production of heterologous microbial lipases by...

Indian Journal of BiotechnologyVol 2, July 2003, pp 346-355

Production of Heterologous Microbial Lipases by Yeasts

Ali Kademi, Byong Lee and Alain Houde*Agriculture and Agri-Food Canada, Food Research and Development Centre

3600 Casavant Blvd West, St-Hyacinthe, Quebec, Canada, 12S 8E3

Received 12 November 2002; accepted 21 February 2003

Lipases (triacylglycerol lipase, EC 3.1.1.3) catalyse the hydrolysis of triglycerides to di-glycerides, mono-glycerides, glycerol and fatty acids. In non-aqueous media, these enzymes also catalyse esterification,interesteriflcation and transesterification. This versatility makes the lipases potential biocatalysts in many industrialapplications. This review describes the recent advances in the use of yeasts as expression systems to produce highlevels of recombinant microbial lipases. Some expression systems such as Saccharomyces cerevisiae and Pichiapastoris are reviewed and compared. Also, cell-surface display systems as novel methods to construct whole cellbiocatalysts are highlighted.

Keywords: microbial lipase, Pichia pastoris, Saccharomyces cerevisiae, expressionsystem

IntroductionAfter many years of fruitful research on structure

and function of different kinds of proteins fromvarious sources, some potentially useful applicationsof these proteins, mainly enzymes, have beenhighlighted. To improve the fundamental andindustrial properties of these proteins and their uses inindustrial applications, high level of expression of thegene must be obtained in a foreign host. Thedevelopment of recombinant DNA technology, whichenabled the production of protein at level greater thanIOO-fold by native enzyme, constitutes a suitable wayto make the target proteins available at a reduced cost.Early successes in the production of heterologousproteins were achieved using Escherichia coli as ahost (Itakura et aI, 1977). E. coli was intensely usedfor the expression of various kinds of proteins.However, the nature of the recombinant proteinsbeing expressed in E. coli became more and morecomplex due to the problems of inclusion bodies,protein folding, and safety issue, leading to theinvestigations of other potential host systems such asyeasts, fungi, plants and animals.

Yeasts as Expression HostsAmong all the potential hosts, yeasts offer many

advantages that makes them a choice for the

*Author for correspondence:Tel: 450-773-1105; Fax: 450-773-8461E-mail: [email protected]

expression of various proteins: 1) yeasts areunicellular micro-organisms that have advantagesover bacterial systems in their ease of handling andgrowth; 2) yeasts are well suited for genetic analysisdue to their maintenance as haploids (one genomecomplement) or diploids (two genome complements),therefore, genetically recessive mutations can beeasily obtained by working with haploid cells, whilegenetic complementation can be simply performed bymating two different haploid mutants; 3) yeastspossess an eukaryotic sub-cellular organizationcapable of accurate post-translational processing andmodifications of many mammalian proteins, and 4)yeasts can be cultivated to a high cell density in acheap medium.

Many different yeast species have been developedinto efficient systems for heterologous geneexpression. Saccharomyces cerevisiae was the firstyeast to be used as a host for the expression ofrecombinant proteins, due to the well-establishedindustrial processes in brewing, baking industries aswell as in industrial alcohol. The human leukocyteinterferon D (LeIF-D) was the first heterologousprotein studied in S. cerevisiae expression system(Hitzeman et al, 1981). Since then, S. cerevisiae hasbeen usually the yeast of choice to host foreign geneexpression. Hepatitis B vaccine was the firstcommercial recombinant vaccine derived from S.cerevisiae (Valenzuela et aI, 1982). However, S.cerevisiae possesses certain disadvantages in

KADEMI et al: EXPRESSION OF MICROBIAL LIPASES BY YEASTS

heterologous proteins production: 1) low productionyields even with a strong promoter; 2)hyperg1ycosylated proteins that might affect theprotein characteristics; and 3) intracellular orperiplasmic gene products with proteins larger than30 kDa.

Other yeasts called non-Saccharomyces wereemployed as alternative production systems. Wang etal (2001) reviewed almost all non-Saccharomycesstrains used for protein expression. Non-Saccharomyces expression systems are mainly themethylotroph strains of Pichia pastoris, P.methanolica and Hansenula polymorpha, the fissionyeast Schizosaccharomyces pombe, the alkane-utilizerYarrowia lipolytica, the lactase-producingKluyveromyces lactis and the amylolyticSchwanniomyces occidentalis.

The methylotrophic yeasts belonging to the generaCandida, Hansenula, Pichia and Torulopsis shared aspecific methanol utilisation pathway (Egli et al,1980). Initial reactions occurred in specializedmicrobodies, the peroxisomes, followed bysubsequent metabolic steps in the cytoplasm.Methanol enters in the peroxisomes where it isoxidized by specific oxidases into formaldehyde andhydrogen peroxide. The latter is degraded to waterand molecular oxygen by a peroxisomal catalase.Formaldehyde generated by the oxidase reactionenters both the cytosolic dissimilatory pathway togenerate energy and the assimilatory pathway to yieldbiomass. For a detailed description of the methanolutilization pathway, refer to Gellissen (2000) andCereghino & Cregg (2000).

