Engineering yeast for high level expression Reinhard Fleer R h 6 n e - P o u l e n c Rorer, Vitry, France As a eukaryotic microbe, yeast remains an attractive host for the expression of a large variety of foreign proteins, including viral antigens, enzymes used as food additives and therapeutic agents. Important progress has been made in the understanding of the critical parameters influencing product yield, and a number of novel tools for the genetic engineering of powerful yeast expression systems have been developed. This review focuses on recent findings in foreign gene expression in the yeasts Saccharomyces, Pichia, Hansenula, and

Kluyveromyces. Current Opinion in Biotechnology 1992, 3:486-496

Introduction Despite important advances in gene expression in mammalian cells and the advent of alternative eukaryotic hosts such as the baculovirus system, yeast has maintained much of its attraction as a microbial host for the expression of foreign genes. In addition to its obvious advantages over bacterial expression systems, such as the absence of endotoxins and the possibility of obtaining proper folding and processing of many foreign proteins due to a secretory pathway closely resembling that of higher eukaryotes, yeast can be easily grown to high cell densities and at much lower cost than any mammalian expression system. Many of the problems encountered previously in the scale-up of heterologous protein production using recombinant yeast cells, such as relatively low yields compared to Escherichia coli or poor strain stability, can be solved b y applying a combination of classical and molecular genetics, screening for strains with high levels of expression following random mutagenesis, or by simply exchanging Saccharomyces cerevisiae for another yeast species. The past year has seen several examples of high level gene expression in yeasts other than S. cerevisiae, the most striking ones being the intracellular production of Bordetella pertussis pertactin (> 3 g 1- 1) [1-] and tetanus toxin fragment C ( 1 2 g l -1) [2-] in Pichia pastoris, the secretory production of guar ~-galactosidase in Hansenula polymorpha (about 2 g 1-1) [3"q and human serum albumin (HSA) in Kluyveromyces lactis (about 3 g 1-1) [4-']. The growing importance of nonconventional yeasts is reflected well in recent reviews on heterologous gene expression [5,6",7"]. The term 'yeast' will therefore be used in its generic form throughout this review, with particular emphasis on Saccharomyces

cerevisiae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis and Yarrowia lipolytica. Recent developments concerning key parameters for high level gene expression will b e addressed, including the quality and quantity of product-specific mRNA (which is affected by promoter strength and transcriptional regulation, accurate transcription termination, mRNA stability, gene dosage, and vector stability), the host strain and species, and the optimization of growth and induction parameters during scale-up. Aspects specific to the secretion of foreign proteins in yeast, such as the role of secretion signals, pro-peptides, processing sites, and glycosylation are not covered in this section due to space limitations. The reader is referred therefore to recent reviews addressing these issues [7"',8"].

Transcription and translation Promoter strength and transcriptional regulation The importance of choosing the right transcriptional promoter for optimal gene expression has b e e n widely documented [7"'] and regulated systems have often b e e n found to be advantageous. The most recent examples include a 5-6-fold increase in interleukin (IL)-6 production by replacing the constitutive 0~-factor (MFoO promoter with the regulated galactokinase (GALl) promoter [9"], a greater than 10-fold increase in porcine pancreatic phospholipase A2 yield by replacing the M / ~ promoter with the inducible galactose transferase (GALT) promoter [10"], and a 20-fold increase in human P450 IIC expression by using the GAL 7 promoter instead of the constitutive alcohol dehydrogenase 1 (ADH1) promoter [11]. Whereas S. cerevisiae offers an overwhelming choice of constitutive and regulated, native and hybrid promoters, the availability of pro-

Abbreviations HBsAg--hepatitis B surface antigen; HIV--human immunodeficiency virus; HSA--human serum albumin; HSE--heat shock element; IL--interleukin; NGF--nerve growth factor; rDNA---ribosomal DNA; RQ--respiratory quotient; tcp--total cellular protein; UTR---untranslated region. 86

