YEAST VOL. 8: 423-488 (1992)

Foreign Gene Expression in Yeast: a Review

MICHAEL A. ROMANOS, CAROL A. SCORER AND JEFFREY J. CLARE Department of Cell Biology, Wellcome Research Laboratories. Beckenham, Kent BR3 3BS, U.K.

Received 14 February 1992; accepted 22 February 1992

CONTENTS Expression in non-Saccharomyces yeasts Introduction Introduction Pichia pastoris Transformation and selectable markers Hansenula polymorpha Auxotrophic selection markers Kluyveromyces lactis Dominant selectable markers Yarrowia lipolytica Autoselection Schizosaccharomyces pombe Episomal vectors Physiology of foreign gene expression ARS vectors Mechanisms of toxicity 2pbased vectors Generation of low-expressingvariants Regulated copy number vectors Large-scale fermentation and Integrating vectors optimization YIP vectors Concluding remarks Transplacement Acknowledgements Integration into reiterated DNA References Transposition vectors Transcriptional promoters and terminators Foreign versus yeast promoters INTRODUCTION Glycolytic promoters Galactose-regulated promoters The yeast Saccharomyces cerevisiae has several Phosphate-regulated promoters properties which have established it as an important Glucose-repressible promoters tool in theexpressionofforeign protein sfor research, Other regulated promoter systems industrial or medical use. As a food organism, it Selection of novel yeast promoters is highly acceptable for the production of pharma- Foreign promoter systems ceutical proteins. In contrast, Escherichia coli has Yeast terminators toxic cell wall pyrogens and mammalian cells may contain oncogenic or viral DNA, so that products Factors affecting intracellular expression Initiation of transcription from these organisms must be tested more exten- RNA elongation sively. Yeast can be grown rapidly on simple media RNA stability and to high cell density, and its genetics are more Initiation of translation advanced than any other eukaryote, so that it can be Translational elongation manipulated almost as readily as E.coli. As a Polypeptide folding eukaryote, yeast is a suitable host organism for the Post-translational processing high-level production of secreted as well as soluble Stability of intracellular proteins cytosolic proteins. Secretion of foreign proteins Most yeast expression vectors have been based on the multi-copy 2p plasmid and contain sequences Introduction Vectors and signal sequences for propagation in E.coli and in yeast, as well as a G1 ycosylation yeast promoter and terminator for efficient tran- Protein folding and transport scription of the foreign gene (Figure 1). The recent Proteolytic processing Correspondenceto: M.A. Romanos (tel: 081-658 221 1 ext. 6410; Strategies to improve secretion fax: 081-650 0725). 0749-503X/92/060423-66 $38.00 0 1992 by John Wiley & Sons Ltd 424 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE rapid expansion in yeast molecular genetics has led substantial section to discussing other yeasts, par- to a great increase in our understanding of these ticularly Pichia pastoris in which there are many components, and as a result there is now a bewilder- examples of high-level expression. ing choice of promoter systems and methods for An area which is frequently ignored by molecular propagating foreign DNA in yeast. In many cases biologists but which must be considered from the ingenious new approaches have been employed, for outset in vector design is the physiology of foreign example in increasing the strength of native pro- gene expression. This includes the physiology of moters or the stability of expression vectors. We will growth to high cell density and promoter induction, attempt to review the choices now available and as well as the effect of expressing a foreign protein on how they relate to different requirements. host cell metabolism. We have gathered examples Insertion of a foreign gene into an expression vec- of the toxicity of foreign proteins and its effect in tor does not guarantee a high level of the foreign causing selection ofgenetic variants expressing lower protein; gene expression is a complex multi-step pro- yields; in many cases these effects can be controlled. cess and problems can arise at numerous stages, from Finally, we will discuss the considerations involved transcription through to protein stability. In the in industrial scale-up and fermenter optimization past, heterologous gene expression has often been through examples with different promoter systems treated empirically-a number of host organisms of S.cerevisiae and with Kluyveromyces lactis and might be tested for successful expression and low P.pustoris. yields or failures would be passed over. Frequently, inappropriate conclusions had been made, for example about relative promoter strengths, simply TRANSFORMATION AND SELECTABLE from knowledge of the input vector and the final MARKERS steady-state level of the foreign protein. However Transformation there is now considerable accumulated experience on foreign gene expression in yeast. In many instances The first methods for the transformation of S. this has led to the identification of a particular prob- cerevisiae involved enzymatic removal of the cell lem at a specific stage in the chain of events, and then wall to produce sphaeroplasts which could take up often to its solution. Among eukaryotes yeast offers DNA on treatment with calcium and polyethylene unparalleled scope for solving such problems on gly~ol.~~,'~~Transformants were then plated out in a account of the power of classical and molecular selective,isotonic top agar for regeneration of the cell genetics combined. We have attempted to draw wall. A more convenient method was later developed together examples of this from the literature, and in which intact yeast cells were made competent by from our own experience. These should be helpful in treatment with lithium ions.'*' This method is now predicting and solving problems with new genes, widely used despite the fact that it gives lower fre- and should be applicable, in many cases, to other quencies; a variation using DMSO increases fre- expression systems, prokaryotic and eukaryotic. quency 25-f0ld.'~~More recently a third approach, The secretion of foreign proteins which are natur- electroporation, has been used, and a highly efficient ally secreted is often necessary for their correct fold- method has been reported by Meilhoc et ing, and is highly advantageous because of the initial The process of transformation appears to be some- purity of the product in the substantially protein- what mutagenic, both for the host and for free culture medium. Although there have been the introduced DNA.72 However the frequency of several commercial successes using yeast, the area mutation is low enough that it should not be a major has frequently presented problems, especially for concern here. Also, Danhash et al.83have reported larger proteins. Despite increased understanding of that transformation induces a heritable slow-growth the processes of secretion, the greatest success in phenotype in Xcerevisiae. improving yields in recent years has been with a An important factor to consider in foreign gene classical random mutagenesis approach. expression is the frequent wide variation in the pro- A number of other yeasts have become important ductivity of different transformants when 2p vectors host organisms for foreign gene expression because are used. This appears to be due to an unexplained of advantages in promoter strength, secretion stable variation in plasmid copy number between efficiency, or ease of growth to high cell density. In different transform ant^.^^"^^^ Clearly it is therefore the future some of these will often be used in pref- important to analyse a number of transformants erence to Sxerevisiae. We have therefore devoted a when optimizing expression. FOREIGN GENE EXPRESSION IN YEAST A REVIEW 425

Table 1. Selectable markers for Sxerevisisae transformation.

Auxotrophic/ Marker dominant Comments

HIS3 A TRPl A Selection possible in CAA LEU2 A LEU2-d for high copy number selection URA3 A (a) Selection possible in CAA (b) URA3-d for high copy number selection237 (c) Counter-selection using 5-FOA37 (d) Autoselection in furl (5-FU-resistant) train^^^,^" L YS2 A Counter-selection using a-amino adipate'7.6'.'2' S.pombe POT A Used in Sarevisiae tpi host; autoselection in glucose2'* Tn903 kan' D Active only in multiple copies unless yeast promoter used; selection using G41 8'54,200,w Cm' D Only effective using yeast promoter; selection using chloramphenicol in glycerol medium only'" Hyg' D Reference 150 CUP1 D Level of Cu2+-resistancedependent on gene dosagels9 HSV TK D Thymidine/amethopterin/sulphanilamideselection; level of resistance dependent on gene dosage418 DHFR D Methotrexate/sulphanilamideselection; level of resistance dependent on gene do~age~~~,~'~

CAA, Casamino acids.

Auxotrophic selection markers with this marker is inefficient and requires the The first and most commonly-used markers for sphaeroplast method, though low levels can be the selection of transformants were LEU2, TRPI, obtained using the lithium method, if cells are incu- URA3, and HIS3 used in corresponding mutant bated overnight in non-selective medium prior to strains which are auxotrophic for leucine, try to selection. Vectors which contain both LEU2-d and phan, uracil and histidine, respecti~ely~~~~~~~~'~~~'another marker, e.g. URA3, can be maintained at (Table 1). Such strains are widely available, and either high or low copy number depending on the some contain non-reverting mutant alleles con- selection used. Loison et al.237used an expression structed to give low background rates in transform- vector containing these markers for the schistosomal ations. Continued selection requires the use of antigen P28-I and obtained product at 3% of total minimal growth media lacking the relevant nutrient. cell protein (t.c.p.) in uracil-deficient medium Or 25% It is worth noting that TRPI and URA3 vectors in leucine-deficient medium. Loison et also con- can be selected in the presence of acid protein structed a promoter-defective URA3 allele, URA3-d, hydrolysates (which lack tryptophan and uracil, which gives high copy number in uracil-deficient e.g. casamino acids) that are often added to semi- medium. defined media in order to enhance growth URA3 and LYS2 are particularly versatile in that rates. there are also methods for counter-selection of the A frequently-used variant of LEU2, LEU2-d,24 marker. Thus uru3- cells can be selected for their has a truncated promoter and is poorly expressed, resistance to the toxic antimetabolite 5-fluoro-orotic so that its selection gives rise to very high plasmid acid,37and lys2- cells can be selected for resistance to copy numbers."' Direct selection of transformants a-aminoadipic a~id.'~,~','~'These methods can be 426 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE

a) Pd bU

AMP-R

pWYG4 5682 bp

cpGALl

Figure 1. 2pbased expression vectors for S.cerevisiae based on galactose- inducible promoters. (a) pWYG4 has the 2p ORI-STB elements, the GALI promoter, and the 2p D gene terminator; an NcoI cloning site containing the ATG is used to insert foreign genes. (b) pWYG7L has intact 2p ORI, STB, REPI and REPZ, the GAL7promoter, and uses theFLPterminator; foreign genes are inserted in the polylinker with their 5' ends at the BmHI or NcoI site. FOREIGN GENE EXPRESSION IN YEAST A REVIEW 427 used either to select mutations in prototrophic Loison et al.236used ura3 furl strains as hosts for strains or to select for plasmid loss in transformants. plasmids containing the URA3 gene. These are non- viable since they are blocked both in the de novo and salvage pathways for uridine 5’-monophosphate Dominant selectable markers synthesis; maintenance of a URA3 plasmid is then Dominant markers are useful in that they increase obligatory for viability even in uracil-containing the range of host strains that can be tested to include media. Since the double mutant without plasmid prototrophic and industrial strains of Xcerevisiae, is non-viable, the transformant was obtained by and can be used for selection in rich medium. Since mating a furl strain with a ura3 strain containing most strains are sensitive to the aminoglycoside the URA3 plasmid, and selecting the plasmid- antibiotic G418, the G418-resistance marker, containing ura3furl progeny. Subsequently, Loison encoded by the E.coli Tn903 transposon, can be et ~1.~~’were able to directly isolate spontaneous used, though it is inefficient in direct selection of furl mutants from ura3 cells transformed with a transformants.200,400Frequencies are acceptable URA3 plasmid by selecting for resistance to 100 pg/ provided transformants are incubated overnight in ml 5-fluorouracil and then screening resistant non-selective medium prior to plating out on G418 colonies for resistance to 300 pg/ml5-fluorouridine. agar.23 Use of glycerol rather than glucose as the We have found that it is possible to generate stable carbon source during selection reduces the number URA3 transformants by a single direct selection on of untransformed G418-resistant mutant colonies 1.3 mg/ml (10 mM) 5-fluorouracil to which onlyfurl that ari~e.”~Multiple copies of the Tn903 G418- mutants should be resistant304(Figure 2). resistance marker are needed to confer resistance in yeast cells unless a yeast promoter is used, in which EPISOMAL VECTORS case it is active in single copy.’54It may be prudent to omit selection with antibiotics which affect ribo- Extra-chromosomal replicons are based either on somes, such as G418, during induction of expression plasmids containin yeast autonomous replication vectors due to the possibility of increased amino sequences (ARS)?’ which function as origins of acid misincorporation. Other antibiotic-resistance replication, or on the native 2p circle of Saccharo- markers that have been used successfullyin yeast are myces.” A comprehensive listing of both episomal hygromycin BI5O and chloramphenicol-resistance.’53 and integrating vectors has been compiled by Parent Copper-resistance in yeast is conferred by multiple et aZ.281 copies of the CUP1gene and can therefore be used as a dominant marker in sensitive (CUPIS)strains. 122It ARS vectors has proved useful on multi-copy vectors, particularly with industrial strains.’59Two other markers which ARSvectors are present in multiple copies per cell can be used for vector copy number amplification by (1 to 20) but aremitotically highly unstable, plasmid- increased drug selection are the herpes simplex virus free cells accumulating at a rate of up to 20% per thymidine kinase gene4’8.419and dihydrofolate generation without selection, due to inefficient red~ctase.~’~ transmission to daughter cells during cell division.264 Even when grown under selection the proportion of Autoselection plasmid-containing cells can be very low, giving a correspondingly low average copy number. ARS A number of ‘autoselection systems’ have been vectors can be stabilized by the addition of yeast devised to ensure that plasmid selection is main- centromeric sequences (CEN), but the copy number tained, irrespective of culture conditions. Bussey is then reduced to 1 or 2 per cell.” In practice ARS and Meaden” showed that expression of a cDNA vectors are hardly ever used for foreign gene encoding the yeast killer toxin and immunity gene expression, and ARSICEN vectors are only used could be used for self-selection of transformants of where low-level expression is desired. laboratory or industrial yeasts since plasmid-free cells are killed by plasmid-containing cells. Another 2p-based vectors system has used the Schizosaccharomyces pombe triose phosphate isomerase gene to stabilize plasmids By far the most commonly-used expression vec- in S.cerevisiae cells lacking the active gene, during tors are E.coli-yeast shuttle vectors based on growth on glucose.208 2p.83u’2812p is a 6.3 kb plasmid present in most 428 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE

Figure 2. Stability of a URA3 vector in wild-type and 5-fluorouracil-resistantstrains. A P-galactosidase expression vector (pWYG4- lac3was introduced into strain KY 117 and spontaneous mutants resistant to 10 m~-5-fluorouracilwere selected.'@' Wild-type (1) and mutant (2) transformants were grown for ten generations in non-selective inducing medium, then plated out on non-selective plates containing XGal and galactose to assay for P-galactosidase expression. White colonies, indicating plasmid-loss, were not present with the mutant strain. This indicates autoselection of the vector in the 5-fluorouracil-resistantstrain.

Form A (FRT) within the inverted repeats, so that cells contain two forms of 2p, A and B. Despite the fact that it confers no known pheno- type and may indeed be slightly disadvantageous to the host ce11,129,251,3972p is stably inherited; plasmid- free cells arise at the rate of 1 in lo4per generati~n.'~~ It achieves this using two mechanisms: (1) by partly FLP overcoming the strong maternal bias in plasmid Form B transmission, and (2) by amplification to correct fluctuations in copy number caused by inefficient Hind 111 transmission. Efficient segregation depends on having the STB Hind 111 ECURI locusincisand the REPl and REP2geneprod~cts.~~'

Hind 111 Amplification overcomes the host regulation which restricts each replication origin to one initiation per STB snaB1 cell cycle; it appears to depend on the inverted repeat sequences and the FLP gene product. According to Figure 3. The two forms of the 2p plasmid. Cis-elements are shown as filled boxes and genes by open boxes, inverted repeat the model proposed by Futcher,128FLP promotes regions are aligned; see text for a detailed description. recombination between replicated and unreplicated DNA so that inversion occurs and two replication forks can follow each other around the circle. Repli- Saccharomyces strains at about 100 copies per cation terminates after a second recombination, and haploid genome1283265,395(Figure 3). The plasmid further recombination generates multiple 2p encodes four genes: FLP (or A), REPl (or B), REP2 monomers. (or C) and D. In addition, 2p contains an origin of The simplest 2p vectors contain the 2p ORI-STB, replication (ORI, which behaves as a typical ARS a yeast selectable marker, and bacterial plasmid element), the STB locus (required in cis for stabiliz- sequences (e.g. pWYG4, Figure l), and are used in a ation), and two 599 bp inverted repeat sequences. 2p+ host strain which supplies REPl and REP2 FLP encodes a site-specific recombinase which pro- proteins.211ORI-STB expression vectors are the motes flipping about the FLP recombination targets most convenient to use routinely in the laboratory FOREIGN GENE EXPRESSION IN YEAST A REVIEW 429 due to their small size and ease of manipulation. though it is not clear whether stability would be They are tenfold more stable than ARS plasmids, retained upon insertion of a high-level expression being lost in 1 to 3% of cells per generation in non- cassette. selective conditions, and are present in 10 to 40 A number of ultra-high-copy number vectors (e.g. copies per cell. In 2p-free cells such plasmids behave pWYG7L, Figure 1) are based on pJDB219 which as ARS vectors. Commonly-used ORI-STB vectors contains the entire 2p B form cloned in the bacterial contain a 2.2 kb EcoRI fragment or a 2.1 kb Hind111 plasmid pMB9 with disruption of FLP.24The select- fragment from the B form of 2p, each having one able marker is LEU2-d whose use results in very high inverted repeat. In order to limit recombination copy numbers (20WOO copies per haploid genome); with 2p, the inverted repeat can be removed, but it the fraction of cells with the highest plasmid copy is important not to remove adjacent STB-distal number are constantly selected, resulting in reduced sequences since these appear to have an important growth rate. Selective growth or use of other markers role in rotecting STB from transcriptional inacti- gives a more normal copy number of about 50 per vationJ6It should be noted that shuttle vectors con- ce11.'09 pJDB219 is best used in 2p-free cells since it taining inverted repeats will exist as a variety of can undergo FLP-mediated recombination with recombinants with thenati~e2p.'~~Inaddition,there resident 2p, leading to loss of the foreign DNA but appears to be some competition between exogenous retention of the LEU2-d marker.98In 2p-free cells 2p vectors and native 2p such that the copy number pJDB219 is very stable due to its high co y number, of both is depre~sed.'~' making it suitable for large-scale cult~re.~',~~~FLP' More complex 2p-based shuttle vectors contain versions of pJDB219, e.g. pXY, have greater the REP1 and REP2 genes in addition to ORI-STB stability in non-selective medium.13' Since the entire and can therefore be used in 2p-free host strains.8 sequence of pMB9 has not been determined, it may They are more cumbersome than ORI-STB vectors be more convenient to use a variant of pJDB219 but stabler and more suitable for scale-up. Many based on pBR322, e.g. ~Cl/l.~~~ examples of this type of vector are disrupted in D Vectors can have very high copy number even in (e.g. pJDB248), and some in FLP (e.g. pJDB219) so non-selective conditions if extra FLP recombinase is that they cannot flip or amplify in 2p-free cells. supplied. For example, induction of FLP from a Nevertheless FLP- plasmids can reach high copy single integrated copy of the gene under control of numbers in 2p-free cells, presumably through asym- the GAL1 promoter leads to a shift in plasmid copy metric segregation. In 2p+ strains they can amplify number from <50 to 20W00.8 Alternatively, the independently if they have two inverted repeats, or autoselective high-copy URA3-d marker can be following integration into 2p if they have only one used in a furl host strain.237 repeat. Recently, vectors have been developed which have all functional regions of 2p intact, by Regulated copy number vectors insertion of the foreign DNA at the unique SnaBI site between ORI and STB.32 These are highly Foreign gene expression could be regulated by stable, but still 20- to 80-fold less stable than native inducing an increase in vector copy number. Two 2p, possibly due to low-level transcription through types of yeast episomal vectors with regulated copy STB. In future similar vectors should find wide use number have been described: (1) vectors with regu- in foreign gene expression. latable centromeres, and (2) 2p vectors in cells with Vectors free of bacterial DNA may be advan- inducible FLP (see above). The first type depends on tageous in foreign gene expression in relation to the observation that CEN elements can be inacti- food and drug regulatory authorities. They can be vated by transcription. Chlebowicz-Sledziewska made in one of two ways: (1) by targetted inte- and Sled~iewski~~constructed vectors containing gration of an expression cassette into native 2p, or the glucose-repressible ADH2 promoter adjacent to (2) by use of a shuttle vector which can remove bac- CEN3 and either an ARS or 2p STB-ORI. In the terial sequences in vivo by excisional recombination. ARS vectors copy number could be increased from Using the latter approach, Chinery and Hin~hcliffe~~1-2 to 5-10 by a switch from glucose to ethanol as constructed 'disintegration vectors' which used the the carbon source. In the ORI-STB vectors copy FLP/FRT system to excise bacterial DNA inserted number could be increased from 1-2 to about 100 at the XbaI site of one of the inverted repeats. Vectors and the vector was very stable. Such vectors could with inserts at the unique PstI site in D or the SnaBI be used to increase the degree of regulation in site between STB and ORI were extremely stable, expressing toxic proteins. 430 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE INTEGRATING VECTORS Integration into reiterated DNA