The oxidase genes have been identified and clonedfrom different yeast species. Pichia pastoris and P.methanolica contain two genes, designated AOXl andAOX2 (alcohol oxidase gene) and AUG] and AUG2(alcohol utilizing gene), respectively (Cregg et al,1989; Raymond et al, 1998). The Aox1 and Aug1proteins are the dominant alcohol oxidases for thesetwo species. For Hansenula polymorpha and Candidaboidinii, only one gene has been identified so far,methanol oxidase (MOX) and alcohol oxidase(AOD]), respectively (Ledeboer et al, 1985; Sakai &Tani, 1992). P. pastoris and H. polymorpha representthe most used methylotrophic yeasts for proteinexpression purposes. For methylotrophic yeasts, keyenzymes in the methanol metabolism pathwayespecially oxidases and dihydroxyacetone synthase

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(DHAS), are undetectable in cells grown on glucose,glycerol or ethanol but are dramatically induced in thepresence of methanol (Roggenkamp et al, 1984). Thesynthesis of these key enzymes is regulated at thetranscriptional level by a control mechanismassociated with the carbon source in the culturemedium.

Since 1985, Pichia pastoris has been successfullyused in the production of over 300 recombinantproteins (Cereghino & Cregg, 2000). The increasingpopularity of P. pastoris is mainly attributed to: (a) itssimple genetic modification, and easy cultivation inlarge-scale; (b) its ability to produce foreign proteinseither intracellularly or extracellularly at high levels;(c) its capability of performing many eukaryotic post-translational modifications, such as proteolyticprocessing, folding, disulfide bond formation andglycosylation; and (d) its ready-to-use availability as akit (Invitrogen Co., CA, USA). P. pastoris is alsousing a respiration metabolism and this representsanother important feature compared to thefermentation metabolism used by S. cerevisiae. In ahigh-ceIl-density culture, the ethanol produced by S.cerevisiae fermentation rapidly reaches toxic levelsand limits further growth. and foreign proteinproduction. On the other hand, P. pastoris can grow atextremely high cell densities without significantethanol accumulation with its respiration metabolism.Since the concentration of a secreted protein isapproximately proportional to the cell density, theability of P. pastoris to be cultivated at high celldensity represents a key property of this species.Methylotrophic yeasts secrete only low levels ofendogenous proteins and this feature representsanother important advantage for P. pastoris in foreignprotein secretion because the target protein is virtuallysecreted in a "purified form". The secretion pathwayis then acting as an important purification step in theseparation of the heterologous protein fromintracellular proteins and other cellular debris.,

H. polymorpha possesses some specific advantagesover methylotrophic yeasts, Candida boidinii and P.pastoris, in being more thermotolerant and capable togrow at higher rates on simple defined media. Therelatively high optimal growth temperature for H.polymorpha (37-43°C versus 30°C for C. boidinii, P.pastoris and S. cerevisiae) may be suitable formammalian (including human) protein production andhas the advantage to reduce the contamination risks inlarge-scale fermentations (Van Dijk et al, 2000).

348 INDIAN J BIOTECHNOL, JULY 2003

Most of the molecular genetic modificationtechniques (DNA-mediated transformation, genetargeting, gene replacement) for non-Saccharomycesorganisms turned out to be similar to those developedfor the well-studied baker's yeast, S. cerevisiae andgenerally lead to similar results.

LipasesThe number of known enzymes is actually around

4000 but only 200 enzymes are commercially used(Sharma et al, 2001). The total sales of enzymesduring 1960s represented only few million dollarsannually but since then this market has grownspectacularly with the development of moleculartechnologies (Godfrey & West, 1996). The majorindustrial enzymes come from microbial originmainly because microorganisms grow quickly andtheir genome size is much smaller than mammaliancells. Some of these commercial enzymes areproduced with recombinant technologies using fungi,bacteria or yeasts as host microorganisms.

Lipases (triacylglycerol lipase Ee 3.1.1.3) areamong the most useful enzymes widely used in theindustry. Their natural function is the hydrolysis oftriglycerides to partial glycerides and fatty acidsduring digestion. Unlike esterases, lipase is activatedin the presence of a water/lipid interface and itsactivity increases sharply as soon as the triglyceridesubstrate forms an emulsion. This phenomenon wastermed interfacial activation (Sarda & Desnuelle,1958) and has been related to the presence of ahydrophobic oligopeptide (the lip or flap), whichcovers the active site of the enzyme in the inactiveform and moving away on contact with the interface.The term true lipase, used to classify a lipolyticenzyme, was based on the interfacial activationphenomenon and the presence of the lid. However, anumber of exceptions were notified in which somelipolytic enzymes have a lid without exhibiting theinterfacial activation phenomenon (Verger, 1997).Therefore, another lipase definition was proposed ascarboxyl-esterases that catalyse the hydrolysis oflong-chain acylglycerols (Ferrato et al, 1997).

Applications of LipasesBesides their natural function of hydrolysing

triglycerides, lipases are capable to catalyzeesterification reaction that leads to ester synthesisfrom a fatty acid and an alcohol in a non-aqueousenvironment. Another type of catalyzed reaction

involves the transfer of an acyl group between twoesters (interesterification), between an ester and anacid (acidolysis), between an ester and an alcohol(alcoholysis) or between an ester and an amine(aminolysis). Lipases can, therefore, synthesize abroad range of molecules of interest. Lipase has thusbecome the enzyme of choice for organic chemists,biotechnologists, microbiologists and enzymologists.Potential applications for lipases concern the agri-food, oil, leather, detergent and pharmaceuticalindustries. Some biotechnological applications oflipases are summarized in Table 1. Despite the highpotential of lipases, their industrial applications aremainly limited to the detergent industry and the finechemistry. This limitation is mainly due to their highproduction cost. The recombinant DNA technologyoffers the possibility of producing these enzymes athigh levels and virtually in a purified form.