© Current Biology Ltd ISSN 0958-1669

Engineering yeast for high level expression Fleer 487 rooters suitable for heterologous gene expression is m u c h more limited in other yeasts. This is especially true for K. lactis w h e r e the inducible ~-galactosidase (LAC4) promoter is the only h o m o l o g o u s p r o m o t e r that has b e e n used for the production of foreign proteins. This has p r o m p t e d the construction of a promoterprobe system adapted to K. lactis and has led to the identification of a number of highly expressed genes w h o s e promoters are currently being investigated (M Bolotin-Fukuhara, personal communication). An interesting and rather novel, highly regulated system that could easily be adapted to any yeast species exploits the observation that mammalian steroid horm o n e receptors can act as transcriptional activators in yeast. Expression of the receptor results in transactivation of a chimeric promoter carrying the cognate response sequences, such that a target gene m a y be expressed in a h o r m o n e - d e p e n d e n t manner. Recent examples of this system include a hybrid of iso-l-cytochrome c (CYC1) and a glucocorticoid response ele m e n t [12"], CYCI and an estrogen response element [13"], and 3-phosphoglycerate kinase (PGK) and an androgen response element [14-]. Addition of increasing amounts of cognate ligand, e.g. dihydrotestosterone in the PGK/androgen response element example, results in an increasing degree of transcriptional activation over a several 100-fold range. The strength of the chimeric PGK promoter was fully maintained and specific transcripts represented 5% of the total mRNA. Other interesting aspects of hormone-regulated systems are: first, the rapid induction kinetics (tl/2 < 10 rain); second, the fact that there is no requirement for carbon source control; third, the absence of observable effects of the steroid ligand u p o n the yeast cell at the low concentrations required for induction (e.g. 10-100nM dihydrotestosterone); and finally, the low cost of the inducer (less than 10 cents per m3). A potential complication in the use of hormone-regulated promoters might arise from the interference of certain h o r m o n e receptors with constituents of the yeast transcriptional machinery. A severe reduction in the growth rate of yeast cells overexpressing one of the glucocorticoid receptor transactivation domains has b e e n reported recently and has b e e n attributed to squelching, i.e. titration of limiting transcription factors n e e d e d for efficient gene activity [15]. Adverse effects on yeast cell growth have also b e e n observed w h e n overexpressing the chicken progesterone receptor A [13"1. Other novel chimeric yeast promoters include hybrids b e t w e e n A D H 2 and GAL 7 upstream sequences [P1], b e t w e e n the invertase (SUC2) and glyceraldehyde3-phosphate dehydrogenase (GPD) promoters [P2"], and b e t w e e n the CYC1 p r o m o t e r and a 242-basepair sequence containing multiple heat-shock elements (HSEs) derived from the nematode Caenorhabditis elegans [16]. Whereas this CYC1/HSE p r o m o t e r has a stronger transcriptional activity than the equivalent CYC1/GAL hybrid, it exhibits a high basal level of activity and induction is only 2-3-fold u p o n heat-shock at 39 "C. On the other hand induction is rapid, and

with the HSE hybrid the same level of reporter protein could be obtained after 45 minutes as with the GAL hybrid after 24 hours. The induction kinetics of a similar CYC1/HSE chimeric promoter as a function of physiological parameters and of the host genetic b a c k g r o u n d has b e e n studied by Kirk and Piper [17], w h o reported an induction range of 30-50-fold. A c o m m o n aim in the optimization of gene expression is the overexpression of transactivators, which often represent limiting factors, especially w h e n foreign genes are expressed from multi-copy vectors. Recent examples of significantly increased product yields due to overexpression of the GAL4 gene, which codes for the transcriptional enhancer of the galactose regulon, include GALl driven IL-2 secretion [18"] and the expression of the light and heavy chain genes coding for a catalytic antibody coexpressed from the bidirectional GAL1-GALIO promoter [19"]. Similarly, gene expression driven by the K. lactis LAC4 promoter can be increased w h e n overexpressing the K. lactis GAL4 equivalent, LAC9 (K Breunig, personal communication). In S. cerevisiae, strong over-expression of the GAL4 gene from an ultra-high copy n u m b e r leu2-d plasmid results in a 'superinducible' vector exhibiting rapid induction kinetics, host-strain independence, and a strong attenuation of glucose repression of a CYC1/GAL hybrid p r o m o t e r carried on the s a m e vector [20]. In spite of a high basal level of expression, a tenfold induction of the ~-galactosidase reporter could b e obtained u p o n addition of 0.5% galactose, even with an excess of glucose, leading to ~-galactosidase yields equivalent to 12% total cellular protein (tcp). This system allows growth on a mixed substrate (glucose :galactose ratio 10:1), thereby significantly reducing the amount of inducer required for maximal gene expression [21-].