YIP vectors A number of strategies based on integration into Chromosomal integration offers a more stable reiterated chromosomal DNA have been used to alternative to episomal maintenance of foreign generate stable multi-copy integrants. At present the DNA. In Saccharomyces integration normally best results in terms of copy number and expression occurs by homologous re~ombination.~~'Integrat- appear to be using integration into the ribosomal ing vectors (YIP) contain yeast chromosomal DNA DNA (rDNA) cluster. The rDNA cluster consists of to target integration, as well as a selectable marker about 140 tandemrepeatsofa9.1 kbunitonchromo- and bacterial replicon. Vectors are usually digested some XII. Lopes et ~1.~~~3~~~have constructed an at a unique restriction site in the homologous DNA integratingvector, pMIRY2, containing aportion of as this promotes high efficiency transformation and the rDNA unit and the LEU2-d marker. Transform- targets integration. Such single cross-over inte- ation with pMIRY2 digested at SmaI or HpaI gave gration results in a duplication of the chromosomal Leu' transformants with 100-200 copies integrated target sequence, so that the vector can subsequently at a non-transcribed spacer of the rDNA locus. Use 'pop out' by excisional recombination. Neverthe- of the LEU2-d or other promoter-defective markers less the integrants are quite stable, the typical rate was important for isolation of high copy integrants. of vector loss being < 1% per generation in the The transformants were highly stable, 80-100% of absence of selection.'68 the integrated copies being retained after 70 gener- For convenience vector integration can be tar- ations, and the levels of foreign protein produced getted to the chromosomal mutant allele of the using the PGK promoter were as high as from 2p selection marker used. However, continued selec- vectors. This approach could also be used as an tion of the resulting transformants is ineffective, alternative to episomal vectors in species where since the duplicated DNA can be excised and a none has been found. wild-type marker retained. In contrast, continued Another reiterated DNA that can be used as a selection is effective where integration is targetted target for integration is the transposable element Ty elsewhere. which is present in 3WO copies per genome in most When high DNA concentrations of integrating Saccharomyces strains. Kingsman et aL216described vectors are used in transformations, tandem multi- the use of a transplacement vector targetted to copy inserts can result, resumably due to repeated replace Ty, whose copy number could be amplified recombination events.' P' Multi-copy integrants are using the LEU2-d selection marker. Levels of inter- relatively stable and have been used, for example, in feron produced from such amplified transformants gene dosage studies.57 were several times higher than from single-copy ARS/CEN vectors but almost ten-fold less than with 2p vectors. Jacobs et ~1.l~~used similar vectors Transplacement in order to co-express the different forms of hepatitis B surface antigen (HBsAg). They obtained trans- An alternative type of integration, transplace- formants containing from one to several copies of ment, makes use of double homologous recombi- the vector without the need for amplification. The nation to replace yeast chromosomal DNA, resulting multi-copy transformants consisted mainly of the in a stable structure without duplications.313Where transplacing DNA at a single Tyl locus and arose by a stable single-copy transformant is required this is an unexplained mechanism. In order to co-express the method of choice. Transplacement vectors con- the different forms of HBsAg in the desired ratio, a tain the exogenous DNA and selection marker and a strains were transformed with each vector, flanked by yeast DNA homologous to 5' and 3' multi-copy integrants selected, and diploids made. regions of the chromosomal DNA to be replaced. Stable diploids with a total of 10 copies were made Prior to transformation the vector is digested with but expression levels appeared to be low. restriction enzymes which liberate the transplacing More recently Shuster et have used vectors fragment with 5' and 3' homologous ends. The fre- that integrate by single cross-over into 6 elements, quency of transformation is low so that the sphaero- which exist either alone or as part of Ty throughout plast method is usually used, and the chromosomal the Sxerevisiae genome. They constructed a vector structure of the transformants must be checked expressing the E.coli IacZ gene with the LEU2 and phenotypically and by Southern blot analysis. CUP1 markers. Leu' transformants were selected FOREIGN GENE EXPRESSION IN YEAST A REVIEW 43 1 following transformation with vector digested by ADHl gene for efficient intracellular expression of XhoI, which cuts in the 6 element within the LEU2 leukocyte a-interferon. 17* marker fragment. These were then selected for Yeast mRNA promoters consist of at least three copper-resistance to isolate multi-copy integrants elements which regulate the efficiency and accuracy which yielded up to ten-fold the j3-galactosidase of initiation of tran~cription:~~’upstream activation level of single-copy strains. Integration into 6, sequences (UASs), TATA elements, and initiator coupled with crossing of haploid integrants, has elements. Many also contain elements involved in been used to generate a 20-copy strain for efficient repression of transcription. UASs, which have some secretion of nerve growth fa~tor.~’’ similarities to mammalian enhancers, determine the activity and regulation of the promoter through specific binding to transcriptional activators (e.g. Transposition vectors GAL4 and GCN4), and act at variable distances 5’ to A different approach to multi-copy integration the initiation site. Some UASs have been mapped to is the use of Ty transposition vectors analogous short regions of DNA, e.g. the GALl-GAL10 UAS to retroviral vectors for mammalian cells. Ty to a 108 bp intergenic region containing four short transposes via a full-length transcript which is sequences of dyad symmetry. These sequences (1 7- encapsidated into virus-like particles and reverse 2 1 bp) are necessary and sufficient for binding of the transcribed to DNA, which can then integrate at GAL4 trans-activator and for galactose-regulation, multiple sites. In transposition vectors a regulated and act synergistically. UASs of some constitutively- promoter, e.g. GALl, is used in place of the Ty 6 expressed genes contain poly(dA-dT) tracts which promoter to generate a transcript encompassing the probably activate transcription by affecting nucleo- foreign gene and selectable marker. Transcription some structure.369 TATA elements (consensus termination signals must be removed from the TATAA) are found 40 to 120 bp upstream of the marker gene for the full-length transcript to be pro- initiation site, in contrast to the more rigid distance duced. The whole unit is placed on an episomal vec- of 25 to 30 bp in higher eukaryotes, and provide a tor for the initial transformation, but can be lost window within which initiation can occur.369The following induction of transposition. Boeke et initiator element, which is poorly defined, directs obtained relatively low copy number integrants (1- mRNA initiation at closely adjacent sites. Yeast 10) by this method, but it may prove feasible to use promoters may be highly complex, extending over the LEU2-d gene for amplification. Although this 500 bp, containing multiple UASs and negative method is not fully developed it could be useful in a regulatory sites, and multiple TATA elements variety of yeasts, since Ty encodes all the functions associated with different initiation sites.369 required for transposition. Most promoters are regulated to some extent, but the most powerful, glycolytic promoters are poorly regulated. This makes them undesirable for use in TRANSCRIPTIONAL PROMOTERS AND large-scale culture, where there is more opportunity TERMINATORS for the selection of non-expressing cells, and unsuit- Foreign versus yeast promoters able for expressing toxic proteins. In such cases it is preferable to use a tightly-regulated promoter so The expression of foreign genes in yeast was that the growth stage can be separated from the examined as soon as transformation procedures expression stage. Despite a severe limitation in became available. The first study was of the rabbit j3- efficiency with multiple copies, GALl has been the globin gene which was found to give rise to aberrant most commonly-used regulated promoter. How- transcripts in which the introns were not ~pliced.’~ ever, there is now a large variety of native or In a few cases the foreign transcript was correctly engineered promoters (Table 3); the right choice is initiated, e.g. with zein?” but in general foreign critical for any one application, especially where a transcriptional promoters were found to ’ve aber- process is to be scaled-up. rant initiation, e.g. Drosophila ADE8,I6’or were totally inactive, e.g. herpes simplex virus thymidine Glycolytic promoters kina~e.’’~Thus for efficient transcription of foreign genes the use of yeast promoters with cDNAs was The first promoters used were from genes en- soon found to be essential. The first published coding abundant glycolytic enzymes, e.g. alcohol example was the use of a 1500 bp fragment 5’ of the dehydrogenase I (ADHl),172 phosphoglycerate 432 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE Table 2. Sxerevisiae vector systems.

Yeast Transformation Copy no. Mitotic Vector sequences frequencf /cell instabilityb Reference

A. Integrating YIP Homologous DNA 102 21 0.1% 168 Transplacemen t Homologous DNA 10 1 stable 313 rDNA rDNA nja' 1oc200 stable 239 integrating TY6 Ty6 DNA nla I20 stable 322,345 B. Episomal Replicating ARS 104 1-20 20% 264 (YRP) Centromere ARSiCEN 104 1-2 1% 75 (YCP) 2p- based ORI, STB, in 2p+ 104 25 2.8 % 130 (YEP) host (YEpl3) ORI, STB, REPI, REP2, 104 50-100 0.&1.8% 130 FLP in 21' host (pJDB248) ORI, STB, REPI, REP2, n/a 200 0.26% 130 in 2p0 host (PJDB~~~)~ ORI, STB, REPI, REP2, D, nla 5c100 0.20% 32 FLP in 2p0host (pJB205) Regulated ORI, STB, ADH2-CEN3 1o4 3-5 to Stablef 69 copy no. 100' aTransformants per pg DNA using sphaeroplast method. bPlasmid-freecells arising per generation during non-selective growth. 'Data not available. dIncreased copy number using LEUZ-d selection. 'On change from glucose to ethanol. '85% after 30h non-selective growth in log-phase. kinase (PGK),'73,383or glyceraldehyde-3-phosphate UAS at -473 to-422 relative to the initiation site, a dehydrogenase (GAP).'77 These were at first heat shock regulatory element, and other features thought to be constitutive but were later found to be contributing to accurate and efficient initiation.*15 induced by addition of glucose, e.g. expression of In contrast, less is known about other glycolytic a-interferon using the PGK promoter was induced promoters, such as GAP. There are three GAP 20- to 30-fold by addition of glucose to a culture genes, of which GAP491 or TDH3 is the most grown on acetate as carbon-s~urce.~~~Glycolytic highly-expressed and whose promoter has been promoters are the most powerful of S.cerevisiue, for used successfully to express a number of protein^.^" example PGK mRNA accumulates to 5% of total. The full-length promoter extends over approx. Despite their poor induction ratio, ADHI, PGK 700 bp,33but there have been a number of reports of and GAP vectors have been used extensive1 in the smaller fragments having full promoter activity, e.g. laboratory, and in some cases industrially. 25r a 198 bp fragment.'69 However, it appears that the The PGK promoter has been studied in some activity of shorter GAP491 promoters is dependent detail: it extends over 500 bp, contains a complex on bacterial vector ~equences.~'' FOREIGN GENE EXPRESSION IN YEAST A REVIEW 433

Table 3. S.cerevisiue promoter systems.

Host Promoter genotype Strength Regulation" Reference

A. Native PGKb, GAP, TPI wt ++++ I20-fold induction by glucose (PGK) 311,383 GAL1 wt +++" 1000-fold induction by galactose 202 ADH2 wt + +d 100-fold repression by glucose 29 1 PHOS wt +/++ 200-fold repression by P, 169 PH05 ph04'~pho80 + 50-fold induction by shift 37°C to 24°C 223 CUP1 wt + 20-fold induction by Cu2+ 113 MFa 1 wt + Constitutive in a cells 42 MFa 1 sir3lS + 105-foldinduction by shift 37°C to 24°C 42 B. Engineered PGK/a2 operator sir3ls ++++ 100-fold induction by shift 37°C to 24°C 398 TPI/a2 operator sir3lS ++++? > 50-fold induction by shift 37°C to 24°C 352 GAPIGAL wt +++? 15&200-fold induction by galactose 35 PGKIGAL wt +++? Induction by galactose 77

GAPI A DH2 wt + + +d? Repressed by glucose 78 GAP/PHOS wt ++++? Two- to five-fold repressed by P, 169 CYCl/GRE' Expressing +++? 5&1OO-fold by deoxycorticosterone 332 glucocorticoid receptor PGKIAREC Expressing ++++ Several 100-fold by dihydrotestosterone 293 androgen receptor

'Induction ratios are reporter-dependent. b5% of mRNA in single copy. '1 % of mRNA in single copy but severely limited in multiple copies. Improved by GAL4 over-expression. dImproved by ADRI over-expression. 'Glucocorticoid responsive element. 'Androgen responsive element.

Galactose-regulated promoters encoded by GALI, the repressor encoded by GAL80, and the GAL UAS (Figure 4). Binding of The most powerful tightly-regulated promoters GAL4 protein to the UAS is necessary for induc- of S.cerevisiae are those of the galactose-regulated tion; GAL80 protein binds GAL4 and acts as a genes GALI, GAL7, and GALIO, involved in repressor unless galactose is added. The regions of metabolizing galactose. Galactose-regulation in GAL4 protein that bind GAL80 and the UAS have yeast is now extremely well-studied and has become been defined, as have the structural features of the a key model system for eukaryotic transcriptional different GAL promoters. regulation (reviewed in reference 201). Many genes GALI, GAL7 and GALIO mRNAs are rapidly are involved in regulation of GAL promoters but the induced > 1000-fold to approx. 1% of total mRNA central interaction is between the trans-activator on addition of galactose.363The promoters are 434 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE ---GAL7 GAL10 GALl -GAL2

GALBO, Chr.xIu -0 1 I I * I:l&&oI;l...... , 1:: pr**: : ...... cl...... Galactose INDUCER 1 J Figure 4. Galactose regulation in yeast. The genes involved in regulation and metabolism and their chromosomal location are shown. Stimulationis indicated by bold lines with arrows and inhibition by lines with bars. strongly repressed by glucose, so that in glucose- other factors also become limiting with multi-copy grown cultures maximal induction can only be promoter^.'^ With multi-copy expression vectors achieved following depletion of glucose. Galactose- GAL4 limitation is exacerbated, for example p- inductions can be carried out in one of three ways: galactosidase expression from a GAL-lacZ vector (1) by growing the culture on a non-repressing, non- increases only two-fold in going from a single-copy inducing ('neutral') sugar, when very rapid induc- to a 2p vector.413Both with single and multi-copy tion follows addition of galactose; (2) by growing vectors, two- to three-fold increases can be obtained the culture in glucose medium but removing the in gal80 strains, but expression then becomes con- glucose by centrifugation and washing the cells ~titutive.~'~Over-expression of GAL4, either by before resuspension in galactose medium (this leads insertion of the gene into the multi-copy expression to a lag of 3 to 5 h in induction'); and (3) by growing vector, or by use of a single-copy integrating ADHI- the cells in medium containing both glucose and GAL4 expression vector, increases product levels galactose, when the glucose is preferentially metab- 2- to 3-fold, but again at the rice of losing tight olized before galactose-induction can occur. The regulati~n.'~.'~~Schultz et ~l?~'usedan integrated first two methods are frequently used for small-scale GALIO-GAL4 expression vector in order to over- but are impractical in large-scale inductions. It produce GAL4 protein in a galactose-regulated should be noted that many commonly-used strains manner. Transcription from the target expression have mutations in the galactose permease gene vector, encoding Epstein-Barr virus (EBV) gp350, (GAL2) and are not inducible. was increased 5-fold. We have tested a similar system The large background of knowledge of galactose- which used an integrated ADH2-GAL4 expression regulation offers the possibility of manipulating the vector in order to over-produce GAL4 protein. This system in a number of ways in order to improve its system alters the regulation of GAL expression vec- characteristics for protein production. Three types tors so that they are induced by glucose-depletion. of manipulations that have been carried out are: (1) Using a constitutive GAL4 gene, i.e. one that does the over-expression of trans-activator; (2) the use of not interact with GAL80 repressor, it was possible mutations in the galactose-regulatory pathway or to increase levels of P-galactosidase produced from in glucose-repression; and (3) the construction of a multi-copy GALl vector by 2- to 4-fold (M.A.R. chimaeric galactose-regulated promoters. and J.J.C., unpublished results). GAL4 protein is present in one or two molecules In order to facilitate galactose-induction of per cell and, moreover, GAL80 re ressor is in excess glucose-grown cultures, the use of mutants defective of this and is galactose-induciblej' Therefore, even in either global or GAL-specific glucose-repression with single-copy promoters, GAL transcription is has been examined. In regl-1 mutants efficient limited by a shortage of GAL4 protein, though galactose-induction occurs with galactose to glucose FOREIGN GENE EXPRESSION IN YEAST A REVIEW 43 5 ratios of 5: 1.248 Hovland et used a novel selec- eglin C. Some hybrid promoters yielded up to twice tion procedure to isolate glucose-resistant mutants, the amount of product as GAP, but the induction and found a reg1 mutant (regl-501) in which efficient ratio was 2- to 5-fold compared to 40-fold for PHOS. induction occurred even with galactose/glucose Since no copy number data were available one can- ratios of 1jlOO. A gall regl-501 strain was made not make conclusions about relative promoter which cannot metabolize galactose so that very low strength. levels of galactose (e.g. 0.2%) could be used, in the Kramer et dZz3constructed a host strain tempera- presence of excess glucose, for efficient induction.I8’ ture-sensitive in the PH04 trans-activator and defec- In an attempt to combine the high activity of tive in the PH080 repressor (pho4‘>ho80) to achieve glycolytic promoters with the tight regulation of phosphate-independent induction of the PH05 pro- GAL promoters, hybrid promoters have been con- moter regulated by temperature. On lowering the structed whereaglycolyticUASisreplaced byaGAL temperature from 35°C to 23°C a 50-fold induction UAS. However, the published results do not suggest of a-interferon was achieved, but the absolute level that these hybrids are more efficient than GAL pro- was only one tenth that in wild-type induced cells. moters. Bitter and Egan3’ have described the use of a GAPIGAL UAS hybrid for the expression of y- Glucose-repressiblepromoters interferon, which is toxic to yeast cells. Insertion of a 55 bp GALI-GAL10 UAS fragment between the Glucose-repression is a global system regulating GAP UAS and TATA element conferred galactose- the expression of a number of genes, including sugar regulation to the promoter. Product levels were fermentation genes, by the availability of glucose. significantly higher than with the native GAP pro- Yeasts preferentially utilize sugars such as glucose moter, due largely to a dramatically increased that enter the glycolytic pathway directly. Genes vector copy number, from 1 to 2&50 per cell. A involved in sucrose or galactose metabolism are similar type of hybrid promoter (PAL) has been transcriptionally repressed by glucose. Typical constructed, by replacing the PGK UAS with a examples of promoters regulated primarily by GAL UAS, and used for regulated production of a- glucose-repression are those of the ADH2, SUC2 interferon and human serum albumin.77However, and CYCl genes, encoding alcohol dehydrogenase no comparison of the efficiency of GAL and PAL 11, invertase and iso- 1-cytochrome c, respectively. promoters has been published; it would be expected The ADH2 promoter is both powerful and tightly that all GAL-UAS hybrid promoters would be regulated and has been used for foreign gene severely limited by GAL4 protein. expression.291Since it is repressed over 100-fold by glucose, it can be used for efficient expression of Phosphate-regulated promoters toxic proteins, e.g. insulin-like growth factor I (IGF-I).343Deletion analysis has identified a 260 bp The promoter of the acid phosphatase gene, region 5’ to the initiation site which contains two PH05, is regulated by inorganic phosphate concen- UASs sufficient for full promoter activity and regu- tration and has been used extensively for foreign lation.26 UAS1 is a 22 bp inverted repeat which gene expre~sion.’~~In one of the earliest studies, a binds the ADRl trans-activator.”’ We have used 1.4 kb PH05 DNA fragment was used to drive pro- the 260 bp region, which is readily assembled from duction of a-interferon, which was induced 200-fold synthetic oligonucleotides, in efficient expression by switching to low-phosphate medi~m.”~More vectors (M.A.R. and J.J.C., unpublished results). recently, the structural features and regulation of In order to maintain repression of ADH2, cells the PH05 promoter have been studied in detail must be grown in excess glucose (e.g. 8%) until (reviewed in reference 394). The promoter, en- induction, which is effected by changing to fresh compassing about 400 bp of DNA, contains two medium containing a non-fermentable carbon UASs which are necessary and sufficient for regu- source, e.g. ethanol, glycerol, raffinose, etc. Alterna- lation. These contain 19 bp dyad sequences which tively, ADH2can be induced by culturing initially in bind the PH04 trans-activator. a lower concentration of glucose (e.g. 1 %) which is Since the PH05 promoter is not very strong, gradually depleted.291Glucose-repressible systems attempts have been made to use the PH05 UAS to have a potentially serious disadvantage in industrial confer regulation to glycolytic promoters. Hinnen fermentations: it is difficult to maintain tight glucose- et ~1.’~~constructed a series of GAP/ PH05 UAS repression under conditions of glucose-limitation, hybrids and tested them in expression vectors for which are required to achieve high cell density. 436 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE Irani et al.ls7showed that transcriptional factors, appears amenable to fine-tuning by use of inter- including the ADRl trans-activator, became limiting mediate induction temperatures. A similar system for ADH2 transcription from multi-copy plasmids. has been developed by insertion of two a2 operators Over-expression of ADRl leads to loss of regulation, in the PGK promoter, achieving a >lOO-fold but ADH2-regulated over-expression of ADRl, induction ratio without any reduction in activity.398 from a single-copy integrating vector, has been used A novel type of promoter system uses the ability of to increase the efficiency of ADH2 expression sys- mammalian steroid hormone receptors to function tems without loss of reg~lation.~~'In this way it has as transcriptional activators in yeast. Schena been possible to increase expression of P-galactosi- et ~l.'~~constructeda CYCZ expression vector under dase from a single-copy ADH2-ZacZ vector by 4- to the control of three tandem 26 bp glucocorticoid 10-fold. The system usually results in about a 3-fold response elements fused upstream of the promoter. increase in yield for intracellular proteins and is used In yeast cells also expressing the glucocorticoid for commercial production (J. Shuster, personal receptor, a CAT reporter gene could be induced 50- communication). to 100-fold to high levels by addition of deoxycor- Hybrid glycolyticlADH2 promoters have been ticosterone (DOC). The degree of induction was constructed by transplanting the ADH2 UAS into titratable over 1 nM to 10 ~M-DOCand was very GAP proximal promoter sequences. Cousens et aZ.78 rapid (t,,*of 7 to 9 mins). A similar system has been fused the ADH2 UAS containing the 22 bp dyad developed using a hybrid PGK promoter containing 320 bp upstream of the GAP TATA element, and androgen response elements.293Addition of increas- were able to achieve tightly regulated production of ing amounts of dihydrotestosterone induced a superoxide dismutase (SOD)-proinsulin fusion to increasing amounts of 13-galactosidasereporter over > 15% t.c.p. However, no critical comparison of the a several hundred-fold range, and the range could be strength of the ADH2, GAP, and hybrid promoters extended to 1400-fold by varying the copy number has been published. of the receptor or reporter genes. In conclusion, steroid-regulated systems appear to be powerful Other regulatedpromoter systems and tightly-controlled, and could be used in yeasts lacking promoters with these qualities (e.g. K.Zactis A few other regulated promoter systems are and Spombe). worthy of comment here: these are of interest The promoter of the CUPl gene, encoding because their induction is independent of culture copper metallothionein, has been used in expression nutrients and can therefore be controlled without vectors.IL3This promoter is tightly-regulated and otherwise perturbing the culture. independent of culture parameters. The concen- A temperature-regulated system based on mating tration of Cu2+ ions for induction depends on the type control has been used by a number of groups. copper-resistance of the host strain from 0.0 1 mM Mutations in the SIR genes de-repress the silent (no CUPl gene) to 0.5 mM (> 6 copies). mating-type loci, so that a or a-specific genes are not expressed. Repression of the MFal promoter is Selection of novel yeast promoters mediated by the MATa2 repressor which binds a 31 bp operator sequence. Brake et al.42first reported A number of selection or screening methods have the use of a MATasir3" strain for secretion of hEGF been used in order to identify new promoters from a using the a-factor (MFal) promoter. Switchingfrom genomic librar~.'~~,~~~Selection procedures can be the non-permissive (35OC) to the permissive (25OC) devised for regulated promoters, e.g. the glucose- temperature led to an induction in secreted hEGF repressible promoters of the GUT2 and PRBl genes from 10 ng/l to 4 mg/l. The a2 operator has been which encode glycerol-3-phosphatedehydro enase transferred to the strong ADHZ and TPZ (triose and vacuolar endoprotease B, respectively.35fm phosphate isomerase) promoters and shown to con- fer temperature re ulation in a MATa ~ir3'~strain. Foreign promoters systems Sledziewski et inserted up to four operators between the UAS and TATA elements of the TPZ Foreign promoters not recognized by yeast RNA promoter and were able to obtain tight regulation polymerase can in principle be used provided the (>SO-fold induction of P-galactosidase) and full cognate RNA polymerase is co-expressed. The activity. Inexplicably, the hybrid ADHZ promoters bacteriophage T7 RNA polymerase is highly active were not tightly temperature-regulated. The system and has been used in a number of prokaryotic and FOREIGN GENE EXPRESSION IN YEAST A REVIEW 437 eukaryotic organisms. T7 RNA polymerase, ideally evidence suggests that a variety of different signals, with an added nuclear targetting signal, can be unidirectional and bidirectional, are used in expressed from a GAL1 promoter and can efficiently yeast. ’’’ transcribe DNA in yeast cell^.^^,^^ However the T7- Terminators from a number of genes have been induced lacZ mRNA is not translated in yeast, due used in expression vectors, e.g. TRP1,‘72ADH1,385 either to absence of 5’ caps and/or polyadenylation, MFl,42etc. In order to simplify vector or to a stable hairpin formed in the 5’ region.29In construction, a terminator from 2~’~’can be used, animal cells a similar problem has been solved using e.g. the FLP’73 (pWYG7L, Figure 1) or D gene the encephalomyocarditis virus leader to promote terminat~r~~(pWYG4, Figure 1). cap-independent translation,Io8but this appears to be inactive in yeast (C.A.S., unpublished results). If the translation problem were solved a yeast T7 FACTORS AFFECTING INTRACELLULAR system would be very powerful, since GALI-driven EXPRESSION expression of the polymerase would act as an Initiation of transcription amplification step. Gene expression is most frequently regulated at Yeast terminators the level of transcription, and it is generally assumed that the steady-state mRNA level is a primary Yeast transcriptional terminators are usually determinant of the final yield of a foreign protein. present in expression vectors for efficient mRNA 3’ The mRNA level is determined both by the rate of end formation. Terminators of prokaryotic or initiation and the rate of turnover. higher eukaryotic genes are not normally active in In most cases the yield of a foreign protein yeast, though there are exceptions such as the expressed using a yeast promoter has been much Drosophila ADE8 gene.I6’ Efficient termination is lower than the yield of the homologous protein probably required for maximal expression: deletion using the same promoter. Using the PGK promoter of ‘termination’ sequences 3’ of the CYCl gene on a multi-copy vector several proteins accumulate resulted in longer mRNA and a dramatic reduction to 1 to 2% t.c.p., whereas with the entire PGK in mRNA le~el.~” gene hosphoglycerate kinase accumulates to over Transcriptional termination of yeast mRNAs is These levels reflect to a large extent the less well understood than that in bacteria and in lower amounts of the foreign versus PGKtranscripts higher eukaryotes. Bacterial transcription termi- for as many as 20 genes,62although there appear to nates at the mature 3’ end of the mRNA. In higher be exception^.'^^ It was suggested that the reduced eukaryotes mRNA 3’ end formation involves levels were due to shorter tIi2sfor the foreign tran- cleavage and polyadenylation, downstream of the scripts, but Mellor et showed that a-interferon signal AAUAAA, of precursor mRNAs that extend mRNA was not unstable but was initiated at a several hundred nucleotides beyond the coding six-fold lower rate. Addition of downstream PGK region. Contrary to earlier ideas, it appears that yeast sequences restored the mRNA level to that for PGK mRNAs follow thesame pattern oftermination, pro- mRNA, suggesting the presence of a downstream cessing and polyadenylation of pre-mRNA as higher activation sequence (DAS), localized to the first eukaryotes. However in yeast these processes are 79 codons, required for maximal transcription. tightly coupled and occur within a shorter distance, Indirect evidence for a DAS has also been found near the 3’ end of the gene.52 with the pyruvate kinase (PYK) gene.292Attach- A number ofconsensus sequences have been impli- ment of the ZacZ gene to the PYKpromoter resulted cated as part of the mRNA ‘terminator’, especially in a 30-fold drop in mRNA molarity, whereas its tIi2 the tripartite se uence TAG..(T-rich)..TA(T)GT..decreased only two-fold, consistent with a 15-fold (AT-rich)..TT?dq6 and TTTTTATA.lm The com- drop in the initiation rate. The putative PYKDAS is monly found tripartite motif shows poor conser- active in single- and multi-copy vectors, has been vation and is tolerant of large sequence alterations, localized to nt 500 to 800 relative to the initiating suggesting that a general feature such as high ATG, and appears to be able to functionally replace AT-content may be critical.279However this can- the PGK DAS (A. Brown, personal communi- not be sufficient since terminators are frequently cation). Evidence for DASs has also been found in absent in AT-rich DNA, and can be unidirectional, the lipoamide dehydrogenase gene,414and in Ty2 implying some sequence specificity. More recent DNA.232 438 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE Table 4. Fortuitous transcriptional termination in foreign genes.