Table l--Some areas of industrial applications of microbiallipases

Industry Application

DairyBakingFat and oils processing

Cheese flavourDough stability and conditioningHydrolysis of oils and fatsReagents for lipid analysisRemoval of subcutaneous fatFlavour synthesisLipid stain removal .Resolution of racemic mixtures

LeatherCosmetics and perfumeDetergentsFine chemistry

Expression of Microbial Lipases in YeastsMany microbial lipases that have been successfully

expressed in yeasts are summarized in Table 2 givingseveral expression strategies especially developed formicrobiallipases.

Rhizopus niveus lipaseRhizopus niveus lipases (RNL) possess a high 1-3

positional specificity and are therefore suitable for fatand oil modification. The RNL I enzyme has a uniquestructure comprising two polypeptide chains, a smallpeptide with a sugar moiety (A-chain) and a largepeptide with a molecular weight of 34 kDa (B-chain).These polypeptides are bound together through a non-covalent interaction. The RNL II enzyme consists in asingle polypeptide chain with a molecular weight of30 kDa. It was suggested that RNL II could be theresult of lipase I limited proteolysis (Kohno et al,

KADEMI et ai: EXPRESSION OF MICROBIAL LIPASES BY YEASTS 349

Table 2--Recombinant microbial lipases produced in yeasts

Strain Enzyme Host strain Promoter Locali- Activity N- Referenceszation* glycosyla-

tion sites

Bacillus subtilis lipase A Saccharomyces ACT] 2.57 U/g prot. Sanchez et al, 2002cerevisiae

Candida lipase B Pichia pastoris AOXl e 25 ug/ml Rotticci-Mulder et al,antarctica 2001

C. rugosa lipllipase P. pastoris AOXl e 150 U/ml 3 Brocca et al, 1998lipllipase S. cerevisiae UASGAL e 5-7 U/ml Brocca et al, 1998

Fusarium lipase S. cerevisiae GAP e 39 ng/ml Nagao et al, 1996heterosporum

Geotrichum lipase II S. cerevisiae a-factor e 7.5 ug/ml 2 Vernet et al, 1993candidum

lipase I S. cerevisiae a-factor e 1 ug/rnl 3 Bertolini et al, 1995lipase A P. pastoris AOXl e 1 to 23 U/ml 2 Catoni et al, 1997lipase B P. pastoris AOXl e 200 U/ml 2 Catoni et al, 1997lipase I P. pastoris AOXl e 56 ug/ml 3 Holmquist et al, 1997lipase II P. pastoris AOXl e 62 ug/ml 2 Holmquist et al, 1997

Geotrichum sp. lipase 1 S. cerevisiae ACT] e 45 U/ml Monfort et al, 1999lipase 2 S. cerevisiae ACTl e 125 U/ml

Humicoia lipase S. cerevisiae TPl e 120 U/ml Okkels, 1996ianuginosa

Rhizopus niveus lipase I S. cerevisiae PGK e 270 ug/rnl Kohno et al, 1998lipase I S. cerevisiae PGK e 200-300 ug/rnl Kohno et al, 1999

Ri oryzae lipase S. cerevisiae UPR-lCL e 2.88 U/ml Takahashi et ai, 1998lipase P. pastoris AOXl e 12.89 U/ml*h Minning et al, 2001lipase S. cerevisiae GAPDH 4.1 U/g dry Washida et al, 2001

cellslipase S. cerevisiae GAPDH 99 mU/ml Matsumoto et al,

2001lipase S. cerevisiae UPR-lCL 474.5 mU/mllipase S. cerevisiae UPR-ICL s 61.3 UI g dry Matsumoto et ai,

cells 2002alipase S. cerevisiae UPR-ICL 350.6 mU/ml Matsumoto et al,

2002b

GAPDH e 880 mU/ml

*i=intracellular; e=extracellular: s=cellsurface


1994). RNL I was expressed in S. cerevisiae strainDBY746 (MATa his3-i1l leu2-3 leu2-112 ura3-52trpl-289) (ATCC 44776) under the control of thephosphoglycerokinase (PGK) gene promoter. Therecombinant lipase I was then produced in the culturemedium at a level of 350 Vlml. In order to increasethe lipase production level, the yeast strain DBY746,harbouring the plasmid YEp352PlipS, was mated tothe strain NA74-3A (MATa Ieu2-3 Ieu2-112 his4-5l9pho9-l canl [cir+]) (!FO 10430) to produce the strainND-12B. The strain NA74-3A lacks the PEP4 genecoding for the Proteinase A located within thevacuole. The diploids obtained were sporulated in asporulation medium and the asci were dissected. Withthe strain ND-12B, the recombinant lipase activityreached ?40 Vlml in the culture medium (Kohno et aI,1998). Optimization of growth conditions increased'the lipase production up to 1370 U1ml (270 ug oflipase proteinlml). However, western blot analysis ofthe recombinant lipase with RNL antibody revealedsome additional bands suggesting that some post-translational modifications occurred in this yeast.

