V o l u m e 291, ~ u m b e r 2, 299-302 F E B $ 10262 © 1991 Federation of European B~ochemtcal Soc~ettes 00145793/91/$3 50 A DOAIIS 001457939100971X

O c t o b e r 1991

Overexpression of alcohol oxidase in Pichia pastoris M J. de H o o p ~, J. Cregg ~, I. Keizer-Gunnink ~, Klaas Sjollerna 3, M Veenhuis ~ and G. A b ~ ~Laboratory o f Btochem~so y, Umver~tty o f Gronmgen, Ntjenborgh 16, 9747 AG Gronmgen, The Netherlands, ZDept o f Chemical and Btologtcal Sciences, Oregon Graduate lnstttute o f Sctence and Technology, 19600 Von Neumann Dr, Beave~ ton 01~ 97006-1999, USA a n d ~Laborato~y for Electron Mtcrostopy, UtItve~stty o f Gronmgen, Kerklaan 30, 9751 N N Gronmgen, The Netherlands Received 8 July 1991 The protein import capacity of perox~somes m methylotroph~c yeasts was stud~ed using Ptch~apastorts containing one or lwo extra cop~es of the gene encoding the pero×~somal protein alcohol ox~dase The alcohol ox~dase overproduced m th~s strata was only partmlly ~mported and assembled rote the active, octamertc term of the protein The excess remmned m the cytosol as protein aggregates composed of monomers These results are discussed m wew of the. possible apphcat~on of perox~somes as storage compartments for heterologous proteins Alcohol ox~dase, Methylotrophtc yeast, Peroxmome

1. I N T R O D U C T I O N Peroxtsomes ate smgle-membrane bounded organelles containing a matrtx of proteins, generally mcludmg catalase and one or more hydrogen peroxtde-producmg oxtdases They are versatde organelles that can vary m number, s~ze and protem content [I,2] In yeast, prohtbtatton of pcroxtsomes ~s dependent on growth condittons; some o f the enzymes mvolved m the metabohsm of particular C- and N-sources m the medium accumulate m the organelle Therefore, the compostt~on of petox~somes can be easdy mampulated m yeast [3-6]. Strong proliferation of perox~somes occurs for example when methylotroph~c yeasts are shifted from glucose to methanol as the sole source of carbon and energy Under certain conditions, these peroxtsomes can occupy up to 80% of the cytosohc volume [7] These perox~somes have a crystalhne matrix composed of alcohol ox~dase octamers, wluch ~s the acttve form of th~s protein [8,9] Also present in the perox~somes, although not as abundant as alcohol oxidase, are 2 other key enzymes of methanol metabohsm, namely catalase and d~hydroxyacetone synthase [10] The ~mport capacity of perox~somes present m methanoi-cultured yeast may offer the possibthty to store hete~ologously expressed protems, eqmpped with the approprmte targettmg stgnals, mto peroxtsomes [11]. Pero×tsomal locahzat~on could have the advantage that the proteins are kept separate from proteolyt~c actwit~es in the cytosol Th~s system may have a potential value for storage of heterologously expressed proteins wluch Abhrevtatton Ilp.AOX, the alcohol oxtdase gent from thu)~enuhtpoh,n~orpha Cotrerl~ondente addrerv G Ab, Laboratow of B~ochem~stry, Umve~s~ty el Gronmgen. N,jenborgh 16, 9747 AG Gronmgen, The Nethe)lands Fax (31) (50) 634165

t'uhh~hcd h) Llcr)k't Sttcnc¢ Puhll~herv B V

are susceptible to proteolysls. In a first attempt to aria* lyze the import capacity of peroxisomes present m methanol-cultured cells, we overexpressed alcohol ox~dase and analyzed to ~,hat extent the enzyme was imported. 2 MATERIALS AND METHODS 2 l Plasmld constntctlon, yeast transformation and genetw analysis A 2 5-kb fragment containing the coding sequence of the alcohol oxldase gene from ltar~senulapolylnorpha (Hp.A OX) [12], flanked by I0 bp 5' and 250 bp 3' untranslated sequences, was inserted rote the BamHl s~te of vector pAOBam Tins ~s An express~on vector derived from pAO804 [I 3] by insertion e r a BamHl hnker (AATTGGATCC) rote the umque EcoRl site In the final construct, the Hp.AOX gene was located between the promoter and terminator of the A OXI gene