AT- Size content RNA Gene (kb) YO analysis" Comments

K.lactis 3.5 73 Truncated Reference 305 plasmid kl ORF2 mRNA Tetanus toxin 1.5 71 Truncated 2 6 terminators, eliminated by fragment C mRNA increasing GC-c~ntent~q~*~'~ HIV gp120 1.5 60 Truncated mRNA Mutation of T,ATA, gave several in P.pastoris longer low-abundance mRNAs not Sxerevisiae Increasing GC-contentbgave full-length mRNA (R.G.Buckholz,personal communication and C.A.S.) SIV gp130 1-6 62 Truncated (C.A.S., unpublished results) mRNA Plasmodium 0.88 74 Multiple (C.A.S., M.A.R. and J.J.C., unpublished falciparum p195 short mRNAs results) (42K fragment) P falciparum 0.9 1 61 No data No complementation (P.Ross- pfc2 (CDC28 MacDonald and D.H.Williamson, homologue) personal communication)

"Nodetectable protein in any example. bBychemical synthesis of the DNA. 'A similar problem in P. pastoris with two bacterial genes (63% and 67% AT) was solved by increasing GC-content (K. Sreekrishna, personal communication).

Since the evidence may now favour the existence expression, but antisense transcripts through the of DASs in certain genes it is possible that they will foreign gene can be eliminated by using bidirectional ,befound in many others. There may be some cir- terminators or by judicious juxtaposition with 2p cumstantial evidence for this, e.g. multi-copy GAL7 DNA (e.g. FLP terminator). gives levels of uridyltransferase of > 15% t.c.p., 13 whereas foreign proteins rarely reach this level. It RNA elongation must be emphasized that many other factors could The elongation of transcripts is not thought nor- account for these differences. If DASs are character- mally to affect the overall rate of transcription, but ized, it may be possible to incorporate them into the yield of full-length transcripts could be affected upstream promoter fragments in order to create by fortuitous sequences in foreign genes which cause more efficient expression vectors. Alternatively, if pausing or termination. These could either act in the DASs prove to be strongly position-dependent, they same way as natural yeast terminators or else by a could be placed within an intron which would be different mechanism. We have found evidence of excised prior to translation. If neither of these this in a remarkably high proportion of the genes we options work, then maximal transcription will only be have examined, especially, but not exclusively, in achievable using fusion proteins, for example to PGK. those with unusually high AT-content. Though not An alternative to all these approaches is to use a widely recognized, this problem could be a very different yeast, such as P.pastoris which does not common reason for low yields or complete failure of appear to have the same problem. foreign gene expression in yeast. Yeast expression vectors frequently give rise to We have listed examples of lack of expression of numerous unexpected transcripts arising from for- AT-rich genes in Table 4. The presence of truncated tuitous promoters in bacterial plasmid DNA.245It is mRNA has not been demonstrated in every case, but not known whether they can affect foreign gene the clearest published example is in the expression FOREIGN GENE EXPRESSION IN YEAST: A REVIEW 439 of the AT-rich Clostridium tetani DNA encoding system is not fully developed in yeast (see Transcrip- tetanus toxin fragment C.307RNA was analysed tional promoters and terminators). If the problem from the native gene and from each of three versions is one of post-transcriptional processing then it of the gene containing progressively more synthetic may be necessary to use an extranuclear expression DNA, starting from the 5’ end (Figure 5). The tran- vector, e.g. one based on the linear AT-rich plasmids script length increased through this series, with only of K.lactis which can also be propagated in S. the fully-synthetic gene giving full-length mRNA as cerevisiae, to segregate the transcripts from nuclear the major species. All the short and full-length tran- processing enzymes.365 scripts were discrete, abundant and polyadenylated. 1.5 C.tetani The kb DNA was shown to contain at stability least six fortuitous polyadenylation sites which were RNA eliminated by increasing GC-content (from 29% to The half-lives of yeast mRNAs range from 1 to 47%). The partially-synthetic genes gave rise to low 100 min and can therefore have a profound effect on levels of run-off translation products, but efficient the steady-state level (reviewed in reference 47). A production of fragment C was only achieved with careful study of 15 randomly-chosen mRNAs the fully-synthetic gene. showed two populations, one with longer t,,2s(40 to The problem with AT-rich genes means that 100 min) and one with shorter tIi2s(6 to 20 min).47 analysis of genomes with high AT-contents (e.g. Within each class there was an inverse relation Plasmodiumfalciparum or Dictyostelium discoideum) between mRNA length and stability. However, there by complementation in S.cerevisiae is possibly not is some disagreement over this division into two worth pursuing as a general strategy. Such studies classes.’62The difference between stable and unstable might be attempted in S.pombe, though recent work mRNAs may be that the latter have destabilizing suggests that the two yeasts have a conserved mech- elements, since sequences from the short-lived URA3 anism for mRNA 3’ end f0rmati0n.l~~More worry- mRNA destabilize PYKmRNA.48A number of the ingly, there is evidence of the same problem with unstable mRNAs have recently been found to genes having an average AT-content similar to that encode ribosomal proteins (A. Brown, personal of yeast (60%). With the HIV-I env gene, truncated communication); the foreign lac2 mRNA has a t1,2 mRNA was found in P.pastoris but not Sxerevisiae. of 27 min.292 Mutagenesis of a sequence resembling a poly- Insertion of premature stop codons in URAl, adenylation consensus inactivated a major poly- URA3 and PGK mRNAs has been shown to cause adenylation site but revealed several weak sites which their destabilization, suggesting that ribosome attach- were removed by increasing GC-content using ment may contribute to mRNA ~tability.”~,~~’,~~~ chemical synthesis (C.A.S. and R.G. Buckholz, in However the same result was not observed for a preparation). PYK and PYK-lac2 mRNA,292suggesting a com- The frequency of the problem means that it would plex relationship between mRNA stability and be desirable to be able to predict its occurrence, translation. Santiago et al.327found no obvious and to solve it without resorting to gene synthesis. correlation between ribosome loading and stability Unfortunately it appears to be very difficult to ident- for ten different mRNAs. ify polyadenylation sites by searching for consensus There appear to be few examples of low mRNA sequences, especially in AT-rich DNA. In the case of stability being a primary factor in poor yields of the fragment C gene, one of the short mRNAs could foreign proteins. Due to the lack of knowledge of have been due to a single TTTTTATA sequence, yeast mRNA degradation mechanisms it is imposs- but a number of tripartite motifs were found in ible to predict whether a foreign mRNA will be regions with no polyadenylation sites.307 stable, though we might expect long mRNAs to be At present the only solution is to increase the GC- somewhat less stable than short ones. AU-rich se- content of offending sections of genes by chemical quences in the 3’-ends of some mammalian mRNAs synthesis. The success of more general solutions confer instability,339but it is not known whether would depend on whether the problem is due to they function in yeast. Where mRNA instability is true premature termination or to uncoupled post- diagnosed as a problem, overall yield might be transcriptional processing. In the former case a improved by (i) using a more powerful promoter, foreign RNA polymerase which uses different ter- (ii) using a promoter with more rapid induction mination signals, e.g. T7 RNA polymerase, might kinetics, or (iii) chemically synthesizing the gene produce full-length mRNA. Unfortunately, the T7 with altered codons or deleting the 3’ untranslated A

Ban1 tt Hinfl EcoRI BamHl i i TET2 d I Sac1I

TET7 Sac1I

TETl 1

TETl5

I1 I1 1111 I Ill I I 0 500 1000 nt

1234

- 1.77 - 1.52 - 1.28

- 0.78

- 0.53

- 0.40

1 pWYG7-TET2 2 pWYG5-TET7 3 pWYG5-TETI 1 4 pWYG5-TET15 Figure 5. Fortuitous transcriptional termination in AT-rich tetanus toxin fragment C DNA. (A) Four genes encoding fragment C are shown, with synthetic DNA of increased GC-content (hatched). Approximate 3’ ends of transcripts generated in yeast are indicated. (B) Northern blot showing fragment C-specific RNA from these genes: only TETlS gave abundant full- length mRNA.307 FOREIGN GENE EXPRESSION IN YEAST A REVIEW 44 1 regiongoain the hope that instability determinants in highly-expressed genes, alteration of the AUG will be removed. context of mRNAs in yeast has at most a 2- to 3-fold effect, in contrast to the much greater effect in mammalian cells.”’71 Initiation of translation As a consequence of these considerations it is Translational efficiency is thought to be controlled preferable to avoid the inclusion of foreign non- primarily by the rate of initiation. This is affected by coding sequences in yeast expression vectors. How- the structure of the 5’ untranslated leader of a ever, where this is inconvenient it should be possible mRNA, and there are examples of poor expression to predict whether a foreign 5’ leader will be deleteri- due to the presence of sub-optimal foreign 5’ ous by examining the sequence for secondary struc- leaders. Using a multi-copy GAP491 promoter vec- ture or runs of Gs or Us. For example, analysis tor to express hepatitis B core antigen (HBcAg) of the sequence 5’ to the HBcAg gene, found by Kniskern et found that the product accumu- Kniskern et to inhibit translation, shows a lated to 0.05% of soluble cell protein when viral 5’ number of predicted secondary structures of up to and 3’ sequences of about 100nt were retained. AG = - 35 kcal/mol (M.A.R., unpublished results). Deletion of the 5’ viral sequence raised the yield to 26%, while deletion of 5’ and 3’ viral sequences Translational elongation raised it to 41%, both without altering mRNA levels. Translational elongation is not thought to affect Initiation in eukaryotes is thought to follow a the yield or quality of polypeptide normally, but scanning mechanism whereby the 40s ribosomal there is now some evidence that it can become limit- subunit plus co-factors (eIF2, eIF3, eIF4C, Met- ing with very high mRNA levels. Codon usage is tRNA and GTP) bind the 5’ cap of the mRNA then known to affect the elongation rate and therefore migrate down the untranslated leader scanning for has to be considered as a potential factor affecting the first AUG codon.222Any part of this process, product yield. In the past it has been discounted on which is affected by the structure of the leader and the basis that some genes containing many rare the AUG context, could limit the initiation rate. codons, e.g. lacZ or HBcAg, are expressed at high Yeast mRNA leaders have an average length of levels; however, these gene products are unusually about 50 nt, are A-rich, have little secondary struc- stable and their yield does not reflect overall ture, and almost always use the first AUG for translational efficiency. initiation (reviewed in reference 70). The length of Despite the degeneracy of the genetic code, a non- the untranslated leader does not seem to be critical: random codon usage is found in most organisms: although there is a 50% reduction in efficiency when whilst the codon usage of most genes reflects the the PGKleader is shortened from 45 to 2 1 nt, further nucleotide composition of the genome, highly- shortening to 7 nt has no effect.389Insertion of long expressed enes show a strong bias towards a subset runs of Gs is very deleterious, however, causing a of ~odonsk’,~~~This ‘major codon bias’, which can complete inhibition of translation; runs of Us have a vary greatly between organisms, is thought to be a partial effect ossibly caused by interaction with the growth optimization strategy such that only a subset polyA lFThe most significant single factor is of tRNAs and aminoacyl-tRNA synthetases is secondary structure: hairpins of AG = - 20 kcal/ needed at high concentration for efficient translation mol can drastically inhibit translation of HZS4 or of highly-expressed genes at fast growth rates.227 CYCZ mRNA in yeast cells,”,7’ in contrast to the Rare codons, for which the cognate tRNA is less much lower sensitivity of translation to leader abundant, are translated at a slower rate in E.coli,282 secondary structure in mammalian cells. but this will not normally affect the level of product The consensus sequence around the initiating from an mRNA since initiation is usually rate- AUG, A/YAA/UAAUGUCU,70 in yeast is differ- limiting. A ribosome finishing translation of one ent from that in higher eukaryotes (CACCAUGG). mRNA molecule is most likely to initiate translation It has been speculated that the mammalian context of a different mRNA species, unless the original reflects an interaction with the sequence GUGG in speciescomprisesa large proportion of total mRNA. 18s rRNA. The prevalence of UCU as the second Thus, the overall rate of translation of an mRNA is codon may reflect the preference for stabilizing not usually affected by a slower elongation rate amino acids according to the N-end rule (see below). unless ribosomes become limiting, which would Although there is a strong bias for A at-3, especially affect all transcripts in the cell. 442 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE In contrast to the normal situation, there is evi- Polypeptide folding dence that codon usage may affect both the yield and quality of a protein when a gene is transcribed to very During or following translation, polypeptides high levels. With very high levels of mRNA contain- must fold so as to adopt their functionally-active ing rare codons, aminoacyl-tRNAs may become conformation. Since many denatured proteins can limiting, increasing the probability of amino acid be refolded in vitro, it appears that the information misincorporations, and possibly causing ribosomes for correct folding is contained in the primary poly- to drop off. Indeed, a high misincorporation fre- peptide structure.IM However, folding comprises quency has recently been observed in a forei n pro- rate-limiting steps during which some molecules may tein produced at very high levels in E.~oli!~~The aggregate, particularly at high rates of synthesis and presence of many very rare codons has been shown at higher There is evidence that cer- to limit the production of tetanus toxin fragment C tain heat-shock proteins act as molecular chaperones in E.~oli.’~’In yeast, Hoekema et al.’” showed that in preventing the formation and accumulation of substitution of a large proportion of preferred unfolded a gregates, while accelerating the folding codons with rare codons in the 5’ portion of the PGK reactions. 10 gene caused a decrease in expression level. However, Due to the intrinsic nature of polypeptide folding these substitutions were made in the region contain- and the low specificity of chaperones, it is very un- ing a putative DAS,253possibly contributing to the likely that foreign cytosolic proteins will accumulate effect on expression levels. Translation of lacZ in yeast in non-nativeconformations, andindeed this mRNA, which contains a high proportion of rare is generally the case. When fragments of proteins or codons, was shown to be limiting at high levels.292 fusion proteins are expressed, however, normal fold- There was a decrease in ribosome loading, possibly ing domains may be perturbed resulting in an in- due to pausing and drop-off, on lacZ mRNA and on soluble product. Nevertheless, fusion proteins that TRP2 mRNA, which also has poor codon bias. are insoluble in E.colimay frequently be soluble when Recently, expression of an immunoglobulin kappa expressed in yeast, e.g. fusions to P-galactosidase chain in yeast was shown to increase 50-fold, with- (J.J.C., unpublished results), glutathione-s-trans- out a change in mRNA levels, using a synthetic, ferase (GST; M.A.R., unpublished results), and codon-optimized gene.220 HBcA~.’~Insoluble proteins can often be renatured Thus the codon content of a foreign gene may in vitro though the techniques for this can be complex influence the yield of protein where the mRNA is and ~npredictable.~~~ produced at very high levels. This may be more likely In contrast to intracellular proteins, naturally- to occur on growth in minimal medium, when the cell secreted proteins encounter an abnormal environ- produces a wide variety of biosynthetic enz mes, ment in the cytoplasm: disulphide bond formation is encoded by genes containing rare cod on^.^^r The not favoured and glycosylation cannot occur. effect on product quality has been difficult tomeasure Though many secreted proteins are insoluble when but requires further attention since it has important expressed intracellularly, e.g. pro~hymosin,’~~ implications for therapeutic proteins.336Proteins human serum albumin,* HIV gpl2O,I9 some are containing amino acid misincorporations are diffi- soluble and biologically-active, e.g. a-interferon,”’ cult to separate and may affect the activity and a,-antitryp~in,~”tumour necrosis factor,359Factor antigenicity of the product. Since small genes are XIIIa.300Factor XIIIa is interesting in that it has nine now frequently synthesized chemically they may Cys residues, none of which forms disulphide bonds. be easily and perhaps profitably engineered to con- In E.coli, foreign proteins are frequently insoluble tain optimal codons for high-level expression in but low temperature has been found to increase solu- yeast. bility in some cases.331This may be due to a decreased mRNA secondary structure, in addition to codon translation rate or to the fact that hydrophobic inter- usage, may affect translational elongation. Baim actions, such as occur in aggregates, become less et a1.I’ showed that a mutation which introduced a favourable. A dramatic increase in the yield of active, hairpin loop (AG = - 19.6 kcal/mol) near the 5’ end soluble protein has been re orted on reducing the of the coding region of CYCl mRNA reduced the rate of induction in E.co1iJ9 Low temperature or amount of protein product five-fold. mRNA levels reduced induction rates may increase product solu- were unchanged and analysis of the distribution of bility in yeast. For example, in the intracellular CYCl mRNA on polysome gradients indicated that expression of the bacterial membrane protein pertac- translation was affected. tin, the proportion of the product that was soluble FOREIGN GENE EXPRESSION IN YEAST A REVIEW 443 Table 5. Effect of penultimate amino acid in determining mature N-terminus of intracellular proteins.