Kohno et al (1999) developed a more efficientexpression system using the strain ND-12B and thehigh multicopy plasmid pJDB219. They reported thatRNL I activity in the culture medium was increasedby 1.2-fold, compared to the previous expressionsystem (Kohno et al, 1998). The protein purificationprocedure revealed two types of recombinant lipases.Analysis by reverse-phase HPLC, N-terminal aminoacid sequence and sugar content showed that thedifference between these two lipase types was mainlydue to their sugar content. Moreover, there were twospecies for each type of lipase. One was processed tothe A-chain and B-chain as in the native lipase whilethe other remained unprocessed. Although thesespurified lipases contained several post-translationalmodifications and different glycosylation patterns,their secondary structures remained similar to nativelipase.

Rhizopus oryzae lipaseThe nucleotide sequence of Rhizopus oryzae lipase

gene revealed a secretion signal sequence of 26 aminoacids, a pro sequence of 97 amino acids and a maturelipase region of 269 amino acids (Beer et ai, 1998).The pro sequence is involved in the proper folding ofthe mature protein and its subsequent secretion fromyeast cells. In S. cerevisiae, no lipase activity wasdetected in the culture medium or in the cell

homogenate when the mature portion of the ROLgene (mROL) was fused to the S. cerevisiae a-factorpre-signal sequence. However, a mROL constructwith its pro sequence (ProROL) at the N-terminus endwas active in both fractions (Takahashi et aI, 2001).The physical linkage (cis) of the prosequence with themature region does not seem to be a prerequisite(Takahashi et aI, 2001). ROL exhibited a high activityon triglycerides of medium chain fatty acids and is a1,3-specific lipase, which is particularly interestingfor fat and oil modification.

Many strategies were attempted for theoverexpression of ROL in yeasts. Takahashi et al(1998) studied the extracellular overproduction ofROL by fusing the sequence encoding theprosequence (ProROL) and the mature lipase regionto the a-factor pre-signal sequence. This constructwas expressed in S. cerevisiae under the control of the5'-upstream region of the isocitrate lyase gene ofCandida tropicalis (UPR-ICL). The VPR-ICL-mediated transcription is tightly repressed by glucoseand induced by non-fermentable carbon sources suchas acetate, oleic acid, ethanol and glycerol/lactate(Kanai et aI, 1996). The recombinant ROL productionwas optimized and reached 2.88 Vlml (28.0 ug/rnl) inthe culture medium at 120 h of cultivation.

Minning et al (1998) used the methylotrophic P.pastoris as a host and only fused the mature ROLlipase sequence in frame with the a-factor prepro-signal sequence from S. cerevisiae under the tightlymethanol controlled AOXl promoter. After an initialglycerol batch-phase to produce biomass, theinduction phase with methanol feeding was started.Methanol feeding was correlated with the dissolvedoxygen concentration (DO) of the culture. This DOcontrol-based strategy allowed a lipase production of500 Vlml (60 ug/ml) in the culture medium. To avoidmethanol toxicity on the cells, increases in methanolfeeding rate were maintained at a moderate level. Thisrecombinant mature ROL was produced in afunctional form, even without the prosequence, asopposed to the experiment with S. cerevisiae.Moreover, the production level in P. pastoris wassignificantly higher than in S. cerevisiae andrecombinant lipase properties were comparable tothose of the native ROL, except for highertemperature stability of the recombinant enzyme fromP. pastoris. However, the indirect control of methanolfeeding rate by DO measurements did not necessarilyassure the maintenance of methanol levels below the


toxicity concentrations (Minning et al, 2001). Theseauthors then used an off-line monitoring of themethanol concentration by gas chromatography tocontrol the methanol feeding rates, instead of theprevious DO control-based strategy. In this newprocess, a transition phase with simultaneous feedingof glycerol and methanol was introduced prior to themethanol induction phase. This optimized processimproved the lipase productivity to 12.89 U/ml/hrrepresenting a 13.6-fold increase compared to theDO-based process (Minning et al, 2001).

The cell-surface display system was well reviewedby Ueda & Tanaka (2000). The yeast cell-surfacedisplay system is of great interest in the generation ofnovel biocatalysts for uses in bioconversions becauseof their safety, their simplicity for geneticmodification and the rigidity of their cell-wallstructure (Lipke & Ovalle, 1998). In the most widelyused yeast-based cell-surface display system, thetarget gene and its secretion signal are fused to the C-terminal half of the a-agglutinin gene which containsthe putative glycosylphosphatidylinositol (GPI)anchor attachment signal. However, when the ROLsequence was cloned in this system, the anchor fusionprotein exhibited no lipase activity because theenzyme active site was very close to the N terminusof the a-agglutinin (Beer et al, 1996). To circumventthis problem, Washida et al (2001) inserted at the 3'-end of the ProROL gene a linker sequence coding fora Gly/Ser repeat motif (spacer) to enhance lipaseactivity by preserving the active site conformationnear the C-terminal end. This linker peptide is oftenused because of its conformational flexibility and itshydrophilic features (Weissman & Kim, 1992;Robinson & Sauer, 1998). The a-factor pre-signalsequence and ProROL gene were fused to the linkersequence and the GPI anchor attachment signalsequence. The authors showed that the lipasedisplayed on the yeast cell wall was active on soluble2,3-dimercaptopropan-1-01 tributyl ester (BALB) andinsoluble triolein. The cell pellet activity towardBALB reached 4.1 U/g of dry cell. Moreover, theyshowed that lipase activity was affected by the linkerpeptide length and was higher on BALB and trioleinwith linker pep tides of 14 and 17 amino acidsrespectively.