el Pwhta pastel ~ A transformation system of the related methylotroph~c yeast Ptclua pasto~,s [14] was used for overexpresslon ofalc0hol oxxdase Transformation of Ptchta pastorts GS115 (ht~4) was perfomaed as described by Crcgg et al [14], and was based on complementat~on of tust~dme auxotrophy To force homologous integration rote the defective hts4 gene, the plasmld DNA was hnearlvedwlth Stul, wtuch cuts approximately m the middle of the HIS4 gene present m pAOBam (Fig I A) Transfon~ants were screened for propel mtegratton of the plasm~d by Southern blotting The blot was s~.rcened w~th random-pr~med [~~'P]dCTP.labelled [15] plasmtd pYM4, which ts pBR322 containing the HI~4 gene of o pa~tort~ [14]

22 Preparanon of cell eatract~, measurement of actvtty and octamert2allotl

A culture of transformed cells, grown on Dffco's Yeast N~trogen Base without amino acids and w~th 0 5% glycerol to an OD,,0o of 2-3, was 10.fold dduted w~th medium containing I~ methanol a~ the sole carbon sourt.e Aftel 16 h. cells were harvested (OD~o=2-2 5) and extrat.ts were prepared [8] Alcohol ox~dase activity was assayed as described [I 6] Monomertc and octamenc alcohol oxtdase were separated by sucrose gradient centrffugatton [8] and v~suahsed by Western blotting [17] The anttbodtes used were etther a polyclonal antibody ratted against denatured alcohol oxldase of Han~enula polymorpha or a monoclonal anttb0dy (OAOI I) The polyclonal antibody recogmzed both alcohol oxldase of P pa~tort~ and ![ polrmorpha, while the monoclonal anttbody was spectfi~, for altohol oxtdase from H polymorpha (M de ilo0p, unpubhshed results)

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::~tnlmo-electronmlcvoscop~

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1 he mtracellular locahzatlon of alcoho! oxtdase and d:hgdrotyacetone synthase was determmed by lmmunogold ldbelhng on thm sechens of Lowlcryl-embedded cells [lo]

3 RESULTS 3 1, Mult~opy mtegratlons m Plchla pastorls GS11.5 To generate a stram that overproduces alcohol 0x1. dase when cultured on methanol, B pastorrs GS 115 was equipped with addltlonal coples of the alcohol oxldase gene from the related yeast H polymorpha The alcohol oxldase gene was placed between the promoter and the terminator of the B pastorrs alcohol oxldase gene The recombinant plasmld was targeted mto the defective his& gene of the host strain GSl15. To favour homologous recombmatlon, the plasmld was hnearlzed m the HIS4 gene prior to transforrnatlon (FIN 1A). DNA from hlstldme prototrophlc transformants was digested with &g/II and analyzed by Southern blottmg using pYM4 as a probe. This plasmld contamed the HIS4 gene and the E cob gene encoding j-lactamase [14] Ilost DNA yielded one band of 3 kb representmg the defective ius4 gene (Fig 1A and Fig. 2, lane 1) Smglecopy integrations m the his4 locus resulted in the replacement of the 3-kb band by a number of new bands (Fig 1B and Fig. 2, lane 2). Knowmg the posltlons of the BgnI sites, we could identify a band of 2 4 kb as the fragment contammg the p-lactamase gene The bands of 4.0 and 4 5 kb hybrldlze because they contam HIS4 sequences plus different parts of the plasrnld An addltlonal band of 5 5 kb and an mtcnslfied 2.4 kb band mdlcated a double-copy mtegrdtlon of the plasmid m the defective h4 gene (Fig. 2, lane 3)

FIN I

Gent tqgmig

36 24

Fig 2 Southern clnalysls of PIC/ZIUpmtorls GSI I5 cells contammg no (I), one (2) or two copies (3) of the Hp-AOX gene mtegrdted In the krs# gene The &fII fragments hybrldlzmg with plasmld pYM4 are shrwn