Met removed Met retained

No W-acetylation Pro, Val, Cys Ile, Leu, Met, Phe, Tyr, Trp, Lys, His, Arg, Gln W-acet ylation Gly, Ala, Ser, Thr Asp, Glu, Asn decreased from 100% to 10% as the expression level Both enzymes are associated with ribosomes and act was increased from 0.1% to 10% t.~.p.~'~ on nascent polypeptides. Similar considerations apply to the assembly of The specificities of yeast MAP and NAT (Table 5) foreign multimeric proteins. Examples of homo- have been determined by N-terminal amino acid polymeric assembly include the HBcAg2I8 and analysis of mutant iso- 1 -cytochrome c or thaumatin HBSA~.~~'The latter is inserted into intracellular polypeptides,1833260and more recently by studies of yeast membranes to form immunogenic 22 nm par- purified MAP.60Thespecificity of MAPis determined ticles resembling those found in the serum of by the size of the penultimate residue. When this is has chronic HBV carriers. In early studies only a pro- a radius of gyration of < 1.29 A (Gly, Ala, Ser, Cys, portion of the HBsAg was found to be immunologi- Thr, Pro and Val) the Met is removed, though with cally-active, and later work showed that mature the larger residues Thr and Val there is only partial particles only formed during extraction, when SH removal if they are followed by Pro. With other residues oxidized to form inter-chain S, bonds.399 penultimate residues Met is retained. These rules More recent work in P.pastoris showed that a much appear to be reliable and highly conserved among higher proportion of HBsAg was correctly folded eukaryotes, so mammalian proteins produced in when produced in a strain growing slowly during yeast should have the normal N-terminal residue. induction, suggesting rate-limiting folding steps Recently it has been shown that fusions ofproteins related to growth rate.81 to the C-terminus of ubiquitin are rapidly processed There are few published examples of heteromulti- in vivo when expressed in yeast, liberating the mature meric assembly in yeast, but a notable one is the protein.3213393This approach is useful since it can co-expression of a and p-globin cDNAs to produce be used to generate a protein with any desired haem~globin.~~~The yield of fully-assembled, N-terminus (apart from Pro), and appears to si soluble haemoglobin, which incorporated yeast nificantly increase the product yield in some cases. I%; haem, was 3 to 5% t.c.p. Another interesting The factors governing W-acetylation are less example is in the simultaneous intracellular ex- clear. It appears that Met-Glu/Asp is sufficient for pression of heavy and light chains of an IgG directed acetylation in eukaryotes.260N-terminal Gly, Ala, against ADH I: these were able to assemble in the Ser, Thr and Met-Asn may also be acetylated, cytoplasm and partially block ADH I activity in vivo, though effects of second and third residues make despite the fact that the inter-chain S, bonds would this less predi~table.~'~However, there is evidence not form.56Similarly, intracellular expression of a that the process is conserved among eukaryotes, so catalytic antibody Fab fragment yielded functional that proteins acetylated in mammalian cells might product at 0.1 % t.~.p.~' be expected to be acetylated in yeast cells.'56 In most cases the structure of the N-terminus Post-translational processing should not affect biological activity of a protein, but there may be exceptions. For example the response Amino-terminal modifications of polypeptides of haemoglobin to physiological modifiers involves are the commonest processing events and occur on the N-terminus, and correct processing of a and p- most cytosolic proteins (reviewed in reference 209). globins in yeast is therefore advantageous over Two types of events normally occur: removal of the expression in E.~oli.~~~W-acetylation of melano- N-terminal Met residue, catalysed by Met amino- cyte-stimulating hormone and of p-endorphin is peptidase (MAP), and acetylation of the N-terminal required for full biological activity (reviewed in residue, catalysed by W-acetyltransferase (NAT). reference 209). 444 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE A variety of other post-translational modifi- reference 96). Unfortunately, we do not know all the cations which are often critical for biological activity determinants nor the relative importance of different appear to be conserved between yeast and higher pathways. Vacuolar degradation is responsible for eukaryotes. The phosphoproteins fos and c-myc are non-selective bulk turnover of long-lived proteins correctly phosphorylated in yea~t.~’*,~*’M yristylation (average t1/,approx. 160 h), whereas short-lived pro- is a co-translational modification of N-terminal Gly teins (tl,* <2.5 h) are degraded in the cytosol by an important for the membrane targetting of certain ATP-dependent pathway involving ubiquitin. proteins, e.g. G proteins, src tyrosine kinases, and A number of proteases, activated by the PEP4 retroviral gag proteins, and this also occurs in and PRBl gene products, are responsible for yeast yeast.”’ Isoprenylation affects an important class of vacuolar degradati~n.’~’Mutations in both genes membrane proteins including G proteins and ras dramatically reduce cellular proteolysis and should proteins:330the Cys ofC-terminalcys-X-X-X-CO,Halso reduce the risk of proteolysis during extraction. is isoprenylated,following which the three C-terminal pep4 mutants are widely used, but do not normally residues are removed and the Cys-CO,H is methyl- appear to offer an advantage in product yield. Indeed esterified. Mammalian proteins such as H-ras p21 a general reduction in protein turnover rate would are isoprenylated in yeast. not be expected to increase the relative accumulation ofaforeignprotein, thoughitmight increasethetotal protein yield. Vacuolar proteolysis is affected by Stability of intracellular proteins culture conditions and increases several-fold during So far, processes affecting the rate of synthesis of N- or C-starvation or in stationary phase.I7’ proteins have been considered, but the ultimate yield In the ubiquitin pathway proteins are marked by is equally affected by the rate of degradation. In fact covalent attachment of ubiquitin, a 76-residue poly- the few examples of very high level expression peptide, and become substrates for rapid degradation (>25% t.c.p.) in S.cerevisiae are of unusually by a cytosolic ATP-dependent pr~teasome.~~In stable proteins, e.g. SOD,’56HBcAg,,I8 and schisto- addition to short-lived proteins, damaged or soma1 GST.237This reflects a difficulty in achieving a denatured proteins conjugate ubiquitin more very high rate of synthesis of foreign proteins in efficiently and are targetted for degradation. Sxerevisiue, probably at the level of transcription. Varshavsky and co-workers have identified one Very low yields are obtained with proteins which component of ubiquitin recognition as the N-terminal are naturally short-lived, such as m~c,~’~or with amino acid (the N-end rule pathway; reviewed in some polypeptides which are naturally secreted, reference 393). The N-end rule was uncovered by the such as insulin366and hEGF.385In some cases fusion production of P-galactosidase variants containing to a stable protein has given high-level accumu- different N-terminal residues, which were generated lation in east, for example with a hybrid TAT-T by the expression and spontaneous processing of particle? a SOD-proinsulin fusion protein, 4 ubiquitin-P-galactosidase fusions; the tIl,s of these fusions to GST (M.A.R., unpublished results), and variantsranged from2 min to > 20 h. The N-end rule a variety of peptides fused to HBcA~.~~Alterna- degradation signal actually comprises two determi- tively, secretion has been used to segregate the nants: a destabilizing N-terminal amino acid and a product from intracellular proteases. In the case of proximal internal Lys. Destabilizing residues are HBsAg particles containing the pre-S2 peptide, classified as Type I (positively charged: Arg, Lys, proteolytic cleavage occurred at a specific site and His), Type I1 (bulky hydrophobic: Phe, Tyr, Trp, could be reduced by using a protease-deficient(pep4) Leu, and Ile in yeast), and Type I11 (small uncharged: strain or by mutation to remove the susceptible Ala, Ser, Thr), the latterarenotdestabilizingin yeast. region.”’ Where these approaches fail, yields might Additionally, the residues Asp and Glu are secondary logically be improved by the following measures: destabilizing residues since they are substrates for post- (i) using a more rapidly-induced promoter, (ii) translational addition of Arg; Asn and Gln are tertiary using additional protease inhibitors to minimize destabilizing residues since they can be deamidated in degradation during extraction, (iii) inducing at vivo. Recognition of a destabilizing N-terminus results lower temperature, (iv) harvesting cells in the in multiple ubiquitination of the internal Lys residue exponential growth phase. which is the prelude to degradation. A number of different pathways of protein degra- In agreement with the N-end rule, almost all cyto- dation exist and therefore there are multiple molecular solic proteins with known N-termini have stabiliz- determinants that confer instability (reviewed in ing amino acids. This might be expected from the FOREIGN GENE EXPRESSION IN YEAST A REVIEW 445 striking inverse correlation between the N-end rule and N-termini that are generated by MAP. (In con- trast most secreted proteins have destabilizing N- termini.) However, recent data suggest that the N-end rule is only one component, possibly a minor one, of the ubiquitinpathway. Mutationsin theyeast gene (UBRZ)for the Type I and I1 recognition com- ponent dramatically stabilized Arg-galactosidase but did not affect either bulk proteolysis or ubiquitin- dependent degradation." A similar result was obtained using inhibitors of Type I (Arg-Ala) or Figure 6. The yeast secretory pathway. Arrows indicate the Type I1 (Leu-OMe) recognition in vivo, resulting in a route taken by proteins through the secretory pathway to either ten-fold stabilization of Arg- or Leu-galactosidase, the vacuole or the plasma membrane. N, nucleus; R, ribosomes; respectively, and > 50-fold increases in accumulated ER, endoplasmicreticulum; G, Golgi; Vac, vacuole; V, secretory protein.'2 Therefore other, undefined, signals are vesicles; PM, plasma membrane; CW, cell wall. more important in the ubiquitin pathway. Another proposed signal for rapid degradation is asparagine-linked glycosyl structures may be added. a variable sequence rich in Pro, Glu, Ser, and Thr The signal for the addition of these N-linked sugars is (PEST) found in the majority of short-lived pro- the same for yeast and mammalian glycoproteins tein~.~"Recently, addition of PEST sequences has (Asn-X-Ser/Thr). 0-linked oligosaccharides may been shown to destabilize DHFR.235It is not clear also be added. Proteins are then transported in whether degradation of proteins containing PEST vesicles to the Golgi where modifications to these sequences involves the ubiquitin pathway. glycosyl structures take place. These modifications differ from those made by higher eukaryotic cells SECRETION OF FOREIGN PROTEINS and, as a result, glycosylation is increasingly regarded as a major drawback to the secretion of Introduction therapeutic glycoproteins from yeast. From the A wide variety of heterologous proteins have Golgi, proteins are packaged into secretory vesicles been secreted from yeast and this approach offers and are delivered to the cell surface (Figure 6). certain advantages over intracellular production. Once in the ER, it is probable that a default path- Although Xcerevisiae secretes only 0.5% of its own way directs a protein to the plasma membrane unless proteins, this level can be increased several-fold so it contains specific signals to cause retention in the that a secreted foreign protein can be almost pure in ER or Golgi, or to target it to the vacuole. Therefore the medium. Many pharmacologically-important it might be thought that if a foreign protein could be proteins are naturally secreted and can often only targetted to the lumen of the ER, it should be success- adopt their correct conformation by folding within fully secreted. However, there are a number of the secretory pathway. As a consequence, intracellu- stages in the secretory process at which problems lar expression is often unsuitable since the product may occur. The yeast proteins which assist in fold- may be insoluble and may also have an incorrect N- ing and disulphide bond formation differ from terminus, although this can be overcome by fusion their counterparts in higher eukaryotes and this to ubiquitin (see 'Factors affecting intracellular may affect folding of foreign proteins. Malfolding expression'). Thus secretion is used mainly for the can result in retention in the ER and degradation. production of correctly-folded, naturally-secreted There are also reports of proteins being retained proteins, but there are, in addition, other instances in the Golgi, again possibly due to malfolding. when secretion may be preferable. For example, Retention in the cell wall has also been a problem, some proteins are unstable or toxic when cyto- especially with larger proteins, although factors plasmically-expressed and these problems may be other than molecular mass are known to be import- circumvented by secretion. ant. In addition to problems of transport, other As in higher eukaryotes, protein secretion in yeast undesirable events such as aberrant processing or is directed by an amino-terminal signal sequence hyperglycosylation may take place during the which mediates co-translational translocation into secretory process. It should be noted, however, that the endoplasmic reticulum (ER). The signal peptide is proteins which are poorly secreted from Sxerevisiae removed by a signal peptidase. In the lumen of the ER may be efficiently secreted from other yeasts, for 446 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE example prochymosin secretion is much more defined medium may result in lower cell density and, efficient from K.lactis and Yarrowia lipolytica in some cases, lower levels of secreted product per than from Sxerevisiae (see ‘Expression in non- cell. Wheat a-amylase secreted from yeast has been Saccharomyces yeasts’). shown to reach much higher levels in rich medium The secretion of heterologous proteins from yeast than in selective, defined medium.314At labora- has been most successful with peptides, many of tory scale, it may be preferable therefore to use a which are commercially important, e.g. epidermal dominant marker such as that for G418 resistance. growth factor (EGF) and insulin. In high-density cultures hundreds of milligrams of these proteins The nature of signal sequences A classical signal may be secreted per litre. The secretion of large pro- sequence comprises a charged N-terminus, a central teins has proved less predictable, nevertheless there hydrophobic core and a consensus sequence for have been some notable successes.186,284,388 The cleavage in the ER by signal peptidase. Some successful secretion of proteins which are not natur- secreted proteins, such as the yeast mating phero- ally secreted, such as HIV- 1 protease has also been mone, a-factor, have additional pro sequences reported,288 but the presence of fortuitous which may aid secretion. glycosylation sites can cause problems.307 As described below, heterologous proteins may be secreted from yeast using either a foreign signal, Vectors and signal sequences often derived from the protein being secreted, or a Promoters and selectable markers Most commonly- yeast signal. Since signal se uences are recognized used secretion vectors are based on high-copy, 2p with low specificity in yeast?” it could be assumed plasmids. Integrated vectors have been reported to that foreign signals would work as efficiently as give higher yields of secreted product than episomal those from yeast, but this is often not the case. vectors; four integrated copies of a prochymosin A number of studies aimed at identifying the expression unit resulted in similar overall expression features of yeast signal peptides have been per- levels but higher secretion yields than were achieved formed. Ngsee et analysed mutated signal with a multi-copy vector.357The reason for this sequences from the yeast invertase gene, SUC2. difference is unclear. However, yields of EGF, which They concluded that the essential feature of a signal in contrast to prochymosin is efficiently secreted, peptide is a hydrophobic core of 6-15 amino acids, were not higher from integrated gene copies.74Sakai which may be interrupted by non-hydrophobic resi- et compared secretion of human nerve growth dues. Additionally, many signal peptides were factor from a 20-copy Ty 6-integrant with that from found to contain one or more basic amino acids a 2p vector. Levels were three- to four-fold higher preceding the hydrophobic core. Small neutral and (3-4mg/l) from the 6-integrant, but the use of a a-helix-disruptingamino acids were often present in constitutive promoter (PGK)may have led to copy the vicinity of the cleavage site. number reduction with the 2p vector. Secretion of a-factor was shown to be drastically A number of the promoters described above (see reduced if the hydrophobic core of the signal was ‘Transcriptional promoters and terminators’) have interrupted by a hydrophilic residue, or a proline been used in secretion vectors, but frequently a pro- which disru ts-helical secondary str~cture.~Ngsee moter and signal sequence from the same gene are and Smith2’ noted that yeast invertase signal se- chosen e.g. MFa1, PH05 or SUC2. Ernst‘“ quence is predicted to have an a-helicalconformation, reported that up to a two-fold increase in somato- whereas that of bovine prolactin has a tendency to medin-C secretion could be obtained by using the form an extended coil. Substitutions in the prolactin weak CYCl promoter rather than the moderate signal which increased the probability of it having an actin promoter. Product toxicity may be more acute a-helical conformation improved its functioning in with powerful promoters and this may work to yeast. Thus there may be more stringent requirements reduce plasmid copy number; the use of weak or for an a-helical secondary structure in yeast signal moderate constitutive promoters, or regulated pro- sequences compared to those of higher eukaryotes. moters which are repressed during early growth Surprisingly, even if the signal peptide is deleted, phases, may minimize this effect. some proteins, e.g. carboxypeptidase Y36and acid The choice of selection marker on a secretion vec- ph~sphatase,~~’may still inefficiently enter the tor may be particularly important since culture con- secretory pathway, indicating that the mature pro- ditions may dramatically affect the final yield in the tein sequence may also contain features which can medium. Selection of plasmid-containing cells in a be recognized by elements of the secretory pathway. FOREIGN GENE EXPRESSION IN YEAST A REVIEW 447 Use of heterologous signal sequences Early attempts Use of yeast signal sequences Owing to the difficulty to secrete foreign proteins from S.cerevisiae utilized of predicting whether a particular foreign signal the protein's own signal sequence, e.g. E.coli sequence will function in yeast, much work has lactama~e,~'~human a- and y-interferon (IFN), P,; been carried out using homologous Scerevisiae mouse immunoglobulin heavy and li ht chainsm signal sequences. The three most widely studied and influenza virus haemagglutinin!" However are those from acid phosphatase, invertase and the expression levels were often very low, with a-factor. only a proportion of the protein being secreted. Acid phosphatase (PHOS) has a classical signal Processing of the polypeptide was not always sequence7 yet there are few examples of its use for correct and, in the case of IFN, degradation of the heterologous protein secretion. When used to secrete preprotein took ~1ace.I~~Nevertheless, some tissue-type plasminogen activator (tPA), less than foreign proteins have been successfully secreted 5% of the total tPA activity was found in the from yeast using their own signal, e.g. Aspergillus medium,16' however the secretion of this protein has awamori glucoamylase,IE6barley a-amylases 1 and been pr~blematic.~~'Human salivary a-amylase has 2358 and human serum albumin (HSA).355 De been secreted using the PHOS signal328which in this Baetselier et al.89have reported the production of up instance gave similar results to the heterologous a- to 3 g/L of active, secreted Aspergillus niger glucose amylase and human gastrin signals and to the yeast oxidase. a-factor leader. The secretion from yeast of Bacillus amylolique- The yeast invertase (SUC2) has been used faciens a-amyla~e~'~using its own signal demon- more widely for foreign protein secretion. Human strated that a prokaryotic signal sequence may also a2-IFN has been secreted using this signal, which efficiently direct secretion. The signal peptide was was correctly cleaved from all secreted molecules59 correctly cleaved, but the a-amylase was core unlike the native IFN signal peptide. 173 The invert- glycosylated in contrast to the native product. ase signal has also been used to secrete human a-l- However, the glycosylated enzyme retained its antitrypsin (a-AT), however approximately 80% of activity. the protein remained inside the The passage Foreign signals may also be used to drive secretion of the a-AT from the ER appeared to be the rate- of other heterologous proteins-the human gastrin limiting step in this case. Production of mouse- signal sequence has been shown to drive secretion of human chimaeric antibody has been achieved using human a-amylase in S.cere~isiae.~~~In general, the the invertase signal to secrete immunoglobulin heterologous signal sequences which function best heavy and light chains.I8' Melnick et al. 255 reported in S.cerevisiae tend to be those from lant or fungal the efficient secretion of human single-chain urinary proteins (e.g. barley a-amyla~e:~~Trichoderma plasminogen activator (scuPA) using the invertase reesei endo lucana~e,~~~Mucor pusillus aspartic signal. proteinase 411). Attachment of a heterologous protein to a yeast The leader sequence from the killer toxin ORF2 signal sequence will usually result in a change in the gene of K.lactis,a yeast closely related to Xcerevisiae, amino acid immediately C-terminal to the signal has been emplo ed to direct the secretion of human peptidase cleavage site. However, there appears to interleukin- 1 ,Iy and diphtheria toxin-hormone be flexibility in recognition of cleavage junctions. fusion proteins.285HSA has also been secreted from Fusions of the invertase signal with proteins which S.cerevisiae using this leader sequence, however it alter the amino acids at the junction, such as HSA or was shown to be less efficient than the native HSA two forms of insulin, are still ~leaved.'~' leader or an Sxerevisiae signal sequence.355 The most extensively used signal sequence for Although there have been some notable successes, heterologous protein secretion from S.cerevisiae is the use of foreign leaders often resultsin intracellular the prepro region from a-factor (MFal),frequently accumulation (e.g. c~-IFN,'~~a- 1-antitrypsinS3), and used with the MFaI promoter. MFal encodes a it must be concluded that the yeast secretory path- 165 amino acid protein, prepro-a-factor, which way has slightly different requirements from higher comprises a signal sequence of 19 amino acids (the eukaryotic secretion systems. Therefore, for most pre region) and a pro region, followed by four cases of heterologous protein secretion from yeast, tandem repeats of the mature 13 amino acid a- it is preferable to use a yeast signal sequence and it factor sequence (Figure 7). Each repeat is preceded may be simpler to do this as a matter ofcourse with a by a short 'spacer peptide' with the structure Lys-Arg- new gene. (Glu/Asp-Ala),,. Processing of prepro-a-factor 448 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE

I I I I I signal K€XZ K€H K€H K€XZ

Glu.Ala.Glu.Ala~Trp.His,Trp.Leu.Gln.Leu.Lys.P~.Gly.Gln.Pro.Mei.Ty~Lys,A~\ tt a-factor tt STE13 KEXl

Figure 7. The structure and processing of prepro-a-factor.The product of the MFaZ gene is shown schematically. The three N-linked glycosylation sites in the pro region are marked (+). The peptide product from the cleavage ofprepro-a-factorby KEX2 is also indicated and sites for further processing by STE13 and KEXl are shown. See text for a description of the activities of the processingenzymes. involves four proteolytic enzymes: (i) the pre region (GM-CSF). Furthermore, prepro fusions to inter- is cleaved by signal peptidase, (ii) the KEX2 pro- leukin-1p were not cleaved whereas pre fusions were tease cleaves on the carboxyl terminal side of the correctly processed."' However, secretion of human Lys-Arg sequence in the pro region and at the junc- insulin-like growth factor I (IGF-I) from yeast is tion of each repeat, (iii) STEl3, a dipeptidyl amino- dependent on the entire a-factor leader; the pre peptidase, removes the spacer residues at the amino region is not sufficient. It may be that the pro region terminus of each repeat and (iv) the KEXl carboxy- aids movement of the IGF-I into the ER or assists peptidase removes the Lys and Arg residues at the folding of the protein in the ER (A. Hinnen, per- carboxyl terminus of the first three repeats (see sonal communication). Additionally, both the pre Figure 7; reviewed in reference 41). and pro regions were required to bring about trans- Fusions of the MFal repro to genes encoding location of a reporter protein, a-gl~bin.~"Reppe mature human al-IFN!' a consensus u-IFN?~ et compared secretion of human parathyroid P-end~rphin~~or human EGF (hEGF)42resulted in hormone (hPTH) directed either by the a-factor pre- efficient secretion of the heterologous protein. Pro- pro region or by the pre region alone. When the pro cessing of the prepro region took place, but the Glu- region was not present, there was a considerable Ala spacer at the N-terminus of the secreted protein reduction in hPTH mRNA levels, with a concomi- was not always removed. 5% of the hEGF and 50% tant reduction in levels of the protein product both of the a1 -1FN was estimated to be in an intracellular in the growth medium and inside the cells. This form. Many foreign proteins have now been secreted suggests that the pro region may additionally play from yeast using the a-factor leader, and this system a role in stabilizing the mRNA or may affect has been demonstrated to be generally applicable. transcription of the gene. Changes can be been made to the MFal prepro to facilitate cloning, such as the introduction of a Xho I Glycosy lation site towards the end of the pro region.74Despite a resultant amino acid change (Asp to Glu), this was Many potential therapeutic proteins are glyco- shown to be fully functional. sylated, including monoclonal antibodies, blood The requirement for the pro region of the a-factor clotting factors, and many IFNs, hormones, growth signal sequence seems to vary. Glycosylation of this factors and viral antigens. The carbohydrate side region appears to be important for efficient transport chains appear to be involved in diverse processes of a-fa~tor.'~Ernst"' reported that the pro region including cell-cell recognition, hormone-receptor was not important for the secretion and processing binding, protein targetting, host-microorganism of aminoglycoside phosphotransferase or human interactions, solubility and stability.295 Glyco- granulocyte-macrophage colony stimulating factor sylation is both organism and cell-type specific and FOREIGN GENE EXPRESSION IN YEAST A REVIEW 449