Matsumoto et al (2002a) have developed anothercell-surface display system by fusing the targetprotein after the C terminus end of the floculationfunctional domain of Flo1 protein. The Flo1

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floculation functional domain recognizes and adheresnon-covalently to cell wall components such as a-mannan carbohydrates causing a reversibleaggregation of cells into floes (Bony et al, 1997). Inthis system, the FS (amino acids 1 to 1099 of Flo1protein) and FL (amino acids 1 to 1447 of Flo1

. protein) gene sequences were fused to the ProROL Nterminus sequence. The fusion proteins wereexpressed under the control of the inducible promoterUPR-[CL. Whole cells expressing the FS-ProROLfusion protein reached a lipase activity of 61.3 U/g ofdry cell after 194 h of cultivation. The anchoredproteins were non-covalently attached to the cell walland these whole-cell biocatalysts were able tosynthesize methylesters from triglycerides andmethanol with a conversion rate of 78.3% after 72 hrsof reaction.

Another way to construct a whole-cell biocatalystsystem consists in the overproduction of intracellularR. oryzae recombinant lipase. Matsumoto et al (2001)constructed a whole-cell biocatalyst system byexpressing the ROL gene with its prosequence underthe control of the UPR-[CL in S. cerevisiae. Theintracellular lipase activity reached 474.5 mU/ml.After an air-drying permeabilization step, thesebiocatalysts successfully converted soybean oil andmethanol to methylesters with a conversion rate of71% after 165 hrs of reaction at 37°C with stepwisemethanol addition.

Geotrichum candidum lipase'Geotrichum candidum produces two different

extracellular lipases. Depending on the strains, theselipases are called I and II for the strain G. candidumATCC 34614 and A and B for G. candidum CMICC335426 (Sidebottom et al, 1991; Bertolini et al,1994). Protein homologies between lipase A and Bwere 84% and between lipase B and I were 97%(Charton, 1991). Despite this high homology, theselipases possess different substrate specificities(Charton & Macrae, 1992, 1993). Lipases I and Bhave a high specificity for cis-~9 unsaturatedsubstrates with long fatty acyl chains as oleic acid.This property could be interesting for some industrialapplications such as the enzymatic restructuring oflipids and oils into products with defined fatty acidcomposition. Various G. candidum lipase isoenzymesfound in the culture medium showed close physicaland biochemical properties but were different forsubstrate specificities. The overexpression of G.


candidum lipases was undertaken in order to isolatethose different lipase isoenzymes.

Vernet et al (1993) studied G. candidum lipase IIgene (GCL II). This gene was first amplified by PCR,cloned and sequenced. Sequencing data revealed thatthe GCL II gene was intron free. The gene sequencewas fused to the a-factor prepro-signal sequence andexpressed in S. cerevisiae under the control of the a-factor promoter and a recombinant enzymeproduction of 7.5 ug/rnl of culture was obtained. TheNHz-terrninal sequencing of the purified enzymerevealed the presence of a 4-amino-acid extensionGlu-Ala-Glu-Ala, Site-directed mutagenesisexperiments showed that the triad Ser217_Glu354_His463was essential for catalytic activity and that the activesite was more tolerant to conservative changes in thecarboxylic side chain within the triad than otherhydrolases with similar catalytic triads.

Bertolini et al (1995) fused the G. candidum lipaseI (GCL I) gene sequence in frame with the a-factorpre-signal sequence. A poly (His) tag and a specificthrombin-cleavage site were added at the N-terminusof the GCL I protein in order to facilitate thepurification of the recombinant protein by affinitychromatography. To compare both lipase isoenzymes(I and 11), the authors also prepared a similarconstruction with G. candidum lipase II gene. Thespecificity studies confirmed the difference insubstrate preference between both isoenzymes andshowed that GCL I was more selective forpolyunsaturated substrates than GCL II, while GCL IIshowed higher specific activity against saturatedsubstrates with long fatty acyl chains. A GCL IIGCLII hybrid molecule was also constructed with 194amino acids from the N-terminal end of GCL I(including the flap covering the active site) linked to350 amino acids from the C-terminal end of GCL II.The hybrid molecule showed a substrate preferencepattern identical to GCL II, suggesting that the flapregion in GCL I was not directly involved in substratedifferentiation although this region was believed totake part in recognition of the interface and in theactivation of the enzyme (Brzozowski et al, 1991;Van Tilbeurgh et al, 1993; Grochulski et al, 1993).

Holmquist et al (1997) used P. pastoris as a hostfor high-level expression of lipases 1 and II with theaim of obtaining lipase variants with altered structurein sufficient quantities for kinetic characterizationexperiments. They have fused the lipase cDNAs withaN-terminal (Hisjs-tag extension sequence and the a-

factor secretion signal peptide sequence of S.cerevisiae under the control of the methanol-inducibleAOX1 promoter. The extracellular activities ofrecombinant lipases I and II were 56 ug/ml and 62ug/ml, respectively. These production yields werehigher than those obtained with S. cerevisiae andconfirmed the high efficiency of this proteinexpression system. Moreover, the purificationprocedure allowed the isolation of 50% of theproduced lipase virtually pure from contaminatingproteins, while less than 15% of the secreted lipaseproduced with S. cerevisiae (Bertolini et al, 1995)could be purified.