3 2 Overexpressron of ulcohol oxrduse Provldmg P pastoru with additional coplcs of the ffp-AOXgene resulted III higher alcohol oxrdasc expresslon levels upon growth on methanol Whereas the increase m activity with the first addltlonal copy was 73%, that with a second copy was considerably smaller (Table I) To examine the locahzatlon of the alcohol oxldase, we performed lmmunogold labehng on Eowlcryl-embedded cell material Fig 3A shows the locallzatlon of endogenous alcohol oxidase m P pastorm GSl15 In these methanol-grown cells, the protein was exclusively located in the peroxlsomes; no alcohol oxiddse could be found outside the peroxlsomes This IS m contrast with GSll5 cells contaming copies of the AOX gent of W polyntorpha. In these cells, dlcohol oxldase-lmmunoreactive material was also found outside the peroxlsomes, often m Irregular, electron-dense structures not surrounded by a membrane (Fig. 3I4.C and D) Extracts of wild-type GSI 15 and GSl15 contammg a double-copy integration of the Hp-AOX gene, cultured on methanol, were analyzed by sucrose gradient centrlfugatlon In GS 115, only octamerlc alcohol 0x1. dose could be detected (Fig 4A) The strum, equipped

of the H pol~morphtr alcohol ox1d.m gcnc Into

the Irrr4 locus of P PotrorrJ Pdncl (A) dcplcts the transfom~dhon vector (pAOBam)contdltrmgthe HI,-AOX gcrw (arrow) bciwccn the ptonlotsr (P) and termmdtor (T) of the P pmmrr> AOX/ gcnc, Homologous rccornbmatkon of the pldsmld, hncdlmcl 111the N/S scqucncc, with the /IIS locus (ddshcd) of tllc P prr\rorr\ gcnomc (stlpplud) 15 mdmkd Pdncl (i3) shows the gcnc configur&on dftcr mtcgrdtlon 111 the /ml

300

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29 1, number

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FEES

LETTERS

Qctober 1991

Fig 5 Western blots of methanol-induced cell lysates from A&~ pas101IS (lane I, 25 fig) or H~i~rerrr~lapolvnorphn (lane 2, 25 pg) probed with polyclonal antrbodles made against denatured alcohol oxldase from Hansetrula pol_wnorplm (A) or the monoclonal antIbody 0,401 I which preferentially recogmzed Ihmetnda po/vtnorphn alcohol oxldase (B)

Fig 3 Immune-electronmlcloscopy of P~lrra puslorrs GSI 15 (A, I5 400x) and a transformant contammg two copses of the Hp-AOX gene (B 3 1 000 x, C. 16 800x. and D. 12 600x) The locahzation of alcohol oxldase on thm sections of methanol-induced transformdnts ISmdrked by IO-run goid pn~l~cles 0rgdne:tcs dre indicated ds follows, m = mltochondrlon, II = nucleus, p = peroxlsome and v = vacuole a = cytosohc aggregate of alcohol oxldase

with two copies of the Hp-AOX gene, contained octamerlc alcohol oxldase as well as alcohol oxldase-lmmunoreactive matellal scdlmcntmg at a rate loughly slmllar to that of bovme serum albumm run in a parallel 1

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oudJsc III P/c hcc ~WOI /Y GSI I5 the HpAOX gcnu Wcstct II blots 01 sucro\c gl,ldlcnt frxtloos (Ltnc 1 bottom. I~nc G lop) of rcll lyadks JIG showr~ A Wcstcrn blot of J.cell lyutc (200/rg) of non-ttdtvfolmcd P/C/UN pc~or IS GSI I5 probed wth polyclon.~l dnttbody .1gm51 .11cohol OYI~J~C 11 Wcstcrn blot cantain~ngccll lycdtc (IOO~g) of ttdw fat m.mt GSI IS owcqmwng .rlcol~ol OXKLISC plobcd with polyclondl .~n~~t~ody~g,~m~t alcohol ovd.w C,Irlcntlcal to (D) but probed with ~O~OLIOI~JI dnlibodtc5 which prcfcrcntlJlly rccogn!zcci ~lcolrol Ol~gorm