YEAST GLYCOPROTEIN I MAMMALIAN GLYCOPROTEIN

MANNAN COMPLEX

W GlcNAc W GkNAc OYul 0 Man A 0.1 + Salk AcM

HIGH MANNOSE & HIGH MANNOSE

Figure 8. Comparison of yeast and mammalian glycosylation. Schematic diagram to compare the structures of oligosaccharide side chains of glycoproteins produced in yeast and mammalian cells. therefore expression of a protein in a heterologous type oligosaccharides. (N-linked glycosylation is system will almost certainly result in a product with reviewed in reference 221). In contrast, Sxerevisiae different modifications from the native material. does not trim back the mannose residues as it lacks This may affect the function or immunogenicity of the Golgi mannosidases present in higher eukary- the protein. It is interesting to note that differences otes. Instead, elongation of the chain may take place in glycosylation of tPA produced in either Chinese through a stepwise addition of further mannose resi- hamster ovary or murine cell lines led to differences dues. These additional mannose units comprise the in the kinetics of its fibrin-dependent activation of outer chain which may be up to 75 residues long, plasminogen.280 with many branch chains. (For a review of protein Oligosaccharides may be either N-linked to glycosylation in yeast see reference 225). asparagine or 0-linked to serine or threonine resi- Many heterologous glycoproteins have been dues. 0-linked oligosaccharides synthesized by secreted from yeast, including Aspergillus gluco- yeast are very different from those of higher arnylase,l8, ~omatostatin,'~~human a-l-anti- eukaryotes, being composed only of mannose resi- trypsin,262porcine urokina~e?'~human erythropo- dues. Many higher eukaryotic proteins have sialy- etinlM and Trichoderma reesei endoglucanase 1 .388 lated 0-linked chains of the mucin-type. N-linked The addition of both N- and 0-linked oligosac- glycosylation in yeast and higher eukaryotes is more charides has been reported (e.g. Aspergillus gluco- conserved, and entails the addition in the ER of a amylaselg6).Analysis has revealed that not all S. core oligosaccharide unit comprising two N-acetyl- cerevisiae N-linked oligosaccharides contain the glucosamine (GlcNAc), nine mannose (Man) and outer chain of mannose residues. Addition of the three glucose residues. The glucose residues are -outer chain to heterologous proteins is regarded as subsequently trimmed from the side chain and one 'hyperglycosylation' because it results in more mannose is also removed. These steps are common extensive glycosylation than is found in higher to yeast, plants and higher eukaryotes. eukaryotic glycoproteins. Additionally, it should be Processing of the core oligosaccharide unit takes noted that glycosylation can be very heterogeneous, place in the Golgi and at this stage there is divergence giving rise to a mixed product with a varying degree between yeast and other eukaryotes (Figure 8). In of oligosaccharide attached. animal cells further mannose residues are removed Some foreign proteins are fortuitously glycosy- and additional sugars may be added. The resultant lated when secreted from yeast. This problem is oligosaccharide side chains are of three types: (i) more likely to occur with proteins from bacteria (for high-mannose (GlcNAc,Man,,), (ii) complex, example see references 307 and 316), in which there which comprises a core of GlcNAc,Man, plus is no glycosylation, or with proteins which are not additional residues including GlcNAc, galactose normally secreted. Interleukin (1L)-la and lp are (Gal), fucose and sialic acid, and (iii) hybrid, contain- naturally-secreted proteins, but do not pass through ing features of both high-mannose and complex- the normal secretory pathway in mammalian cells, 450 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE and are therefore not glycosylated. However, when Protein folding and transport secreted from yeast using a signal sequence, both proteins are glycosylated. Although this has no si - Folding of secreted proteins occurs in the ER and nificant effect on the biological activity of IL- 1a,2 4 involves accessory proteins such as BiP (heavy chain the activity of IL-I p is reduced fi~e-fold.'~~ binding rotein, the product of the KAR2 or GRP78 Although many glycoproteins produced in yeast gene27233g),and protein disulphide isomerase.126 are active, yeast glycosylation, especially hyper- Nascent proteins bind to BiP co-translocationally glycosylation, may give rise to problems, depend- and are released upon folding, assembly and glyco- ing on the intended use for the protein. In some sylation; malfolded proteins bind permanently and instances, extensive glycosylation may inhibit are retained in the ER. The assemblyof an oligomeric reactivity with antibodies, as was the case for EBV bacterial enterotoxoid in S.cerevisiae has recently gp350 secreted from S.cerevisi~e.~~~This is been shown to be dependent on BiP, which acts as a obviously an undesirable effect for potential vaccine molecular chaperone.333During the secretion of antigens. Furthermore, al,3-linked mannose units, foreign proteins, problems might arise either from which occur in large numbers in the outer chain, saturation of these accessory proteins or from their appear to be immunogenic'6and thus yeast-derived inability to aid the folding of heterologous proteins. glycoproteins may be unsuitable as therapeutics. Glycosylationof a protein can aid both generation Problems with yeast glycosylation of foreign pro- of the correct conformation and passage of the teins may be circumvented by mutating the glycosy- molecule through the secretory pathway. Inhibition lation site(s), as was done in the case of SCUPA~'~to of glycosylation with tunicamycin results in the yield a fully-active, non-immunogenic product. intracellular accumulation of inactive invertase.136 Alternatively, the use of glycosylation mutants At lower temperatures, unglycosylated protein may allows the production of more homogeneous pro- be secreted: at 25"C, 50% of unglycosylated inver- teins with limited glycosylation. Mutants in mannan tase is secreted.*l6Similarly, active, unglycosylated biosynthesis (mnn ) may be useful for the production acid phosphatase can be secreted from tunicamycin- of heterologous proteins; mnn9 mutants do not add treated cells at 20°C or 25"C, but not at 30°C.259 the extensive outer chain of mannose how- Unglycosylated glycoproteins are transported only ever immunogenic terminal a l ,3 mannose linkages if they achieve the correct conformation and the are still present. Use of the mnnl mnn9 strain, which lower temperatureallows this to happen. Progressive additionally lacks these residues, may enable the elimination, by mutation, of each of the 12 glyco- production of non-immunogenic, non-hyperglyco- sylation sites of acid phosphatase resulted in increas- sylated proteins. However some mnn mutants, e.g. ing irreversible retention of the malfolded protein in mnn9, exhibit slower growth and greater osmotic the ER as more sites were removed.2wAt lower sensitivity than wild-type cells. Sledziewski et temperatures under-glycosylated or unglycosylated created a strain in which MNN9 expression was protein could be secreted. temperature-regulated, using the a2 repressor sys- Sato et studied the expression of a series of tem in a MATa sir.?' strain (see 'Transcriptional human salivary a-amylase mutants which either promoters and terminators'). Cultures were grown lacked regions encoding cysteine residues or con- to high density at the permissive temperature for tained mutations at cysteine codons. The preven- MNN9 expression (25"C), then switched to the non- tion of the possible formation of a disulphide bond permissive temperature (35°C). Glycoproteins pro- abolished secretion. Elimination of either of the duced at the higher temperature were shown to have two glycosylation sites did not affect secretion and the shorter, homogeneous mannose side chains indeed a-amylase is secreted from tunicamycin- characteristic of mnn9 mutants. treated cells in a fully active form. Thus, at least for Some 'super-secreting' mutants are also hyper- a-amylase, the unique conformation of the protein glycosylation-deficient and may be suitable host appears to be much more important than glyco- strains for glycoprotein production. pmrl mutants sylation for secretion in yeast. This phenomenon (previously called sscl; see below) possess a defec- indicates that attempts to secrete protein fragments tive Ca2+ ATPase ion pump and their growth is or fusions could lead to intracellular accumulation ~alcium-dependent.~~'The mutation is thought to if normal folding and disulphide bond formation cause secreted proteins to by-pass the Golgi. Pro- cannot take place. teins secreted from this strain are core-glycosylated In addition to requiring successful targetting and only. folding in the ER, a foreign protein also has to Pass FOREIGN GENE EXPRESSION IN YEAST. A REVIEW 45 1 through the secretory organelles in order to be vivo reconstruction system provides a useful new released into the culture medium. Transport from approach for the study of signal transduction the ER to the Golgi has often been shown to berate- pathways. limiting. Hepatitis B virus large surface protein is The presence of the cell wall complicates the retained in the ER, provoking the enlargement of secretion process in yeast. Permeability may be a this ~rganelle.~’This retention may be due to mal- limiting factor and it is notable that most success has folding or to a specific retention signal. a-l-anti- been with very small proteins. Some proteins have trypsinZ6’and erythropoetinIo6are also retained in been reported to be localized mainly in the cell wall the ER. Soybean proglycinin, although expressed when secreted from S.cerevisiae, e.g. a-IFN4” (166 to high levels and correctly processed, was mostly amino acids, 20kDa). Although it has been insoluble and accumulated in Golgi-like struc- reported that only molecules with a molecular mass t~re.~~~Insolubility was due to interaction of the below 760 are able to diffuse freely through the cell acidic re ion of the polypeptide with cellular com- wall, a number of very lar e proteins such as EBV ponents!86 A Golgi or post-Golgi bottleneck was membrane glycoprotein3F (842 amino acids, also postulated to re resent a major obstacle in the approximately 400 kDa) and cellobi~hydrolase~~~ secretion of IGF-I.38 Retained material may have (up to 200 kDa) have been shown to be capable of been malfolded and this is likely to be a common passing through the cell wall and therefore this reason for intracellular accumulation of proteins property is not related simply to the size of the within the secretory pathway. molecule. It may be that there are a few large holes in A blockage or bottleneck in the secretory pathway the cell wall with the average pore size being small. It caused by foreign protein secretion may also inter- is difficult to draw conclusions from observations of fere with the secretion of host proteins. This was permeability since many other factors, including observed for acid phosphatase during the secretion strain, growth phase and composition of the medium, of tPA’69and may result in toxicity. may have an effect on cell wall porosity (reviewed in Correct folding, assembly and transport are reference 91). Additionally, glycosylation and the especially important in the production of multimeric charge on a protein are thought to affect its passage proteins. The first multimeric protein to be secreted through the cell wall. mnn9 mutants have been from yeast was a mouse-human chimaeric antibody. reported to have increased porosity and release Co-expression of immunoglobulin heavy and light invertase octamers, which are normally retained.373 chains resulted in the secretion of properly folded krel mutants, which have altered (1,6)-P-D-glucan and assembled antibody.’” Both whole antibody in the cell wall, over-secrete secreted yeast proteins, and Fab fragments were functional. The pentameric possibly due to the cell wall being more leaky.51 Torpedo californica nicotinic acetylcholine receptor with a stoichiometry a$yG has also been pro- Proteolytic processing duced in east by co-expression of the four sub- units. ‘9994’y These integral membrane proteins There are two ways in which proteolytic process- entered the secretory pathway and were processed ing is relevant to heterologous protein secretion in and glycosylated. However, no functional receptor yeast. Firstly, correct processing of the signal pep- was detected, possibly due to improper folding or tide or prepro region must take place so that the assembly. mature product is secreted. Secondly, fortuitous, Membrane proteins often cause problems when undesirable processing events may occur as a result secreted due to non-specific insertion into intra- of cleavage by processing proteases. cellular membranes (see ‘Physiology of foreign gene Yeast signal peptidase (SPase) is a polypeptide expression’). EBV membrane glycoprotein gp350 complex which includes the SECIZ gene product was highly toxic, but could be secreted in a mem- and a glycopr~tein.~’~Although eukaryotic SPases brane anchor-minus form.335Nevertheless, some and the E.coli SPase I are disparate, it is interesting membrane proteins have been successfullyproduced to note that homologous overproduction of the in yeast. The human P,-adrenergic rece tor was co- E.coli protein resulted in increased efficiencies of expressed with a mammalian G protein!I4 Coupling export and maturation of two poorly-processed hy- of these components to each other and to down- brid secretory proteins.392This indicates that, at stream effectors of the yeast mating signal transduc- least in E.coli , the availability of the SPase can be a tion pathway was demonstrated, indicating that limiting factor. Indeed, S.cerevisiae strains carry- correct folding and targetting took place. This in ing multiple copies of the wild-type PH05 gene 452 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE accumulate unprocessed precursor, suggesting that aspartyl protease ( YAP3) has been identified which there is saturation of some com onent of the allows KEX2-independent processing of the a- secretory pathway, possibly SPase. IR factor precursor.Io2 This could be over-expressed Deletion of the SPase cleavage site in yeast acid when KEX2 processing was inefficient and limiting. phosphatase leads to unprocessed, core glycosylated Over-expression of STE13 could also improve protein which accumulates inside the cell.”’ How- inefficient processing; wild-type cells carrying mul- ever, even where processing of the signal does take tiple copies of the MFal gene produce mainly place, cleavage may be aberrant, giving rise to incompletely processed a-factor, indicating that the heterogeneous product. 64% of human IFN dipeptidyl aminopeptidase is rate-limiting.204 secreted from yeast using its own signal was prop- In addition to inefficient proteolytic processing, erly processed, but 36% contained an additional problems may also be caused by aberrant processing three amino acids of the pre sequence.’73Further- at internal sites in the protein. In the extreme case more, 90% of the total IFN produced was not of P-endorphin, no complete mature protein was secreted and this intracellular material also included secreted into the medium;34 two trypsin-like a third form which retained eight amino acids of pre cleavage sites were observed after internal lysine sequence. It is unlikely that molecules which residues. Use of the vacuolar protease mutant retain part or all of the pre sequence will be secreted pep4-3 did not reduce the degradation, suggesting since the hydrophobic core may be retained in the that proteolysis was taking place durin passage membrane. through the normal secretory pathway?’ Internal The processing of the prepro sequence from a- processing was also a problem in the expression of factor has been described above and is a more com- hPTH in which cleavage, probably by the KEX2 plex process involving the KEX2 and STE13 gene gene roduct, was observed after two basic resi- products in addition to signal peptidase (reviewed in dues.” A mutant form of hPTH which lacked one reference 49). The KEX2 protease cleaves on the of the basic residues was no longer subject to carboxyl side of the dibasic residues, Lys-Arg and internal proteolytic pro~essing.~~’The mutant form Arg-Arg. In a fusion of the a-factor prepro and a retained full biological activity and was produced in heterologous protein, these residues are at the junc- significantly higher yield. Differences in proteolysis tion, and cleavage by KEX2 liberates the hetero- between strains and/or growth conditions may exist logous protein from the leader region. The Glu/ since two reports of secretion of GM-CSF identi- Asp-Ala spacer residues, which provide a hydro- fied different aberrant processing events. Miyajima philic environment at the KEX2 cleavage site can be et al.257reported cleavage after the arginine residue dispensed with in certain cases, circumventing the at position 4 of the mature protein. Price et problem of incomplete STEl3 processing, e.g. for however observed both the full-length mature hEGF4* and al-IFN.350However, removal of the species and the product of cleavage after the proline spacer peptide does sometimes lead to a failure in at position 2 of the mature protein. Differences were KEX2 processing, as observed for interleukin-6 (IL- also apparent in the efficiency of a-factor leader pro- 6)ls2where the presence of a proline residue on the cessing. This indicates that it may be possible to carboxyl side of the cleavage site inhibited cleavage. eliminate or to minimize internal cleavage of heter- However, accurate recognition and cleavage ologous proteins by optimizing growth conditions occurred when the construct was modified to include and selecting the best strain. alanine N-terminal to the proline. Expression of the An analysis of secreted hirudin (hir) revealed full- modified gene fusion had then to be carried out in a length hir65, but also two C-terminally degraded stel3 mutant, to avoid trimming at the N-terminus products, hir64 and hi1-63.~~~Use of the protease of IL-6 by the STE13 protease. mutantsprcl and kexl showed that this degradation Another solution to the problem ofinefficient pro- was due both to yscY and ysca activities. Similarly, cessing is to over-express the processing enzyme secreted EGF was found to be C-terminally genes. This approach was successfully employed in trimmed .7471 38 the expression of transforming growth factor a Mutants in the gene for the extracellular protease (TGFa).I9 Inclusion of the KEX2 gene on the same SKI5 were shown to give higher yields of secreted multi-copy plasmid as the TGFa gene eliminated the yeast proteins due to decreased degradation in the presence of unprocessed forms of the a-factor leader- medium.51 TGFa fusion protein and resulted in increased levels An interesting developmentin protein production of secreted TGFa. Additionally, a novel Sxerevisiae has been the exploitation of yeast processing FOREIGN GENE EXPRESSION IN YEAST A REVIEW 453 enzymes to cleave heterologous precursors. Pro- enabled identification of mutant forms of scuPA insulin is converted to insulin as the result of cleavage with either decreased or increased activity com- at two dibasic sequences separated by a spacer pep- pared to the parental form. The assay uses fibrin- tide. Theseprocessingreactionscan becarried out by agar and colonies are scored by measuring zones of the KEX2 protease, and various forms ofproinsulin/ clearing. insulin have been secreted from yeast.376Human pro- Other workers have employed a similar approach albumin has also been shown to be processed after to identify super-secreting strains. A plasminogen- the Arg-Arg sequence by KEX2 to yield mature casein assay for secreted tPA was used to isolate albumin." Aspergillus glucoamylase secreted from secretor strains;I4' previous attempts to secrete tPA yeast using its own signal sequence was shown to be from yeast had not been successful.'69~231A general cleaved both by SPase and by KEX2, which screen for secreted proteins based on a visual anti- removed an additional six amino acids from the N- body precipitation assay has been described.354 terminus.Ig6This yielded a product with the same Repeated rounds of mutation and selection N-terminus as that of the Aspergillus enzyme and enhanced the secretion of HSA six-fold and the showed yeast to be capable of this additional resultant strains were also able to produce higher processing step. levels of internally-expressed a- 1-antitrypsin and human plasminogen activator inhibitor 2. This screen is suitable for any protein for which anti- Strategies to improve secretion bodies are abundantly available. Multiple rounds of mutagenesis and selection were also used to isolate a Several Sxerevisiae strains with a 'super-secreting' strain which secreted 70-fold more endo lucanase I phenotype have been isolated by screening for than the original wild-type parent strain. PAgain, the mutants with increased secretion of a particular mutant strain was found to secrete elevated levels of product. Such methods have yielded strains which other proteins. show a general increase in heterologous protein By expressing a gene fusion of HSA and hygro- secretion. On finding that less than 1% of the pro- mycin B phosphotransferase, Chisholm et chymosin made in yeast was secreted, Smith et selected for increased expression on the basis of the employed a mutagenesis approach coupled with a level of resistance to hygromycin B. This method rapid screening assay to isolate super-secreting was initially used to isolate strains with increased colonies. The secreted material, which was activated intracellular expression of the HSA fusion protein. by the low pH of the medium, was assayed by over- However, when cured of the intracellular expression laying the surface of the plate with a mixture of milk vector and retransformed with a HSA secretion vec- and molten agarose. The chymosin clots the milk tor, these strains also showed significant increases and the speed of appearance of the opaque clotted in secretion. Genetic analyses suggested that mul- regions and their size and intensity indicate the level tiple mutations were responsible for the observed of prochymosin secretion. Two super-secreting effects. strains in particular were identified and designated Selection may be used to identify mutants which sscl andss~2.~~~Theeffectsappeared to beadditivein are resistant to the toxic effects of a foreign protein. thedoublesscl ssc2mutant. The SSCl gene was later The slow growth rate of IGF-I-expressing cells was found to beidentical to PMRI, whichencodesaCa'+ exploited by Shuster et who isolated fast- ATPase3I5(see above). The pmrl mutation signifi- growing IGF-I-resistant mutants which gradually cantly increased the secreted levels of prochymosin, accumulated in a population of IGF-I-expressing bovine rowth hormone and scuPA by five- to 50- cells under selection. Mutations at a single locus, fold.357,984 However secretion of a-1-antitrypsin, designated HPXl (for Heterologous Protein which was efficientlyexported in the wild-type strain, expression) were found to confer both resistance was not further improved, indicating that the pmrl to the toxic effects of IGF-I and its increased mutation may be most effective at improving the production. secretion efficiency of proteins that are secreted In summary, the screening approach has fre- poorly by wild-type cells. quently proved successful in the isolation of super- In addition to using colony assay screens to find secreting strains, even in cases where the initial yield the most efficient secretors, Moir and DavidowZ6' was high. Therefore, for industrial applications described a screening procedure which involved in when a high yield is required, this approach deserves vitro mutagenesis of scuPA genes on plasmids. This consideration. 454 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE Table 6. Comparison of the expression of two genes in Y.lipolyticu are both industrial yeasts which have E.coli, Baculovirus, S.cerevisiae and P.pastoris. been examined largely because of their capacity for high-level secretion. K.luctis is similar to S.cerevisiue Expression level (YO) and has well-developed molecular genetics. Unlike Expression system Fragment C Pertactin the other systems discussed here it has the advant-

~~~~~~ age of highly stable episomal vectors. Finally the E.coli 24241a 30242a distantly-related fission yeast, S.pombe, has very advanced genetics but its use in foreign gene Baculovirus lo#,@" > 40* expression has mainly been in isolating and studying homologous mammalian genes, rather than protein Xcerevisiae GAL7 2-3307 0.1306 production. The following sections will briefly P.pastoris 284,73 107,306 review these non-Saccharomyces heterologous expression systems. A summary of the various pro- #Estimate; yield variable and greatly reduced on scale-up. teins that have been expressed in each system is *Estimate; yield variable and greatly reduced on scale-up; I.G. given in Table 7. Charles, personal communication. gScale-up without loss of yield to > 12 g/l. 7Scale-up without loss of yield to >4 g/l. Pichia pastoris The methylotroph P.pustoris has now been used EXPRESSION IN NON-SACCHAROMYCES to express high levels of many different intracellular YEASTS and secreted proteins. Strains were originally devel- oped for the large-scale, high-yield production of Introduction single-cell rotein using defined medium containing In some ways S.cerevisiae could be regarded as a methanol!' This fermentation technology subse- non-optimal host for the large-scale production of quently provided the basis for an efficient expression foreign proteins due to drawbacks such as the lack system which uses the promoter from the tightly- of very strong, tightly-regulated promoters, the regulated AOXI gene.*' AOXI encodes alcohol oxi- need for fed-batch fermentation to attain high-cell dase, which catalyses the first step in the assimi- densities, and hyperglycosylation. Although these lation of methanol, and can be induced to give levels have been addressed by exploiting its sophisticated of up to 30% of t.c.p. by the addition of methanol.76 molecular genetics, another important approach Since P.pastorishasno native plasmids (other than has been to develop expression systems in other linear DNAs, K. Sreekrishna, personal communi- yeasts. Most of these alternative systems are based cation) expression vectors designed for chromo- on commercially-important yeasts that have been somal integration have been developed. These selected for their favourable growth characteristics vectors have used the HIS4 gene for selection, but a at industrial scale, or on yeasts which have other limited number of alternative selection markers are favourable intrinsic properties (e.g. high-level now available, e.g. ARG4 from S.cerevisiaeso or secretion). P.pustoris (J. Cregg, personal communication), the The most extensively developed system is based Tn903 G418-resistance gene (K. Sreekrishna per- on Pichiapastoris. An efficient and tightly-regulated sonal communication), and the S.cerevisiae SUC2 promoter coupled with very straightforward tech- gene361which can be used as a dominant marker since niques for high-biomass cultivation make this a P.pastoris cannot use sucrose as the sole carbon powerful expression system. It is currently the source. Multi-copy episomal vectors containing the simplest of eukaryotic systems to scale up, and there P.pastoris ARS sequences, PARS1 and PARS2, are now several comparative studies suggesting it have also been con~tructed.~~However, for maxi- can be used to avoid limitations on transcription mum stability, particularly in large-scale inductions, which are sometimes encountered with S.cerevisiae the expression cassette has been transplaced into (see Table 6). Hunsenula polymorphu has similar AOXI, or integrated by single cross-over into AOXI properties and there are some promising examples or HIS4 (see Figure 9). It is worth noting that, using of its use. A potential problem however, especially HIS4 selection, HIS4 integrants are the least stable with toxic products, may be that under the con- type of transformant since recombination can result ditions normally used for high-density growth, in excision of the expression cassette leaving behind expression is significantly derepressed. K.luctis and a wild-type marker. Although transplacement into FOREIGN GENE EXPRESSION IN YEAST A REVIEW 455 AOXl results in gene disruption, transformants are differences between MutS and Mut' strains during still capable of slow growth on methanol due to the induction may in some instances influence the presence of a second alcohol oxidase gene, AOX2, expression of foreign genes. which is less well expressed." The abundance of alcohol oxidase led to the The unusually high level of alcohol oxidase assumption that single-copy integrants would yield together with evidence for transcriptional regu- sufficient levels of foreign proteins using the AOXl lationlo7 suggested a very powerful and efficiently promoter. However, with single-copy vectors it has regulated promoter suitable for foreign gene not been possible to obtain yields of heterolo ous expression. This was first tested by isolating the proteins that are as high as alcohol ~xidase.'~~~,~" AOXl promoter, and also that from theco-regulated This is not due to the presence of DAS elements since, dihydroxyacetone synthase ene (DHAS), and unlike the S.cerevisiae PGK gene2s3,replacement of fusing them to the lacZ gene 4' In single-copy the the AOXI coding region with foreign sequences does AOXl-lacZ hybrid gene was tightly repressed by not affect mRNA levels73 (R. Buckholz personal glucose or glycerol and was efficiently induced by communication). Rather, higher levels of alcohol methanol; with multi-copy PARS vectors some oxidase are probably due to its exceptional proteo- expression in glycerol occurred. The AOXl pro- lytic stability which may be enhanced as a result of moter has subsequently been used to produce a sequestration to peroxisomes. Multi-copy integrant variety of foreign proteins. Promoters from several strains are now used for increased yields of foreign other P.pastoris genes have been isolated, e.g. from proteins. These can be obtained usin vectors con- the constitutive GAP and PGK genes, and from the taining multiple expression cassettes39 or, for very AOX2 gene,*' but there are no published examples high copy number, by screening for multiple inte- of their use. gration event^.^^.^^,^" Surprisingly, very high copy A number of factors may potentially affect number integrants can be isolated from transform- expression levels using P.pastoris integration vec- ations using DNA fragments designed for single- tors. In particular, disruption of AOXl by trans- copy transplacement. Sreekrishna et observed placement might be expected to have important a high degree of variation (from 1-30% t.c.p) in the physiological consequences which may influence level of tumour necrosis factor (TNF) expressed in foreign gene expression. During induction aoxl different MutS transformants and found this was transformants do not simultaneously produce high due to differences in gene copy number. levels of alcohol oxidase and heterologous protein, The mechanism of this 'multi-copy transplace- and they also grow more slowly ('rnethanol-utilization ment' was initially unclear and it was not apparent slow', MutS), and have a much lower 0, demand whether this was a general phenomenon, nor than wild-type (Mut') strains. Indeed, in the first whether high-level expression would invariably report of foreign gene expression in Pgastoris, a occur as a result. However, multi-copy transplace- higher proportion of immunogenic HBsAg particles ment has now been observed in several other were produced in a Muts host compared to Mut', instance^.^^,'^^^" The use of such strains has fre- although expression levels were similar.8' This was quently resulted in remarkably high yields which attributed to one or more events during assembly compare very favourably with other expression sys- being rate-limiting in fast-growing cells, though there tems (see Table 6). These studies also show that is evidence that particle maturation in Xcerevisiae multi-copy transplacement strains are stable during occurs during protein extracti~n.~~The parameters high-density growth and induction. The clonal vari- affecting foreign gene expression in P.pastoris were ation in vector copy number and protein yield investigated in a study using tetanus toxin fragment among transplacement transformants expressing C.73In a direct comparison, similar amounts were pertactin is illustrated in Figure 10. produced in MutS and Mut' hosts, even at levels Multi-copy transplacement occurs at a variable approaching 28% t.c.p. Expression levels were also frequency, and is observed in about 1-10% of MutS independent of the site (HIS4 versus AOXl) and transform ant^.^^^^^^^" To understand the mechan- type (single versus double cross-over) of integration. ism involved and the factors controlling this, Clare However, in another study, a MutS integrant et a1.73carried out a detailed DNA analysis of frag- expressed Bordetella pertussis pertactin at 2% t.c.p. ment C-vector transformants. An interesting find- in shake flasks and 10% t.c.p. in fermenters, while a ing was that 75-95% of transformants had not Mut' strain gave 54% t.c.p. in shake-flasks or undergone a transplacement event (i.e. were Mut') fermenter~.~'~Thus, the significant physiological but were single or multiple integrations at AOXl or Table 7. Production of foreign proteins in non-Saccharomyces yeasts.