Cantoni et al (1997) studied G. candidum ATCC335426 lipases A and B in P. pastoris. The sequencesencoding the mature lipases were fused in frame withthe a-factor signal sequence and expressed under thecontrol of the AOXI promoter. Depending on theclones, the extracellular production of lipases A and Bvaried from 1 U/ml to 23 U/ml and 1 U/ml to 50U/ml, respectively. Optimization studies of therecombinant lipase B production increased theactivity to 200 U/ml of the culture medium. Thelipase B amino acid sequence contained two commonN-glycosylation sites and a carbohydrate content of3%.

Candida rugosa lipaseCandida rugosa lipases (CRLs) are among the

commercial lipases most often employed inhydrolysis and synthesis of a wide range of esters ofcommercial interest. The potential applications ofthese lipases were already reviewed (Benjamin &Pandey, 1998). C. rugosa produced several closelyrelated lipases. Five genes coding for lipases (lip1 tolip5) have already been cloned and sequenced (Lottiet al, 1993). The recombinant production of CRLisoenzymes is the most promising strategy for futureindustrial applications of pure/defined CRLisoenzyme preparations. Furthermore mutagenesisallows the generation of new CRL derivatives forfurther industrial applications as well as for studies onstructure-function relationships (Ferrer et al, 2001).

The gene product from lip1 constitutes the majorlipase isoenzyme of C. rugosa and is the bestcharacterized since its crystallographic structure hasbeen solved (Grochulski et al, 1993). The matureprotein encoded by lip1 contains 534 amino acidsafter the cleavage of a 15-residue leader sequence.The protein is glycosylated and has a molecular


weight of about 60 kDa (Longhi et al, 1992). C.rugosa and some other Candida species have anunusual codon usage in which the triplet CTG, theuniversal codon for leucine, was read as a serine(Kawaguchi et al, 1989). In this species, the CTGtriplet accounts for about 40% of the total serinecodons (Lotti et al, 1993). This hampers thefunctional expression of genes derived from this yeastin a conventional heterologous host. In the lipl gene,CTG triplets encode 20 out of the 47 serine residues,including the catalytic Ser209

• As consequence, whenlipl was expressed in S. cerevisiae using either theenzyme endogenous leader sequence or the signalpeptide of the Kluyveromyces lac tis killer toxin, therecombinant lipase was correctly targeted to theendoplasmic reticulum but did not process furtheralong the secretory pathway and remained entrappedin an inactive form in the membranes (Fusetti et al,1996). To circumvent this problem, the first attempttried by Brocca et al (1998) consisted in replacing bysite-directed mutagenesis the CTG codons by otherserine universal codons. A group of eight Serresidues, believed to play a major role in the proteinstructure and function, was targeted. Four mutantgenes, containing an increasing number of restoredSer residues (2, 3, 5, 8), were cloned intopEMBLyex4, a shuttle expression vector for S.cerevisiae with the inducible VAS GAL sequence. Theexpressed lipases, produced only in the intracellularfraction at a level of 10-20 ug/ml of culture, were alsoinactive meaning that these substitutions were notsufficient to produce a recombinant active protein. Asalternative approach, lipl gene has been completelysynthesized with an optimized nucleotide sequence.The synthetic gene (slipl) showed 77% nucleotidesequence identity with the natural gene. sLipl wasexpressed in S. cerevisiae under VAS GAL promoter.The synthetic gene was also fused to the a-factorprepro-signal sequence (pp-slipl), to the a-factor pre-signal sequence (p-slipl) or to the natural leadersequence of the lipl gene (nl-slipl) and thosecontructs were expressed under the control of theAOXl promoter in P. pastoris. The recombinantclones with p-slipl and pp-slipl were more activethan those with nl-lipl sequence. In l-L bioreactor,the lipase production in the culture medium for theclones harbouring the sequence pp-slipl reached 150U/ml. With S. cerevisiae, the lipase activity in theculture medium ranged around 5-7 U/ml regardless ofthe signal sequence used. The recombinant lipase

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contained three potential N-glycosylation sites inpositions 291, 314, and 351 and a carbohydratecontent of 5%. The physicochemical and catalyticproperties of the recombinant lipase were similar tothose of commercial C. rugosa lipase, showing thatlipl encodes the major isoenzyme in commercial CRLpreparations.

ConclusionLipases are amongst the most potential biocatalysts

for industrial applications. Recombinant DNAtechnology constitutes a suitable way to produce highlevels of lipases with reduced cost. Several reportsrevealed that P. pastoris constitutes a very efficientexpression system for the production of recombinantmicrobial lipases at high levels. Yeast expressionsystem can be successfully engineered to constructwhole cell biocatalysts expressing recombinant lipaseintracellularly or in a cell surface display system. Thelatter approach will gain much attention in the futuredue to their potential applications such asbioconversions in organic media. The futuredevelopment in recombinant DNA technology mainlythe use of more efficient expression systems should,in combination with the directed evolution, allow toovercome the problems limiting the use of theseenzymes in the industry.