5' and 3' untranslated mRNA sequences

The sequence and structure of the 5' leader and 3' trailer m a y influence the efficiency of translation initiation, mRNA stability and the efficiency of transcription termination, all parameters that influence gene expression in yeast. The most striking example of increased product yield as a result of modification of the 3' untranslated region (UTR) has been reported by Demolder et al. [18.]. Expression of a prepro-MF0c/murine IL-2 fusion under control of different yeast promoters in S. cerevisiae gave tenfold higher yields b y deleting the major part of the mammalian 3' UTR, which app e a r e d to be responsible for rapid degradation of the murine IL-2 mRNA [18"]. A stabilizing effect of local structural alterations in the 3" UTR of the S. cerevisiae P G K gene, resulting in a twofold longer mR.NA halflife, has also b e e n reported [22"]. It would be interesting to test whether this modified trailer structure is able to confer enhanced stability on a heterologous transcript. An example of reduced gene expression as a result of point mutations in the 3' UTR has b e e n described for the S. cerevisiae A D H 2 gene [23"]. Furthermore, a cellfree yeast system developed for studying the turnover of individual mRNAs has allowed the identification of

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Expression systems sequence motifs involved in the endonucleolytic cleavage of P G K t r a n s c r i p t s [24°]. The existence of fortuitous signals within the o p e n reading frames of genes foreign to yeast appears to be one of the factors that significantly influence expression levels (see below). As far as the 5' UTR is concerned, attempts to increase the translational efficiency of the yeast P G K or E. coli ~-galactosidase transcripts in S. cerevisiae, by replacing their 5' UTR by plant viral leader sequences derived from alfalfa mosaic virus or tobacco mosaic virus, have not met with success [25]. This is in marked contrast to the k n o w n stimulatory effect of these leader sequences on translation in mammalian, plant and bacterial in vivo and/or in vitro systems. Another approach aiming for increased translational efficiency is the reconstitution of the consensus sequence found upstream of the initiator AUG of highly expressed yeast genes. A threefold increase in the secretion of guar 0t-galactosidase, as well as in the intracellular expression of the poliovirus capsid protein VP2 in S. cerevisiae, following the introduction of the consensus sequence A A A A A C A A T G (instead of CTCAAAAATG) into the 5' UTR of the GAL 7 promoter, demonstrates that this approach can be beneficial [26"].

Synthetic genes revisited: codon bias versus fortuitous transcription termination There has b e e n m u c h debate about the usefulness of engineering genes so that their sequences reflect the small subset of codons used in highly expressed yeast genes. Theoretically, c o d o n usage is not often likely to prevent a high level of gene expression [27",28]. Nevertheless, there are n o w a n u m b e r of examples that point to i m p r o v e d synthetic gene performance w h e n the genes have b e e n engineered to match the yeast codon bias, as c o m p a r e d to their cDNA counterparts. Most recently, Kotula and Curtis [29q have described a 50-fold increase in expression of an immunoglobulin ~ chain in S. cerevisiae by using a synthetic, codon-optimized gene. The higher level of the polypeptide was not a result of significant changes in the steady-state level of mRNA, or of a difference in plasmid copy number. Less dramatic but still significant was the threefold increase in guar ~-galactosidase yields in S. cerevisiae b y using a gene with optimized codons [26"]. W h e n changing the primary sequence of a gene, however, one interferes simultaneously with a n u m b e r of parameters other than codon bias, the most obvious being the primary and secondary structure of the resuiting mRNA, which might in turn affect translatability, mRNA stability and 3' end formation (see above). It is difficult, therefore, to attribute an observable effect on product yield to a specific feature, such as codon usage. A striking example of this is the expression of the Clostridium tetani gene encoding tetanus toxin fragment C, a candidate subunit vaccine against tetanus. Expression of this gene in S. cerevisiae led to very low product yields (0.001% tep) which could be increased 2000 to 3000-fold (2-3% of total soluble pro-