Yeast Protein” Locationb Promoter‘ Reference

Pichia pastoris P-galactosidase I AOXl, DHAS 380 HBsAg I AOX 1 81 Tetanus toxin fragment C I AOX 1 73 Pertactin I AOX 1 306 TNF I AOX 1 359 Streptokinase I AOX 1 154a SOD I AOX I 43,375 HIVgpl20 1, s AOX 1 C.A.S., R. Buckholz, unpublished results S.C.invertase AOX 1 381 Bovine lysozyme AOX 1 97 Human EGF AOX 1 43 Murine EGF AOX 1 74 Aprotinin AOX 1 375 HSA AOX 1 K. Sreekrishna, personal communication

Hansenula polymorpha P-lactamase 1, s MOX, FMD, DAS 198 HBsAg PERI MOX, FMD 340,197 PreSl -S2-HBsAg PERI MOX 197 a-galactosidase S MOX 115 Glucoamy lase S FMD 137 HSA S MOX 174 S.C.invertase S MOX 198

Kluy veromyces lactis Prochymosin S LAC4 39 I IL-IP S S.c.PHO5,S.c.PGK 119 HSA S LAC4,S.c.PHOS,S.c.PGK 120 HSA-CD4 S S.c.PGK R. Fleer, personal communication Schw.0. a-amylase S Homologous 368 tPA S ? 41 1 TIMP S ? 41 1

Yarrowia lipoly tica P-galactosidase LEU2 134 S.C.invertase XPR2 270 Bovine prochymosin XPR2, LEU2 124 Porcine IFN XPR2 164.271

Schizosaccharomyces Polyoma middle-T Ag I S.c.PGK 27 pombe P-galactosidase I 54/ 1, fbp, adh, GRE, CaMV35S 224,176,287 CAT I nmtl, HCGa, CMV', SV40*,GRE 249,379,287 Human epoxide hydrolase I adh 192 Factor XIIIa I adh 45 IBD virus VP3 I adh,S.c.ADHl 194 E.coli P-glucuronidase I CaMV35S 289 Single-chain Ab I adh 88 Bacterio-opsin PLM adh 166 STPl glucose transporter PLM adh 329 S.C.invertase PERI Homologous 263 S.diu. glucoamylase PERI? Homologous 112 S.C.a-mannosidase CWALL? Homologous 226 S.C.exoglucanase CWALL? Homologous 226 S.C.endochitinase CWALL? Homologous 226 Antithrombin 111 S S.c.ADH1,S.c.CYCl 46 Schw.0. a-amylase S Homologous 368"

"S.C.,Saccharomyces cerevisiae; Schw.o., Schwanniomyces occidentalis; S.dia., Saccharomyces diastaticus. bLocation of expressed protein: I, intracellular; PERI, periplasmic; PLM, plasmamembrane; CWALL, cell wall; S, secreted. 'Promoters given are native to the organism except: S.C.,Saccharomyces cerevisiae; *, viral; homologous, homologous to the gene expressed; GRE, glucocorticoid response elements; HCGa, human chorionic gonadotrophin a. 458 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE

3’AOXl

hr

A 2 3’AOXl -Chi

cut with StuI single croiso~winto HIS 4 IAOXI’I IMUT+) C 1

hr Figure 9. Plasmid map of the P.pustoris expression vector pPIC3 showing the three different modes of chromosomal integration. (A) Digestion with BgnI yields a fragment with ends that are homologous to the 5’ and 3’ regions of AOXl that is targetted to transplace into AOXl. (B) Linearizing with Sac1 targets integration by single cross-over into AOXl. (C) Linearizing with SruI targets single cross-over integration into HIS4.

HIS4, or lacked vector sequences and were presum- For the routine isolation of high-copy integrants, ably HIS4 gene convertants. The multi-copy trans- mass screening methods based on colony hybridiz- formants predominantly contained head-to-tail ation have been (Figure 10). More recently, tandem arrays of the transplacing fragment located vectors containing the G418-resistance marker have at AOXZ. Both these and the other structures been used to identify high-copy integrants by resist- observed could be explained by intramolecular ance to increasing concentrations of the drug (K. ligation of transplacement cassettes in vivo, prior to Sreekrishna, C.A.S.,unpublished results). Thus, the repeated single cross-over integration into AOXZ, most efficient procedure for isolating high-copy HIS4 or into previously transplaced vector. Inter- integrants is probably to use these vectors in single- molecular ligation also occurred at low frequency cross-over integrations and to select clones, from since integrants containing the entire vector or amongst primary His+ transformants, that are head-to-head repeats were also found. resistant to high concentrations of G418 (e.g. 2 mg/ This mechanism of multi-copy formation (i.e. ml). Multi-copy MutS clones can be obtained by repeated single cross-overs of circularized trans- using a strain carrying a disrupted copy of AOXZ placement cassettes) suggests that DNA fragments (e.g. KM7180). designed for targetted single cross-over integration With some foreign proteins there is a direct corre- rather than transplacement could be used to gener- lation between gene dosage and expression level, ate multi-copy transformants more readily, since although the yield per expression unit is usually higher transformation frequencies can be achieved. reducedat very highcopy number. Thiscan beclearly 1 2 3 4 5 678 9 10 11 12 a b

C d e 1 23 4 567

* Figure 10. Screening for high copy number Pichia integrants. Mut' transformants of a pPIC3 vector expressing pertactin were isolated prior to screening for high copy number.'" (A) DNA dot blot of intact whole cells which were grown in microtitre wells then lysed on nitrocellulose. Most dots correspond to single-copy transformants (e.g. al) while some are multi-copy (e.g. a4). (B) Western blot showing variation of pertactin expression levels among transformants showing a correlation with vector copy number. Track (1) native pertactin, (2) single-copy Pichiu transformant, (3) 13-copy,(4) 3O-copy, (5) 12-copy,(6) 21-copy, (7) XcerevisiaepWYG7-based vector. 460 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE

1 2 3 4 5 6 7 8 9 10 11 121314 copy no.

Figure 11. The correlation between gene dosage and the level of expression of tetanus toxin fragment C in Mut' P.pustoris transformants. Closed circles correspond to high density inductions in the fermenter, open circles represent shake-flask inductions.

seen in Figure 1 1, which shows fragment C expres- they are also mannosylated to a lesser extent than sion at varying copy number. The nature of the gene those of S. cerevisiae-about 35% of N-linked and its protein product may determine the gene oligosaccharides having less than 14 mannose dosage at which expression becomes limited. For units.'49 This suggests that P.pastoris may be a toxic proteins increasing gene dosage will not necess- better host for the production of heterologous arily improve yields, e.g. increasing the copy number glycoproteins, since extensive outer chain structures from one to three did not result in higher levels of the can alter the enzymic or immunogenic properties of secreted enzyme bovine lysozyme, although the the product (see 'Secretion of foreign proteins'). amount of mRNA was proportionally increased.375 However, hyperglycosylation of certain foreign Indeed, the secretion level was reduced and the proteins secreted from P.pastoris has been observed enzyme accumulated intracellularly, suggesting a (e.g. HIV gp120, C.A.S., unpublished results). blockage of the secretory pathway. Several other proteins have been secreted at high In addition to efficient expression of intracellular level from P.pastoris, including bovine lysozyme products, P.pastoris is also capable of secreting high (0.55 g/197), HSA (3 g/l, K. Sreekrishna, personal levels of foreign proteins. This was first demon- communication), aprotinin (0.93 g/1375), hEGF strated using the S.cerevisiae SUC2 gene product, (0-4 g/143)and mEGF (0.45 In some of these invertase, which accumulated to 2.5 g/1 in the examples the corresponding signal peptide was medium.38' The P.pastoris-secreted product had used. The prepro leader sequence of Xcerevisiae a- much shorter N-linked carbohydrate side chains factor has also been used, and is efficiently processed than that of S.cerevisiae (8-14 mannose units com- by a KEX2 protease-like activity in P.past~ris.~~,~~~ pared to >50). In addition, these did not contain Interestingly, Thill et have reported that S. the terminal a 1,3-mannose linkages379awhich are cerevisiae invertase is secreted by Pichia with much present in Scerevisiae glyco roteins and have been faster kinetics using the a-factor prepro sequence shown to be immunogenic.'F Studies of bulk endo- than with its own secretion signal. This may be an genous glycoproteins from P.pastoris revealed that important factor in determining the gene dosage at FOREIGN GENE EXPRESSION IN YEAST A REVIEW 46 1 which secretion of a particular protein becomes LEU2 genes, although integration has subsequently limited and may also influence the glycosylation been shown to The bacterial plasmid pattern of the final product (J. Tschopp, personal sequences may promote autonomous replication communication). since the 2p replication ori in probably does not A problem that has occurred with several proteins function in X.polymorpha!' The URA3 vector, secreted by P.pastoris is proteolytic instability in the YIPS, gave a low frequency of very unstable trans- culture medium, e.g. mEGF,74 hEGF,43 HSA (K. formants that occasionally gave rise to stable Sreekrishna, personal communication), HIV gp120 derivatives in which the plasmid had randomly (C.A.S., unpublished results). This can be mini- integrated.302The transformation frequency was mized by altering the pH of the culture medium significantly increased by the presence of two inde- upon induction (e.g. raising to pH 6~0,~~or lowering pendently isolated chromosomal DNA sequences, to pH 3.0, G. Thill, personal communication), or by HARSl and HARSZ, which also conferred unstable using a strain deleted in the PEP4 homologue (M. autonomous replication. Following prolonged Gleeson, personal communication). In the long propagation of such transformants in non-selective term it would be desirable to identify the specific medium, stable Ura+ segregants could be isolated extracellular proteases responsible in order to which contained multiple (up to 79,tandemly inte- construct strains lacking these activities. grated copies of the vector. Although the mech- An advantage of the Pichia system is the ease of anism of multi-copy integration is unclear, this high-density growth and scale-up without any procedure can be used to generate stable, high-copy reduction of specific productivity.8 This feature is strains for the expression of foreign ~r0teins.I~~The especially advantageous for secreted products since use of the dominant G418-resistance marker in the concentration in the medium increases with cell H.polymorpha has also been des~ribed.'~,'~~ density. In fact shake-flask inductions are normally The H.polymorpha system has been used for the sub-optimal, and there is often a large improvement efficient expression and assembly of HBsAg par- in productivity using controlled fermenter~~~,~~,~~ticles containing preS2 sequence^.^^ More than probably due to the high 0, demand of the 95% of the protein produced could be recovered as organism. particles, over half of which were secreted to the periplasm and could be released into the medium by Hansenula polymorpha inducing cells in the presence of P-glucanase. The overall yield in these permeabilized cells was several The expression system developed in the methylo- fold higher than in untreated cells, suggesting that troph, H.polymorpha,is similar to that of P.pastoris. the intracellular accumulation of articles inhibited The gene encoding the peroxisomally-located further synthesis. Janowicz et a1.l' produced mixed enzyme, methanol oxidase (MOX), has been iso- particles containing preS 1-S2-HBsAg and HBsAg lated229and the promoter used to express foreign by co-expression with two promoters (from the genes. As with P.pastoris AOXI, the MOX gene is methanol-regulated formate dehydrogenase gene, highly-expressed, giving methanol oxidase levels up FMD, and from MOX) and by selection with to 37% t.c.p.,I4, and its transcription is tightly URA3 and G418. Multiple integration of both genes regulated. One important difference, however, is was achieved using HARS vectors by the method that expression of the H.polymorpha gene is signifi- described above. By obtaining strains with different cantly derepressed during glucose limitation or in copy numbers of each gene, composite particles the absence glucose, e.g. usin substrates such as containing various ratios of preSl-S2 and S antigen glycerol, sorbitol or ribose.'0394O4 Thus, tight regu- could be produced, with a total yield of 2-8% lation of the promoter is lost in the conditions t.c.p. normally used for high-biomass fermentation^.'^^ The secreted plant enzyme, a-galactosidase has Transformation systems were developed using been produced in H.polymorphd.''SThe Sxerevisiae the LEU2 and URA3 genes from S.cerevisiae as invertase signal sequence was used, which gave selectable markers. The LEU2 vector, YEpl3, gave efficient secretion and was correctly processed. a low frequency of transformants which maintained Attempts to isolate high-copy integrants with the the plasmid autonomously, but with low mitotic YEpl3-derived expression vector, by passage of ~tabi1ity.I~~No integration was observed, even transformants in non-selective medium, resulted in with linearized vector DNA, suggesting a limited a strain containing two copies integrated at MOX homology between H.polymorpha and S.cerevisiae and one at LEU2. This low integration frequency 462 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE and copy number may be because 2p sequences pre- promoters. The difficulties in manipulation can be vent integration or are unstable when integrated,348 overcome by targetted integration of foreign DNA or because the plasmid is less stable than HARS into native kl. If conventional nuclear selection vectors and is lost before multiple integration can markers are used, e.g. LEU2, this results in linear occur. The three-copy strain secreted 42 mg/l, nuclear plasmids containing telomeres.206However, which was equivalent to about 5% t.c.p. The H. by fusing such markers to kl promoters, recombi- polymorpha-derived material was over-glycosylated nant linear plasmids which are cytoplasmic and compared to the native enzyme and had a lower stable can be generated.207*374It should be possible to specific activity. Full activity could be restored on use this system for foreign gene expression, although treatment with endoglycosidase H. the kl/k2 promoters appear to be rather weak and As with P.pastoris, H.polymorpha can be grown further development, e.g. the use of the bacterio- to high density (100-130 g/l), resulting in very high phage T7 transcription system, may be necessary. volumetric yields of secreted proteins. Using an Stable high-copy K.1acti.s expression vectors integrant containing four copies of a Schwannio- have been constructed based on the Kluyveromyces myces occidentalis glucoamylase ene, up to 1-4g/1 drosophilarum plasmid, pKDl . Although there is of secreted enzyme was obtainedB7 The yield from little sequence similarity, pKDl is organizationally an eight-copy integrant was much lower suggesting very similar to the 2p plasmid of S.cerevisiae. It a blockage of the secretory pathway at the higher gene encodes analogous replication, amplification and dosage. The secretion of several other foreign pro- segregation functions,6' which are also active in teins in H.polymorpha has been reported including K.lacti~.~~Several different types of vector based on HSA174,invertase and p-lactamase.198 pKDl have been constructed which behave simi- larly to the analogous 2p vectors.30Vectors carrying Kluyveromyces lactis just the cis-acting replication element, located near one of the inverted repeats, can be maintained in KJactis has been used in the food industry for host strains which have resident pKDl . However, many years in the production of P-galactosidase vectors containing the entire pKDl plasmid are (lactase). Thus, its large scale cultivation has been significantly more stable and can be maintained in extensively studied, and it is well accepted for the any K.lactis host strain.@ The unique EcoRl site production of proteins for human use. The ability adjacent to one of the inverted repeats can be used to to grow on cheap substrates, such as lactose and insert forei n DNA without interruption of plasmid whey, further increases its potential as a host for the functions8Such vectors are highly stable in pKDlO production of heterologous proteins, especially for strains, even in the absence of selection, although low-value products. stability is somewhat reduced in pKDl' hosts, per- Transformation systems were initially developed haps due to incompatibility or competition. These by isolating K.lactis ARS sequences, since neither the vectors are currently used for optimal foreign gene S.cerevisiae ARSI nor 2p replicates in K.la~tis.~~,~~~expression in K.lacti~."~'~~~An other reported However, as with S.cerevisiae, K.lactis ARS vec- method of producing highly stable pKD1-based tors are highly unstable and are of limited use in vectors is to transform pKDl' hosts with plasmids expression systems. A number of selection markers containing one of the inverted repeats, in addition are available for K.lactis, e.g. S.cerevisiae TRPIg4 to the desired foreign DNA. Once introduced, these and IYRA~,~'K.lactis TRP1,365 URA3346 and can recombine with resident pKDl at relatively LAC4,84and the G418-resistance gene,847362 high frequency, due to the pKDl-encoded FLP The two cytoplasmic linear plasmids, kl (8.9 kb) recombinase, giving stable recombinant vectors.402 and k2 (13.4 kb), present in killer strains of K.lactis A small number of promoters have been used in have been considered as a potential vector system. K.lactis expression vectors. The best-characterized They are stably maintained at 100-200 copies per cell K.lactis promoter is that of the LAC4 gene, en- and the regions of kl that encode killer toxin can be coding P-galactosidase, which is induced up to 100- deleted without affecting maintenance (reviewed in fold by lactose or galactose. Its regulation parallels reference 364). However, their 5'-termini are the S.cerevisiae GAL system, though there are covalently linked to protein, hindering manipulation significant differences: the KJactis GAL genes, in vitro and amplification in E.coli. Additionally, including LAC4, show no glucose repression, and these plasmids encode their own cytoplasmic tran- the GALI,7 and 10 genes are only induced five- to scription system which does not recognize nuclear ten-f~ld.~~~Some of these differences can be ascribed FOREIGN GENE EXPRESSION IN YEAST A REVIEW 463 to the fact that LAC9, the GAL4 homologue of active. Plasmid stability was significantly reduced in K.lactis, is not involved in glucose repression in IL-10 expressing cells, but this could be improved by many strains. Several S.cerevisiae promoters are replacing the constitutive promoter (S.cerevisiae active in K.lactis and have been used for foreign gene PGK) with a regulated one (S.cerevisiae PH05). expression, e.g. PGK'" and PHO5.'I9 In future, The glycosylation pattern of two other hetero- promoters from the recently isolated K.1acti.s enes logous proteins secreted in K.lactis was examined by encoding GAP344and alcohol dehydrogena~$~~>'" Yeh et al?" Variants of tPA and the tissue specific may be used. inhibitor of metalloproteinases (TIMP) were A number of studies show that K.lactis can effici- secreted using their own secretion signal or that of ently secrete foreign proteins, including prochymo- the killer toxin. In each case the secreted material sin which is only poorly secreted by S.cerevisiae. was reported to be over-glycosylated and was not Signal peptides derived from HSA, the K.lactis immunoreactiveunless treated with endo H. killer toxin a-subunit, and the prepro peptides of a- factor from both K.lactis and S.cerevisiae have been Yarrowia lipolytica used. In marked contrast to S.cerevisiae, prochymo- sin was efficiently secreted by K.lactis in a fully Y.lipolytica has been investigated for use in a soluble, acid-activatable form, using a single-co number of industrial processes, including the pro- integration vector carrying the LAC4 promoter.YJ duction of a various metabolites (e.g. citric Remarkably, about 80% of the prochymosin pro- 2-keto gl~tarate,~~~erythrit01,~~ mannit01,~~ iso- duced was secreted even when expressed without a propyl malate92),the bioconversion of alkanes and signal peptide, though the overall level was reduced. fatty acids into alcohol, and the production of single The highest levels were obtained using the native cell protein from n-paraffin~.'~~,'~~In addition, this leader peptide or the a-factor prepro sequences from yeast secretes a variety of high molecular weight either K.lactis or Xcerevisiae. The stability of the proteins including acid proteases,48 lipases,' a ribo- integrated expression cassette was sufficient to allow nuclease,@ and an alkaline extracellular protease scale-up to 41,000 litres, and the product is used (AEP).377Under optimal conditions AEP can be commercially in the manufacture of milk products. induced to levels of 1-2 g/1 and is the major compo- Other heterologous proteins have been secreted nent of the culture supernatant.377This inherent using pKDl -derived vectors. Fleer et described capacity for high-level secretion, plus the ability to the secretion of HSA using its own secretion signal, grow to high cell density at industrial scale, have or that of the K.lactis killer toxin a-subunit. The level prompted the investigation of Y.lipolytica as a host of HSA produced was highly strain-dependent and for heterologous gene expression. this was largely due to differences in stability of the Early studies of Y.lipolytica genetics were vector. Using the S.cerevisiae PGK promoter, the hampered by its low sporulation frequency and low highest-expressing strain produced about 300 mg/l spore viability. Indeed, these characteristics led to HSA in shake flasks. In high density (80-90 g/l dry its original classification as Candida lipolytica, since weight of cells), fed-batch fermentations several g/1 no sexual cycle was demonstrated until 1970.'"'3 HSA were produced from cultures of up to 1000 However, improved techniques for mating, sporu- litres. This system has also been used for the produc- lation, and ascospore recovery, combined with tion of a HSA-CD4 fusion protein designed as a improved strains derived from inbreeding pro- potential therapeutic agent for use in HIV infection grammes have led to the development of a genetic (R. Fleer, personal communication). map, comprising at least five linkage The secretion of interleukin 1p (IL- 10) by K.lactis Y.lipolytica is a dimorphic yeast, being unicellu- has also been reported, using the toxin a-subunit lar in minimal medium containing glucose or n- signal, either with or without the pro region derived hexadecane, forming mycelia in minimal medium from HSA.'I9 These signal peptides were accurately containing olive oil or casein, and giving a mixture of and efficiently processed, but only about 20% of the both formsincomplexmedium. Mutants which form product was secreted (80 mg/l in shake flasks). As in smooth colonies containing only yeast-phase cells Sxerevisiae, the K.lactis-derived IL- 1p was for- have been i~olated'~~.~~~but the molecular events tuitously glycosylated and largely inactive, though involved in the regulation of growth morphology are full activity was restored by digestion with endo- uncharacterized. glycosidaseH. A mutant form of IL-1 p which lacked Transformation of Y.lipolytica was first achieved the unique N-linked glycosylation site was also fully using two different selectable markers. Davidow 464 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE et a1.85used the homologous LEU2gene to transform of the secreted invertase remains in the periplasm, cells permeabilized by lithium acetate. Transform- preventing crossfeeding of untransformed cells. ation occurred by integration into the chromosomal The XPR2 promoter, which is tightly regulated LEU2locus via homologous recombination. Linear- by pH and by carbon and nitrogen sources~75*276 ization of the vector within the LEU2 gene resulted and the XPR2 secretion signal sequence have been in a 1000-fold increase in transformation frequency. used to produce other foreign proteins in addition to Gaillardin et ~1.l~~used vectors containing random invertase. The XPR2 gene was independently cloned Y.lipolytica genomic fragments inserted into the by three rou s and found to encode a prepro- upstreamregionoftheS.cerevisiaeLYS2genefor the enzyme.873'4727 ' ' The mature protease is produced selection of transformed sphaeroplasts. Transfor- by a series of processing events which sequentially mants commonly contained several tandemly inte- remove the presequence and two or three pro- grated copies of the vector suggesting that the regions.247The presequence is cleaved on trans- L YS2 gene was poorly expressed. Unexpectedly, no location into the endoplasmic reticulum and, after sequences capable of supporting autonomous repli- trimming of N-terminal X-Pro and X-Ala dipep- cation were isolated. Wing and Ogrydziakm5were tides, the pro sequences are then cleaved by a KEXZ also unable to isolate ARS sequences using a similar like activity, most likely encoded by the XPR6 gene. strategy. This was subsequently found to be because Deletion analysis suggests that the roregion is ARSs occur relatively infrequently in Y.lipolytica, required for efficient secretion of AEP. p33 and because plasmids containing these sequences are The AEP prepro sequences have been used to unusually ~tab1e.I'~Thus, such transformants were direct the secretion of two commercially-important probably erroneously scored as integrants. Using proteins, bovine pro~hymosin'~~and porcine a- mutant host strains in which replicatin vectors were interfer~n.'~,~~'Franke et ~l.''~fused prochymosin fortuitously less stable, Fournier et aLh3isolated the cDNA to five positions within the AEP coding Y.lipolytica ARS elements, ARS18 and ARS68. sequence, resulting in the expression of prochymo- Vectors containing these sequences were present in sin fusion proteins containing the presequence only 1-3 copies per cell and were very stable, being alone, the preproI region, the preproIpro11 region, lost at a rate of 0-5-5% per generation. Since only and the preproIproI1 region plus either 14 or 90 two different ARSs were isolated, a maximum fre- amino acid residues of the mature AEP. Each of quency of only one element per 1000 kb of genomic these fusion proteins was efficiently secreted and DNA can be calculated. These characteristics released active chymosin on treatment with acid. suggested that the ARSl8 and ARS68 elements Thus, the AEP proregion was not necessary for the could also contain centromere function, and this has secretion of prochymosin. In the case of the pre- been demonstrated geneti~a1ly.l~~Expression vec- proIproII fusion, the AEP secretion and processing tors based on the ARS18 element have now been signal sequences were shown to be accurately de~eloped.'~' recognized and cleaved. Porcine a-interferon was In addition to LEU2, several other Y.lipolytica efficiently secreted when fused to either the preproI genes have now been cloned and used as selection or the preproIproII regions of AEP1649271although markers for transformation, e.g. L YS1, L YS5 and some incorrect processing was reported133.The final ADE1,1639407HIS1 and URA3.86Y.lipolytica is resist- level of a-interferon secreted was increased about ant to many of the antibiotics commonly used for two- to three-fold using an ARSl8 rather than an S.cerevisiae, including G4 18 and chloramphenicol, integrating vector.271However, this improvement but alternative dominant selection markers have was less than the six-fold increase found with AEP been developed. The LEU2 promoter was fused to since a-interferon expression resulted in reduced the phleomycin-resistance gene from Tn5 and used vector stability. Similar results were obtained for for the direct selection of tran~f0rmants.l~~A high prochymosin which gave only 1.3-fold higher levels proportion of resistant colonies were found to be using the ARS vector. Regulation of the XPR2 pro- untransformed mutants, but this could be reduced moter was also impaired using this vector, since by introducing an expression phase prior to plating. basal AEP levels were found to be 50-fold higher An alternative dominant transformation marker is than normal under repressing conditions. the SUC2 gene of S.cerevisiae, which was fused to the promoter and secretion signal sequence from Schizosaccharomycespombe the AEP gene, XPR2.270 Direct selection on Aside from S.cerevisiae, the fission yeast, sucrose medium was possible, probably since most S.pombe, is the most intensely studied and well FOREIGN GENE EXPRESSION IN YEAST A REVIEW 465 characterized of the yeast species. This is largely from S.cerevisiue genes generally function poorly, because, like budding yeast, its life cycle and growth giving inefficient and aberrant initiation of transcrip- characteristics are particularly suited to genetic and ti~n.