ReferencesBeer H 0 et al, 1998. Cloning, expression, characterization and

role of the leader sequence of a lipase from Rhizopus oryzae.Biochim Biophys Acta, 1399,173-180.

Benjamin S & Pandey A, 1998. Candida rugosa lipases:Molecular biology and versatility in biotechnology. Yeast,14, 1069-1087.

Bertolini M C et al, 1994. Polymorphism in the lipase genes ofGeotrichum candidum strains. Eur J Biochem, 219, 119-125.

Bertolini M C et al, 1995. Expression and characterization ofGeotrichum candidum lipase I gene: Comparison ofspecificity profile with lipase II. Eur J Biochem, 228, 863-869.

Bony M et al, 1997. Localization and cell surface anchoring of theSaccharomyces cerevisiae flocculation protein Flolp. JBacteriol, 179,4929-4936.

Brocca S et al, 1998. Design, total synthesis, and functionaloverexpression of the Candida rugosa lipl gene coding for amajor industrial lipase. Protein Sci, 7, 1415-1422.

Brzozowski A M et al, 1991. A model for interfacial activation inlipases from the structure of a fungal lipase-inhibitorcomplex. Nature( Land), 351, 491-494.

Cantoni E et al, 1997. Overexpression of lipase A and B ofGeotrichum candidum in Pichia pastoris: High-levelproduction and some properties of functional expressedlipase B. Biotechnol Tech, 11, 689-695.


Cereghino J L & Cregg J M, 2000. Heterologous proteinexpression in the methylotrophic yeast Pichia pastoris.FEMS Microbiol Rev, 24, 45-66.

Charton E, 1991. Purification et caracterisation de deux lipases deGeotrichum candidum CMICC 335426. Ph D Thesis, InstitutNational Agronomique Paris-Grignon, Paris.

Charton E & Macrae A R, 1992. Substrate specificities of lipasesA and B from Geotrichum candidum CMICC 335426.Biochim Biophys Acta, 1123, 59-64.

Charton E & Macrae A R, 1993. Specificities of immobilizedGeotrichum candidum CMICC 335426 lipases A and B inhydrolysis and ester synthesis reactions in organic solvents.Enzyme Microb Technol, 15, 489-493.

Cregg J M et al, 1989. Functional characterization of the twoalcohol oxidase genes from the yeast Pichia pastoris. MolCell Bioi, 9, 1316-1323.

Egli T et al, 1980. Methanol metabolism in yeasts: Regulation ofthe synthesis of catabolic enzymes. Arch Microbiol, 124,115-12l.

Ferrato F et al, 1997. A critical re-evaluation of the phenomenonof interfacial activation. Methods Enrymol, 286, 327-347.

Ferrer P et al, 200 I. Production of native and recombinant lipasesby Candida rugosa. Appl Biochem Biotechnol, 95, 221-255.

Fusetti F et al, 1996. Effect of the leader sequence on theexpression of recombinant Candida rugosa lipase bySaccharomyces cerevisiae cells. Biotechnol Leu." 18, 281-286.

Gellissen G, 2000. Heterologous protein production inmethylotrophic yeasts. Appl Microbiol Biotechnol, 54, 741-750.

Godfrey T & West S, 1996. Introduction to industrialenzymology. in Industrial Enzymology, edited by T Godfrey& S West, 2nd edn. Stockton Press, New York. Pp 1-8.

Grochulski P et al, 1993. Insights into interfacial activation froman open structure of Candida rugosa lipase. J Bioi Chem,268, 12843-12847.

Hitzeman R A et at; 1981. Expression of a human gene forinterferon in yeast. Nature( Land), 293, 717-722.

Holmquist M et al, 1997. High-level production of recombinantGeotrichum candidum lipases in yeast Pichia pastoris.Protein Express Purif, 11, 35-40.

Itakura K et al, 1977. Expression in Escherichia coli of achemically synthesized gene for the hormone somatostatin.Science, 198, 1056-1063.

Kanai T et al, 1996. A novel heterologous gene expression systemin Saccharomyces cerevisiae using the isocitrate lyase genepromoter from Candida tropicalis. Appl MicrobiolBiotechnol, 44, 759-765.

Kawaguchi Y et al, 1989. The codon CUG is read as serine in anasporogenic yeast Candida cylindracea. Nature(Lond), 341,164-166.

Kohno M et al, 1994. Purification, characterization, andcrystallization of two types of lipase from Rhizopus niveus.Biosci Biotech Biochem, 58, 1007-1012.

Kohno M et al, 1998. Cloning of genomic DNA of Rhizopusniveus lipase and expression in the yeast Sacchromycescerevisiae. Biosci Biotechnol Biochem, 62, 2425-2427.

Kohno M et al, 1999. High-level expression of Rhizopus niveuslipase in the yeast Saccharomyces cerevisiae and structuralproperties of the expressed enzyme. Protein Express Purif,15, 327-335.

Ledeboer A M et al, 1985. Molecular cloning and characterizationof a gene coding for methanol oxidase in Hansenulapolymorpha. Nucleic Acids Res, 13, 3063-3082.

Lipke P N &. Ovalle R, 1998. Cell wall architecture in yeast: Newstructure and new challenges. J Bacteriol, I~O, 3735-3740.

Longhi S et al, 1992. Cloning and nucleotide sequences of twolipase genes from Candida cylindracea. Biochim BiophysActa, 1131,227-232.