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gradient (data not shown) suggestmg that it represented monomeric alcohol oxidase (Fig. 4F3) The amount of the rnonomerlc alcohol oxldase was estimated to be 20-30% of the total alcohol oxldase. Dlscllmmatlon of the import system against heterologous alcohol oxldase did not OCCUI This can be concluded from a Western blot analysis using a monoclonal antibody which 1s spec~fic for alcohol oxidase from Hansenula and does not react with dkohol oxldase from Plckla (Fig 5). Clearly the alcohol oxldase of Hmsmuia assembled into octamers (Fig 4C) as did the endogenous alcohol oxidase from P. pastorzs (Fig. 4A). Moreover, we have found earher that peroxlsomes of P pas&w are able to recogmze and accumulate alcohol oxldase from H polymot-pha When the Hp-AOX gene was expressed m a P&a strain m which both endogenous alcohol oxldase genes were disrupted, all alcohol oxldase was localrzed exclusively mslde peroxisomes and was m the active octamellc form (M de Hoop, unpublished results). Fig 4C shows that the alcohol oxldase of Hansenula was able to assemble into octamers m P pastor IS Since octamers ale normally present mslde peroxlsomes only [8,16], we conclude that the octamerlc alcohol oxldasc from Hansetrula IS localized mslde the organelle The ovelexprcsslon of alcohol oxidase had no effect on import of other matrix proteins, c.g dlhydroxyacetone synth&e After lmmunogold-labelhng with antlbodles against dlhydroxyacctone synthase, gold particles were exclusively confined to the peroxlsomal matrix (data not shown)

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4 DISCUSSION Methylotrophlc yeasts are an attractive host for the expression of heterologous proteins for several reasons Firstly, methanol 15 a cheap C-source Secondly, methylotrophlc yeasts can be grown to high cell densllics (130 g dry ccl! weight/l) in contmuous culture [IS]. Thirdly, the promoter of the llighly expressed and tightly regulated alcohol oxldasc gene can be used to drive synthks ljf a foreign ptotcm Fmally, mtegl sting cxpresston vcctors yielding stable transformants have been deslgned 301

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FEBSLE-M-ERS

[13]. The pcroxlsomes present m methanol-cultured yeast might add yet another advantage to this expression system, because the organelles may be used for storage of heterologously expressed proteins known to be susceptible to proteolytlc cleavage. The results presented here show that alcohol oxldase expressed from several gene copies m P pastom is not fully imported and assembled into peroxlsomes Obvlously, the balance between alcohol oxldase synthesis and its post-translatlonal import IS disturbed m these cells, suggesting that saturation of either the import capacity of the peroxisomal space or of the peroxlsomal nnpolt machinery had occurred Both sltuatlons should result in a cytosolic accumulation of all peroxlsomal matrix protcms. A similar situation exists m peroxlsome-deficlent cells, where peroxlsomal matrix enzymes are located m the cytosol. In these cells cytosohc crystals composed of active alcohol oxldase are observed [19]. Such alcohol oxldase crystallolds were never observed m the cytosol of strains described m this study and also dlhydroxyacetone synthase was St.111 only detectable m the matrix and was not observed m the cytosol. Therefore, we believe that the rate of alcohol oxldase import versus synthesis IS hmltlng, rather than the capacity of peroxisomes for protein storage The fact that peroxlsomes can oscupy 80% of cell volume m methanolhmlted continuous cultures [20] demonstrates that pcroxlsomes should have a larger storage capacity than seen m the multi-copy gene experiments described here Even under the condltlons where peroxlsomes do not prohferate such as glycerol 1161or glucose [21], single organelles appear to have the ability to internalize the high amounts of alcohol oxldase synthesized m transformants containing multl-copy plasmids Because m our experiments alcohol oxldase IS being expressed from multiple copies of the strong alcohol oxidase promoter, it is reasonable to assume that the rate of alcohol OXIdase synthesis IS excepttonally high m these strains Why would a rapid rate of synthesis cause saturation of the Import system and result m aggregation m the cytosol? Too high an expression I ate might oversaturatc the capacity of cytosolic unfoldases [22] m theil task of holding alcohol oxldase monomers in an Import-competent conformation. This might result m the formation ofalcohol oxldase aggregates as observed m our studies Once aggregated. the alcohol oxtdasc IS probably no longer suitable for import Concerning the prospects to compartmentalize hetcrologous proteins in peroxlsomes. one has to take into account that pcroxisomes have a large storage capacity, but that the efficiency of Import may be inversely rclated to rate of synthcsls of peroxisomal proteins FUI thcrmorc, It IS also hkely that different hctclologous proteins are rmported at different rates and thcreforc. it may bc necessary to adjust cxprcsslon rates for optlmal compclrtmcntalizstion of each protcm To implove lmpol t of heterologous protcms into pero302