~'~,~'~Nevertheless, the S.cerevisiae PGK pro- biochemical analysis. The early development of a moter has been used to express polyoma middle T transformation system2*has led to the cloning and antigen,27and the ADHI and CYCl promoters used characterization of a large number of genes, and to to produce biologically active, secreted antithrom- the development of an array of genetic manipu- bin III.& In addition, the S.cerevisiue genes for three lation techniques comparable to those used in S. glycosidases (a-mannosidase, exoglucanase and cerevisiae (for review, see reference 319). Such endochitinase226)and the Saccharomyces diastaticus studies have highlighted how distantly related these glucoamylase gene'I2 have all been expressed in two yeasts are. In fact, sequence comparisons show S.pombe using their respective promoters. However, that for many S.pombe genes, the mammalian the SV40 early promoter and the promoter from the homologues are only marginally more divergent S.pombe alcohol dehydrogenase (udh) gene have than corresponding S.cerevisiae gene^,^" and some been most commonly used. The adh gene is constitu- are actually less divergent (e.g. ra~'~~).The utility of tive and gives alcohol dehydrogenase levels of about S.pombe in isolating mammalian genes by comple- 0.5-2% t.~.p.~~'Withthe SV40 early promoter tran- mentation of corresponding mutant homologues scripts initiate at the same position in S.pombe as in has been dem~nstrated,~~'and this remains the most mammalian cells203although it is weaker than the important use of this expression system. In contrast, adh promoter. Toyama and Oka~ama~~'used the the use of S.pombe for protein production has been chloramphenicol acetyltransferase gene to test a limited since little fermentation technology has been number of other mammalian promoters for activity developed, and because relatively few inducible in S.pombe. The human chorionic gonadotrophin a promoters were available. and human cytomegalovirus promoters were about Transformation of S.pombe has been described ten-fold stronger than the SV40 promoter and using sphaeroplasts,22lithium salts,'65and electro- several others, although weaker, were also func- poration."' A highly efficient method uses lipofec- tional. Other constitutive promoters that have been tin to enhance uptake of DNA by sphaer~plasts.~A used are the cauliflower mosaic virus (CaMV) 35s number of selectable markers have been described pr~moter,'~~.~~'which gave expression levels similar (see reference 409) but the LEU2 and URA3 genes to the adh promoter, and a promoter isolated from from S.cerevisiae, which complement the S.pombe random S.pombe genomic fragments, called 54/1 ,224 leu1 and ura4 mutations, are most commonly which gave P-galactosidase levels of 5% t.c.p. used. The corresponding S.pombe genes have also Regulatable expression systems for S.pombe have been and dominant selection using the also been described. One uses the promoter from the G418 and bleom cin resistance genes has been S.pombe fructose bisphosphatase @p) gene which is demon~trated.~~~,~Y expressed at very low levels in cells grown on 8% S.pombe expression vectors normally contain glucose and is derepressed 100-fold in media con- sequences derived either from the 2p plasmid of S. taining non-repressing carbon sources.176However cerevisiae or from the S.pombe arsl. The 2p it should be noted that, even in 8% glucose, thefbp sequences that have ARS activity in S.pombe do not promoter is partially induced on entry into station- include the complete 2p ORI and do not depend on ary phase. A second regulated system uses mam- any 2p-encoded f~ncti0n.l~~These vectors have malian glucocorticoid response elements to drive relatively low copy number (5-10 per cell) and expression. These are induced up to 70-fold by mitotic stabilit (30-45% loss per generation with- glucocorticoid hormones when the hormone recep- out selection 16Y). The arsl vectors behave similarly tor is co-e~pressed.~~~The fully-induced expression but have higher copy number (about 30/~e11'~~).level was about 20% of that using the adh promoter. Stability and copy number of arsl vectors is greatly A promising regulated system uses the promoter enhanced by the presence of an S.pombe-derived from a highly-transcribed nmtl gene which is sequence called stb, which appears to provide a par- strongly repressed by thiamine.249This was used to titionin f~ncti0n.l~~These arsllstb vectors, e.g. regulate a multi-copy chloramphenicol acetyl- pFL20,84U have a copy number of about 80 and are transferase (CAT) reporter gene, and gave a 200-fold lost at a rate of 13% per generati~n.'~~ thiamine-dependent repression of CAT expression. A number of promoters have been used for the In addition to the examples already described expression of foreign genes in S.pombe. Promoters above, a diverse selection of other heterologous 466 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE proteins have beenexpressed in S.pombe. The human growth is over many generations. With regulated liver microsomal enzyme, epoxide hydrolase, was systems only highly toxic products should be prob- expressed in an active form that could be isolated lematic, because of their ability to rapidly affect host from S.pombe microsomal fractions. lg2 Active factor cell metabolism. XIIIa was produced at 2mg/l using a high copy number, adh expression vector derived from Mechanisms of toxicity PFL~O.~~The expression of functional single-chain Toxicity is diagnosed by a difficulty in obtaining antibody molecules capable of binding their cog- transformants with constitutive expression vectors nate hapten, the aromatic dye fluorescein, has been or a reduction in growth rate when regulated vectors described.88Jagadish et al. lg4 expressed the large are induced. Which metabolic process is predomi- polyprotein of infectious bursa1 disease virus, which nantly affected depends on the foreign gene and was processed to give stable VP3 protein. Strasser vector system. The maintenance of ultra-high-copy et al.368 expressed the a-amylase gene from 2pILEU2-d plasmids alone is sufficient to reduce the Schwanniomyces occidentalis. The bacterial pro- host cell growth rate,397.'6gwhereas no effect would teins, /3-glu~uronidase,~~~xylose isomerase5* and be expected with integrated vectors. Multi-copy pro- bacterio-opsinlM have also been produced in moters may compete for transcription factors and S.pombe. The latter is correctly processed by cleav- inhibit expression of co-regulated genes: thus multi- age at the N-terminus, is inserted into membranes, copy GAL7 vectors inhibit GAL10 gene transcrip- and forms photoactive bacteriorhodopsin pigment tion, and could affect galactose metaboli~rn.'~ when cells are rown in the presence of its chromo- High levels of mRNAs containing rare codons phore, retinalJ6 Another integral membrane pro- could deplete cognate tRNAs and inhibit the trans- tein expressed in active form by S.pombe is the lation of host mRNAs containing rare codons. This glucose transporter from Arabidopsis thaliana appears to be the effect of lacZ mRNA in yeast, encoded by the STPl gene.329 which at high levels is toxic, especially during The secretion of foreign proteins is a relatively growth on minimal medium.292 unexplored area of potential interest since, like Toxicity is a relatively common problem with higher eukaryotes, S.pombe is known to galactosy- secreted proteins, especially complex ones. This may late lycoproteins (e.g. in~ertase?~~acid phospha- be due to the complexity of the secretory pathway, tase 9f). However, Sxerevisiae invertase was not offering a number of possible bottlenecks, or else to galactosylated when expressed in S.p~mbe,~~~and the possibility that foreign secreted proteins may be both this protein and homologous ones are highly frequently malfolded and cause a blockage in the glycosylated. In addition, antithrombin I11 was pathway. Examples of highly toxic secreted proteins hyperglycosylated when secreted by S.pombe.46 are tPA,16' IGF-1,343EBV g~350.~~'In the case of tPA, acid phosphatase secretion is reduced, suggest- ing a general block in secretion. Membrane proteins, PHYSIOLOGY OF FOREIGN GENE e.g. E.coli ompA,lg6 influenza virus haemaggluti- EXPRESSION nin,lglpolyomavirusmiddleTantigen~7areparticu- High-level expression of a foreign gene can place larly likely to be toxic, possibly due to non-specific a significant metabolic burden on the host cell, insertion and disruption of yeast intracellular mem- reducing its growth rate and affecting the efficiency branes. This may be the mechanism of toxicity of of gene expression. Expression of some genes causes HBsAg, since constitutively-expressing strains have a more acute effect, either through a severe effect on reduced viability on freezing and thawing (M.A.R., metabolism or by direct toxicity. In constitutive sys- unpublished results). tems where growth and expression are linked there Certain proteins have specific, acutely toxic is then strong selection for cells with reduced foreign effects. As an extreme example, induction of intra- gene expression, so that low-expressing variants cellularly expressed A-chain of ricin instantaneously arise. Variants may also be selected without growth, kills yeast cells, and this has been used as a selection for example through effects of the product on cell for non-toxic A-chain mutants. 125 Over-expression viability during freezing or long-term storage on of proteases, such as HIV-1 protease, may be toxic. agar. Therefore it is highly desirable to use tightly- Mammalian transcriptional trans-activators are fre- regulatedpromoterswhere thegrowthandinduction quently toxic, presumably through their ability to phases can be largely separated. This is particularly sequester components of the yeast transcriptional important with large-scale fermentations where apparatus. FOREIGN GENE EXPRESSION IN YEAST A REVIEW 467 A number of approachescan be used in an attempt reduced product yields. Mutant cells were detected to overcome product toxicity. Secretion may be used using a chromogenic plate assay for secreted acid to segregate the product from the site of toxicity even phosphatase expressed from the chromosomal if it is not normally secreted, e.g. HIV-1 protease.288 PH05 gene (Figure 12). The problem was elimi- Alternatively, fusion to other proteins may inhibit nated by a combination of measures: (i) using a biological activity and toxicity. The approach of freezing protocol which gave higher viability to selecting for mutants resistant to toxicity has been reduce enrichment of mutant cells and (ii) plating used successfully with IGF-1 .347 Toxin-resistant out stock cultures and using the plate assay to moni- mutants have been used to express diphtheria toxin tor for non-expressing colonies. The mutants did not fragment A, which normal1 kills yeast cells, and arise at detectable frequencies without the enrich- chimaeric diphtheria toxins.25 ment that occurred during low-viability freezing. This example illustrates the type of problem that can Generation of low-expressing variants occur on scale-up of aconstitutive system expressing a mildly toxic product. To a certain extent it is possible to limit the gener- ation of variants having reduced expression levels, Large-scale fermentation and optimization which can arise with constitutive systems. The most common response to toxicity with 2p vectors is In the development of a large-scale industrial fer- reduced copy number, which can reach a level as low mentation process, the first step is transfer to small as 1 per cell even with selection.35However, when fermenters. These offer the possibility of controlling LEU2-d selection is used the drop in copy number a number of culture parameters, resulting in much that can occur without affecting growth is limited, greater reproducibility and the ability to reach very and other mechanisms such as structural rearrange- high cell densities. However, a significant effort is ment predominate. Where the selection marker is frequently required in order to maximize product flanked by 2p DNA, recombination can result in its yield, though in theory it should be possible to con- transfer to native 2p with loss of the expression trol conditions so as to give higher yields than in cassette.99One way of preventing this is by using shake-flasks. High-density fermentation is generally plasmids such as pJDB219 in 2p-free strains. desirable in order to minimize capital expenditure Another mechanism for plasmid loss involves on fermentation equipment; it is particularly valu- homologous recombination between the selection able with secretion, where the concentration of marker and chromosomal allele to generate a secreted protein in the medium can increase almost prototroph. This can be prevented by using a host proportionately to cell density.’I3 strain with a deletion in homologous chromosomal In the case of recombinant S.cerevisiae, glucose is sequences or by use of recombination-deficient almost always used as the carbon-source. The two mutants (e.g. ra~i.52’~). extremes of glucose-utilization are fermentation, An unusual plasmid mutation was observed which yields ethanol as a by-product, and respi- during the constitutive expression of polyomavirus ration, which is much more efficient for conversion middle T antigen.27Transformants grew very slowly, to biomass. However, growth to high density on and there was either plasmid loss or spontaneous glucoseis problematic since Xcerevisiae has a limited generation of plasmids expressing a truncated anti- capacity to metabolize glucose oxidatively, accumu- gen, which lacked the membrane-spanning domain lating ethanol, which eventually inhibits growth, and was non-toxic. Mutant plasmids arose by above a specific growth rate of 0.2 to 0.25 h-I. ‘I7 deletion of one G.C base pair in a run of nine, Secondly, glucose concentrations above approx. causing a frame-shift 5’ of the DNA encoding the 0.1 % repress re~piration.’~~In aerobic batch cul- hydrophobic region. tures, such as shake-flasks, growth on glucose is We have observed an example where a mutation diauxic: the glucose is rapidly fermented to ethanol in a host cell gene inactivated the promoter used in and CO,, and the ethanol is metabolized in a second the expression vector. The system was a PH05 vec- growth phase once the glucose is exhausted. tor expressing HBsAg in a constitutive pho8O back- In order to prevent the conversion of glucose to ground (M.A.R., unpublished results). Mutant ethanol and maximize biomass, bakers’ yeast is non-expressing cells were enriched during freezing grown using a fed-batch process: molasses and nutri- and thawing of stock cultures, and out-grew the ents are slowly fed to the fermenter, the feed rate expressing cells in fermenters, resulting in greatly increasing as the cell density increases, so as to obtain 468 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE

Figure 12. Accumulation of non-expressingcells during fermentationof an S.cerevisiaepho80 strain containing a PHOS vector for HBsAg expression. Samples were taken at the end of 10 litre fermenter runs of the constitutively-expressing strain, plated on non- selectiveagar and assayed for secreted acid phosphatase (Pho phenotype) by overlayingwith agar containing a chromogenicsubstrate. (A) High-yield fermenter run contained Pho+ cells only. (B) Low-yield run contained a high proportion of Pho- cells in which the PH05 promoter is inactive. Low-yield runs resulted from enrichment of mutant non-expressers during low-viability freezing. Improved freezing protocols resulted in high-yield runs. an almost constant specific growth rate. Similarly, found severe plasmid-loss using a PGK vector fed-batch using calculated glucose feed rates has expressing y-interferon, and were unable to achieve been used for hi h density culture of recombinant high cell densities. However, an optimized process S.~erevisiae."'.'~8-More refined methods have also has been developed for secretion of pro-urokinase been applied: computer control has been used to from a super-secretin pmrl strain using the PGK couple glucose feed rate to respiratory quotient or TPZ promoters.38% Accumulation of secreted (RQ = CO, evolved/(>, consumed; maintained at product was growth-associated but was not as high 1-0 to 1.2) using on-line gas ana~ysis,"~,~'~~~~~or to in an RQ-controlled fed-batch culture (77 g/1 dry ethanol concentration using a bio~ensor,~resulting mass) as in a perturbed batch fermentation (45 g/l), in cell densities of 80 to 200 g/1 dry mass. These where glucose was pulsed into a batch culture. This results have been achieved with semi-synthetic difference may be due to activation of glycolytic media containing protein hydrolysate (casamino promoters by high concentrations of glucose. In acids) or yeast extract. batch fermentations the secretion of human lyso- With an ideal tightly-regulated system, biomass zyme, using the enolase promoter, was increased accumulation would be separated from foreign pro- four-fold by increasing starting glucose concen- tein induction, and the two could be optimized separ- tration from 2 to lo%, even though growth was ately. In constitutive systems this is clearly not ~naffected.'~~In view of these observations another possible, but even with regulated systems acombined approach would be to use glycolytic promoter sys- growth/induction process must usually be developed. tems in two-stage fermentations, an initial glucose- We will illustrate some principles of process develop- limited stage for biomass accumulation followed by ment by referring to Sxerevisiae systems using an increased glucose feed for induction. three types of promoters: constitutive glycolytic, Glucose-repressible expression systems present glucose-repressible, and galactose-inducible. problems because of the conflicting requirement to As described above, product toxicity is a serious maintain high glucose in order to repress the pro- problem with constitutive systems: Fieschko et moter, and to limit glucose in order to maximize FOREIGN GENE EXPRESSION IN YEAST: A REVIEW 469 biomass. However, a fed-batch process for the pro- recombinant K.lactis strain expressing secreted duction of a SOD-proinsulin fusion protein using a HSA under the control of the Xcerevisiae PGK pro- GAPIADH2 promoter has been developed, though moter has been grown to high density (80 to 90 g/l) the cell density achieved was only 30 g/l.378Initial using glucose-limited fed-batch at the 1000 1 scale, experiments indicated that a glucose excess was yielding HSA at several g/I.'*O A commercial process important to repress the promoter and maintain for the secretion of prochymosin has been scaled-up plasmid stability. Quite high yields of product to 41,000 lit re^.^^' ( > 0.5 g/l) were obtained in simple batch cultures Ppstoris is particularly suited to high-density with glucose concentration starting at 5%, but bio- culture and can be grown to > 100 g/l on simple mass was low. A fed-batch procedure was developed defined media containing glycerol without the need using a constant, empirically-determined glucose for complex fed-batch procedures." The tight regu- feed which led to glucose excess during most of the lation of the AOXZ expression vectors means that growth phase but to very low glucose at later stages the growth phase is identical for different protein as the cell density increased. Sub-optimal feed-rates products, and scale-up to large fermenters does not would have resulted either in plasmid loss or in affect biomass or product yield." Additionally, incomplete induction. Apparently the residual glu- contrary to the experience with S.cerevisiae, an cose (20 mg/l) was partially repressing, since immediate improvement in percentage yield of changing from glucose to a final ethanol feed yielded foreign protein is generally seen on going from a two-fold improvement to 1.2g/l of product shake-flasks to fermenter~.~~.~~.~~~Th'is appears (increasing to 1.6 g/1 at 26OC). to be due, at least in part, to the high 0,-demand The first published study of galactose induction in of the organism, which is not satisfied in a high-density fermentation involved the expression shake-flasks. of y-interferon using the GAPIGAL hybrid pro- Figure 13 shows the results of a high-density fer- moter."' Although biomass yields of 200 g/1 were mentation using a Pichia Muts recombinant con- possible using RQ-controlled glucose-limited fer- taining 14 integrated copies of a tetanus toxin mentations, biomass was restricted to c 100 g/1 fragment C expression vector.73 The culture was because of 0,-transfer limitation. Three induction started with an initial batch growth in glycerol; methods were compared: (i) 10 g/1 pulses of galac- when this was used up a slow glycerol feed was tose, (ii) replacement of the glucosefeed by galactose, initiated, during which growth was glycerol-limited (iii) (i) followed by (ii). The third method gave the and the AOXZ promoter derepressed, allowing highest yields and this was rationalized as being due rapid induction during the subsequent methanol to the partial induction of galactose-metabolizing feed. Most of the growth occurred during the enzymes during galactose pulsing, preventing tem- glycerol feed; product accumulated for about 30 h porary carbon-source starvation on switching the into the methanol feed, during which time there was feeds. However, in another study galactose was no cell doubling, reaching a level of 27% t.c.p. (cf. rapidly metabolized in glucose-limited culture^.^ A 10% in shake-flasks) or approx. 12 g/l. In contrast, further improvement (to 2 g/1 of product) was made Mut + strains continue to grow rapidly during the using a diploid host strain, however the yield of y- methanol feed and are fed methanol at a higher rate. interferon was only 3 to 5% t.c.p. compared to 10% Using similar protocols high levels of several com- in shake-flasks. mercially-important proteins have been achieved: More recently, the production of P-galactosidase e.g. SOD (1.3 g/1375),HBsAg (0.4 g/I"), tumour was examined using a galactose-inducible, hybrid necrosis factor (10 g/13@),Bordetella pertussis per- CYCIIGAL pr~moter.~It was found that galactose tactin (3 g/1306),secreted HSA (3 g/l; K. Sreekrishna, added to glucose-limited cultures was rapidly personal communication), secreted EGF (0-45 g/l;74 metabolized and depleted, since the cells were G. Thill, personal communication). Mut' strains derepressed, and it was preferable to feed a glucose/ can also be grown in continuous culture to increase galactose mixture so that glucose was preferentially fermenter prod~ctivity.~~ metabolized. In this way P-galactosidase was induced Apart from the growth and induction protocols, a to 7% t.c.p. at a cell density of 100 g/l; addition of a number of other variables have been found to affect final 50% galactose feed increased the yield to 8%, yields and should be looked at systematically. considerably higher than the level in shake-flasks. Lower temperatures may increase yield by reducing Expression systems based on several yeasts prote~lysis.~~'Added phosphate in the medium has other than Sxerevisiae have been scaled-up. A increased heterologous secretion in at least two 470 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE

- 30

- 25

-20 x 8

-3- 10 J- 0 -5 e 9 -0

MW v)-W -15 0 2 4 6 8 24 28 30 32 52 82 96 -3 hours into induction

k Figure 13. Fermentation of a 14-copy Pichia Mut' transformant expressing tetanus toxin fragment C. (a) Cell density (A,), dissolved oxygen (do,), and fragment C as per cent of cell protein are plotted against time (0 h = start of induction). Fermentation conditions were as described elsewhere.'? Timepoint A, addition of shake-flask inoculum, B, glycerol-limited feed, C, methanol feed. (b) Coomassie blue-stained SDS polyacrylamide gel showing protein extracts from cells taken at different times during induction. The arrow indicates the position of fragment C. For comparison, a sample from the same transformant induced for 48 h in a shake flask is shown in lane 3. and extract from an untransformed control strain is shown in lane 2. ca~es."4,~~~Buffering to high pH with S.cerevisi~e~~~CONCLUDING REMARKS and high or low pH with P.p~storis'~(K.Sreekrishna, personal communication) has reduced proteolysis of In its early stages the use of yeast for heterologous secreted proteins. Increased growth rate increases expression was highly favoured due to the obvious plasmid stability and can result in greater product advantages of a microbial eukaryotic system. How- yield.82Finally, different strains of Sxerevisiue may ever, interest waned with the discovery of unforseen have dramatically different growth and induction problems and with the advent of powerful alterna- properties. tive systems such as baculovirus. Yeast expression FOREIGN GENE EXPRESSION IN YEAST. A REVIEW 47 1 systems are now in a period of resurgence, for a in increasing yields of secreted proteins has been number of reasons. One of the major disadvantages made using the powerful empirical approach of compared to E.coli and baculovirus has been the random mutagenesis and screening. The secretion generally lower yield of product, frequently due of pharmaceutical glycoproteins remains problem- to the difficulty in obtaining high-level transcrip- atic due to the differences in glycosylation between tion of foreign genes. This problem has now been yeast and mammals. Even glycoproteins which are addressed in S.cerevisiae in a number of ways: not hyperglycosylated contain antigenic mannose for example, by over-expressin transcriptional linkages: an engineered strain which could over- trans-activators (e.g. with ADH2%I ), by construct- come this problem has been reported,353though no ing glycol tic promoters with superimposed results on product antigenicity are available yet. An regulation?’ and by random screening for alternative possibility is P.past0ri.s which does not super-expressing mutant strains.354 The use of appear to add the antigenic al,3-linked terminal yeasts such as Pichia pastoris, which naturally manno~e.~~~~The production of glycoproteins for have powerful, tightly-regulated promoters, has non-pharmaceutical use, e.g. glucoamylases and provided an alternative solution. Table 6 (see xylanases, has been highly successful and presents ‘Expression in non-Saccharomyces yeasts) shows no such problems. data for two proteins produced in four expression In the last few years there have been many systems, S.cerevisiae, P.pastoris, E.coli and baculo- successes in the production of therapeutic proteins virus, illustrating the comparative strength of from yeast, for example the recombinant subunit the P.pastoris multi-copy AOXI vector system. vaccine against hepatitis B virus, human proinsulin, Another factor has been the accumulation of experi- EGF, HSA, etc. There have also been developments ence in the high-density growth and induction of in the food industry, such as the experimental use recombinant yeasts, comparing favourably with of recombinant yeast secreting glucoamylase in the difficulties encountered in scaling up other brewing,157and the production of chymosin from eukaryotic systems. Pichia is particularly suited to K.l~ctis.~~IHowever, in future the requirements of scale-up and should continue to become more an expression system may be increasingly to provide widely used for industrial production. reagents for research and drug discovery rather than A number of other areas have shown significant in the production of therapeutic agents. For rapid advances, for example the development of more isolation of such products it may be advantageous stable 2p vectors, of rDNA and Ty multi-copy inte- to make use of fusion proteins or ‘tags’ which allow grating vectors, and the use of autoselection simple affinity urification: a variety of systems are markers. The development of episomal vectors for now a~ailable.4)~’”~~Since many pharmacological Kluyveromyces lactis is also worthy of mention target proteins may be toxic when expressed to high since this yeast appears to be particularly efficient in levels, or may have short half-lives, it may be necess- secretion. However, there are areas where improve- ary to accept low yields and concentrate products ments are needed. In S.cerevisiae it appears that using an affinity tag. foreign genes are frequentlypoorly transcribed using A highly significant development has come from certain promoters, possibly due to a requirement for the realization that many pharmacological target intragenic yeast sequences (DASs) for maximal proteins can function in yeast cells in vivo. It has been transcription. Unfortunately, it is not clear how reported that adrenergic receptors can be expressed general the problem is, nor is there yet direct evi- in yeast and coupled to the signal transduction path- dence for DASs. In P.pastoris there appears to be no way for a-factor; using a lacZ reporter fused to the such problem using the AOXI promoter since very a-factor regulated FUSl promoter, a colorimetric high levels of foreign transcripts can be obtained assay that could be used for drug screening was with multi-copy integrant~.~~ developed.’I4 There is intense commercial interest in Despite several important successes in secreting such systems because of their advantages over mam- proteins, especially unglycosylated polypeptides, malian cells in setting up robust drug screens, but this is an area which can present problems. It is still there is scope for improvement at present. Other not clear why some foreign proteins are not correctly examples of systems that have been or could be folded and transported. The current rapid progress used for in vivo pharmacological screening are: in our understanding of protein folding and chaper- HIV translational frameshifting,404HIV TAT ones may eventually provide explanations and poss- trans-activation,lW steroid receptor binding and ibly solutions. Recently, however, the most progress trans-a~tivation.*~~*~~* 472 M. A. ROMANOS, C. A. SCORER AND J. J. CLARE Finally, yeast expression technology is now cursor of repressible acid phosphatase contains a important in the genetic analysis of organisms whose signal peptide. Nucleic Acids Res. 11,1657-1672. genetics are less well developed. A number of mam- 8. Armstrong, K. A., Som, T., Volkert, F. C., Rose, A. malian genes have been isolated by their ability to and Broach, J. R. (1989). Propagation and expression complement mutant yeast homologues, e.g. the of genes in yeast using 2-micron circle vectors. In Barr, P. J., Brake, A. J. and Valenzuela, P. (Eds), human homologue of the S.pombe cdc2 gene.230 Yeast Genetic Engineering. Butterworths, pp. S.pombe may frequently be more suitable than 165-192. S.cerevisiae for screening human cDNA expression 9. Bach, M.-L. (1987). Cloning and expression of the libraries because of its closer phylogenetic relation- OMP decarboxylase gene ural from the yeast ship, and because mammalian expression vectors Schizosaccharomyces pombe. Curr. Genet. 12, using the SV40 promoter are active in S.pombe. 527-534. 10. Baim, S. B., Pietras, D. F., Eustice, D. C. and Sherman, F. (1985). A mutation allowing an mRNA ACKNOWLEDGEMENTS secondary structure diminishes translation of Saccharomyces cerevisiae iso- I-cytochrome c. Mol. We would like to thank the following people who Cell. Biol. 5, 1839-1846. have helped us in writing this review by helpful dis- 11. Baim, S. B. and Sherman, F. 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