Lotti M et al, 1993. Cloning and analysis of Candida cylindracea. lipase sequences. Gene, 124, 45-55.

Matsumoto T et al, 200l. Yeast whole-cell biocatalyst constructedby intracellular overproduction of Rhizopus oryzae lipase isapplicable to biodiesel fuel production. Appl MicrobiolBiotechnol, 57, 515-520.

Matsumoto T et al, 2002a. Construction of yeast strains with highcell surface lipase activity by using novel display systemsbased on the Flolp flocculation functional domain. ApplEnviron Microbiol, 68, 4517-4522.

Matsumoto T et al, 2002b. Preparation of high activity yeastwhole cell biocatalysts by optimization of intracellularproduction of recombinant Rhizopus oryzae lipase. J MolCatal B: Enzymatic, 17, 143-149.

Minning S et al, 1998. Functional expression of Rhizopus oryzaelipase in Pichia pastoris: High-level production and someproperties. J Biotechnol, 66,147-156.

Minning S et al, 200l. Optimization of the high-level productionof Rhizopus oryzae lipase in Pichia pastoris. J Biotechnol,86,59-70.

Monfort A et al, 1999. Expression of LIP I and LIP2 genes fromGeotrichum species in baker's yeast strains and theirapplication to the bread-making process. J Agric Food Chem,47,803-808.

Nagao T et al, 1996. Expression of lipase cDNA from Fusariumheterosporum by Saccharomyces cerevisiae: High-levelproduction and purification. J Ferment Bioeng, 81, 488-492.

Okkels J S, 1996. A URA3-promoter deletion in a pYES vectorincreases the expression level of a fungal lipase inSaccharomyces cerevisiae. Ann NY Acad Sci, 782, 202-207.

Pignede G et al, 2000. Autocloning and amplification of LIP2 inYarrowia lipolytica. Appl Environ Microbiol, 66, 3283-3289.

Raymond C K et al, 1998. Development of the methylotrophicyeast Pichia methanolica for the expression of the 65kilodalton isoform of human glutamate decarboxylase. Yeast,14, 11-23.

Robinson C R & Sauer R T, 1998. Optimizing the stability ofsingle-chain proteins by linker length and compositionmutagenesis. Proc Natl Acad Sci USA, 95, 5929-5934.

Roggenkamp R et al, 1984. Biosynthesis and regulation of theperoxisomal methanol oxidase from the methylotrophic yeastHansenula polymorpha. Mol Gen Genet, 194, 489-493.

Rotticci-Mulder J C et al, 200l. Expression in Pichia pastoris ofCandida antarctica Lipase B and Lipase B fused to acellulose-binding domain. Protein Express Purif, 21, 386-392.

Sakai Y & Tani Y, 1992. Cloning and sequencing of the alcoholoxidase-encoding gene (AOD1) from the formaldehyde-producing asporogeneous methylotrophic yeast, Candidaboidinii S2. Gene, 114, 67-73.

Sanchez M et al, 2002. Engineering of baker's yeasts, E. coli andBacillus hosts for the production of Bacillus subtilis lipase A.Biotechnol Bioeng, 78, 339-345.


Sarda L & Desnuelle P, 1958. Action de la lipase pancreatique surles esters en emulsion. Biochim Biophys Acta, 30, 513-521.

Sharma R et al, 2001. Production, purification, characterization,and applications of lipases. Biotechnol Adv, 19,627-662.

Sidebottom C M et al, 1991. Geotrichum candidum producesseveral lipases with markedly different substrate specificities.Eur J Biochem, 202, 485-491.

Takahashi S et al, 1998. Extracellular production of activeRhizopus oryzae lipase by Saccharomyces cerevisiae. JFerment Bioeng, 86, 164-168.

Takahashi S et al, 2001. Function of the pro sequence for in vivofolding and secretion of active Rhizopus oryzae lipase inSacchromyces cerevisiae. Appl Microbiol Biotechnol, 55,454-462.

Veda M & Tanaka A, 2000. Cell surface engineering of yeast:Construction of arming yeast with biocatalyst. J BiosciBioeng, 90, 125-l36.

Valenzuela P et al; 1982.' Synthesis and assembly of hepatitis Bvirus surface antigen particles in yeast. Nature(Lond), 298,347-350.

355

Van Dijk R et al, 2000. The methylotrophic yeast Hansenulapolymorpha: A versatile cell factory. Enzyme MicrobTechnol, 26, 793-800.

Van Tilbeurgh H et al, 1993. Interfacial activation of the lipase-procolipase complex by mixed micelles revealed by X-raycrystallography. Nature(Lond), 362, 814-820.

Verger R, 1997. 'Interfacial activation' of lipases: Facts andartifacts. Trends Biotechnol, 15, 32-38.

Vernet T et al, 1993. Cloning and expression of Geotrichumcandidum lipase II gene in yeast. J Bioi Chem, 268, 26212-26219.

Wang T-T et al, 2001. Transformation systems of non-Sacchromyces yeasts. Crit Rev Biotechnol, 21,177-218.

Washida M et al, 2001. Spacer-mediated display of active lipaseon the yeast cell surface. Appl Microbiol Biotechnol, 56, 681-686.

Weissman J S & Kim P S, 1992. The pro region of BPTIfacilitates folding. Cell, 71, 841-85l.

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