October 1991

xlsomcs, elther the cxpresslon of endogenous pcroxlsornal proteins has to be reduced, or the import machinery must be further increased by e.g. overexpresslon of chaperones, like members of the HSPSQ family [Z&231 ~ckno&e~fgernerrrs We thank Dr M A C Gleeson for his interest and stlmulatmg dIscussIons and are grateful to Prot Dr W Harder for cntlcally readmg the manuscript A plasmld contammg the I-/p-AOX gene was kmdly provided by Drs B Dlstel and A Ledeboer Part of this work WJS carned out at the Salk Instltutc Biotechnology and Industrial AssoLlates, Inc (SIBIA), Ln Jolla. CA, USA m the group of Dr G Thlll We would like to thank him for glvmg M de Hoop this opportumty This was supported by a Wood/Whelan Research Fellowship from the International Umon of Blochemlstry and the ‘Gronmger Umverslteltsfonds

REFERENCES 111Bremer, J , Osmundsen, H , Chnstlansen,

R Z and Borrcbaek, B (1981) Methods Enzymol 72, 5Ofj-520 PI Tolbert, N E (1981) Ann Rev Blochem 50, 133-157 131 Fukul, S , Tanaka, A , Kawdmoto, S . Yashura, § , Terdmshl, Y and Osuml, M (197.5) J Bdcterlol 123, 317-328 [41 Zwnrt, K , Veenhuls, M , van DUken, J P and Harder, W (1980) Arch Mlcroblol 126, 117-126 PI Vecnhuls, M , Mateblowskl, M , Kundu, W H and Harder, W (1987) YedSt 3, 77-84 Kl Veenhuis. M and Halder, W (1987) m Peroxlsomes in Biology and Medlcme (F&m-u, I-l D and Sles, H eds) pp 436-458, Sprmger, Berlm-Heldelbelg PI Veenhuls, M , van Dijkcn, J P , PIIon S A F and Hdrder, W (1985) Arch Mlcroblol 143. 153-163 PI Goodman J M , Scott, C W , Donahue, P N and Atherton, J P (1984) J BIOI Chem 259, 8485-8493 PI Vecnhuls. M , Harder, W , van Dqken, P . Mayer, F (1981) Mol Ccl1 BIOI 1. 949-957 PO1Doumd. A C, Veenhuls, M , dc Konmg. W , Evers, M and Harder, W (1985) Arch Mlcroblol 143, 237-243 [I 11Gould, S J , Keller, G A, Hosken N , Wllkmson. J and Subramdm, S (1989) J Cell BIOI 108, 1657-1664 [I4 Ledeboer, A M , Edens. L , Mdat, J, Vlsser, C , Bos. J W, Vcrrips. C T , Jdnowrcz, Z , Eckart, M , Roggenkamp, R and MolIcnberg, C P (1985) Nucleic Auds Res 13, 3063-3082 [l31 Dqdn. M E , Tschopp. J , Grmnd, L , Lair, S V Crdlg, W S . Vehcelebl, G , S&gel, R , Davis, G R dnd Thlll. G (1988) Dev lnd Mlcroblol 29, 59-65 Crcgg. J M , Balrmgcr, K J , Hessler. A Y dnd Mdddcn, K R (1985) Mol Cell Btol 5. 3376-3385 Fanberg, A P dnd Vogel\lcm, I3 (1983) And1 Blochcm 132, 6-13 Roggcnkdmp. R , DIdIon. T dnd Kowdlhk, K V (19S9) Mol Ccl1 B~ol 9. 988-994 Burncttc. W N (1981) And1 Blochcm 112, 195-203 Crcgg, J M dnd Mdddcn. K R (1987) in B~olog~cdl Rescdrch on lndustri~d Ycdsts. volume II (Stewart. G G , Russell. I , Klcm. R D and Hlcbsch. R R , cdr) pp l-l 8, CRC Prcbs. Boca Raton WI dcr KIcI, I (I991 ) Thc\~s. Umvcrr~ty of Gronmgcn, The Ncthcrldnds Vccnhuls. M + vdn Dykcn, J P dnd Hdrdcl, W (1983) Adv Mtcroblol Phys~ol 24. l-82 D~$tcl, U , WII dcr LCIJ.I , Vccnhu~s, M dntl Tabdk, H F (1988) J, Cell 0101 107, 1G69-1675 Dcshd~c~. R J , Koch. I3 D . Wcrncl -Washburnc, M , Crq, E A dnd Schckm,in. R (1989) Natulc 332. X00-805 Alva~cs, K , Cdrllllo, A , Yu.m. P M , Kawano, H , Mormtoto. I7 1. Kcddy, J K (1990) Proc Ndtl Acdd SCI USA 87, S2935297