 was calculated from the weight loss.

b Aerobic fermentation in a bioreactor.

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on 10% glucose. Moreover Yocum and Johnston (26) andTorchia et al. (23) showed that disruption or deletion of theGAL80 gene does not eliminate glucose repression of thegalactose pathway enzymes. Hence, glucose repression ap-pears to be a direct effect on galactose uptake that isindependent of the GAL4-GAL80 system.Our results show that expression of the secreted 3-galac-

tosidase can be readily used as a dominant marker for theselection of recombinant polyploid yeasts. There is selectivepressure to conserve the plasmid during growth on lactose,and therefore this would allow selection for transformantssimply by growing transformed cells on lactose medium andscreening the resulting colonies on X-Gal plates. Moreover,the recombinant polyploid distiller's yeast appears to pro-duce a larger halo on plates than the laboratory yeast,indicating that the level of secreted enzyme is much higherthan the haploid laboratory yeast (17). This could be due tothe increase in the copy number of the plasmid with theincrease in ploidy of yeast cells (19). Plasmid pVK1.1 couldbe used in cotransformation experiments with polyploidyeasts for the selection of other plasmids. Alternatively, thisplasmid could be used as a vehicle for other heterologousgenes whose expression in an industrial yeast host is desir-able, e.g., glucoamylase genes to aid starch fermentations orheterologous genes for vaccines or pharmaceutical proteins.

ACKNOWLEDGMENTS

This work was supported by internal Imperial College funds. S.R.was the recipient of a Commonwealth Scholarship Award-UK.

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2a.Bradford, M. M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72:248-254.

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