rein, 60-90 m g 1-1 in shake flasks, or > I g 1-1 in high cell density fermentations) w h e n using a fully synthetic gene [30"']. It was found that the native AT-rich gene contained at least six fortuitous polyadenylation sites which gave rise to truncated mRNA species. When increasing the GC-content from 29% to 47%, full-length mRNA and efficient production of fragment C could be obtained concomitantly [30"]. In their recent review o n heterologous gene expression in yeast, Romanos et al. [7"] cite several examples of genes that give rise to truncated mRNA species w h e n expressed in S. cerevisiae. They suggest that fortuitous transcriptional termination might represent a very c o m m o n reason for low yields or complete failure of foreign gene expression in yeast, and might not be limited to AT-rich genes. It should b e stressed that this p h e n o m e n o n is not restricted to S. cerevisiae as truncated mRNA species have also b e e n observed in P. pastoris [7"] and in K. lactis (N Amellal and R Fleer, unpublished data).

Gene dosage and strain stability One of the single most important parameters for high level gene expression is the n u m b e r of expression cassettes introduced into the host cell, be it on replicating vectors, e.g. derived from the multi-copy 2/.t or pKD1 plasmids, or b y integration at chromosomal locations into the host genome. Whereas it remains true that an increase in c o p y number d o e s not lead automatically to higher expression levels, especially w h e r e protein secretion is intended (e.g. [31]), some of the most spectacular improvements in strain productivity over the last year have b e e n achieved by approaches designed to increase g e n e dosage [1"',2"',4"',32",33"',34"',35"]. The following will address aspects of both episomal and integrative expression systems.

Episomal vectors and autoselection systems One of the disadvantages frequently encountered w h e n expressing foreign genes from episomal vectors is a decrease in copy n u m b e r and the eventual loss of the expression vector, due to the selective advantage of plasmid-free host cells o v e r their recombinant counterparts. For S. cerevisiae, t w o n e w autoselection systems have b e e n reported which ensure the maintenance of plasmid selection irrespective of the m e d i u m composition. The first system relies on the use of a host with a disrupted chromosomal C D C 9 gene, coding for DNA ligase, which is transformed with a vector carrying a functional c o p y of this essential gene. Use of the temperature-sensitive mutant allele cdc9-1 leads to a system that allows temperature-dependent copy-number amplification of 2~t plasmids by up to threefold [36]. The second system is b a s e d on the cloning of the SRB1 gene, which determines osmotic shock stability in S. cerevisiae [37"]. This gene has b e e n used as an autoselection marker in srbl-1 hosts which normally require osmotic stabilization for growth [38",P3"]. W h e n cul-

Engineering yeast for high level expression Fleer 489 tured in a non-stabilized medium, cells having lost the expression vector will lyse spontaneously and cease to grow, thus preventing accumulation of non-productive cells. The efficiency of this system has been d e m o n strated with a 2bt-based SOD expression vector, which was maintained stably over 100 generations of growth in continuous culture in the absence of an osmotic stabilizer. The control plasmid harboring SOD, but not the cloned SRB1 gene, was completely lost after 42 generations. Rather surprisingly, the p h e n o t y p e of srbl:: TRP1 disruption mutants differed from that of the srbl-1 ochre mutant in a n u m b e r of aspects. Cells carrying the disruption allele lost the slow growth p h e n o t y p e of the mutant as well as the d e p e n d e n c e u p o n osmotic stabilization for growth. Nevertheless, quite unexpectedly, srbl ° disruption strains conferred autoselection on an expression vector harboring a functional SRB1 gene [P3"]. Furthermore, they maintained the lyric ability u p o n osmotic shock, which makes srbI ° disruptants a potentially useful tool for the commercial production of intracellularly expressed proteins such as the hepatitis B antigens (HBsAga) or other virus-like particles. As regards yeasts other than S. cerevisiae, an autoselection system has b e e n described for K. lactis that allows the stable maintenance of expression vectors b a s e d on the plasmid pKD1 [39"]. This system relies on the use of host ceils with the chromosomal P G K gene deleted. The disruption mutants exhibit normal growth on media containing glycerol and ethanol, but cannot grow on media containing glucose, lactose or other sugars as the sole carbon source. Expression vectors harboring the HSA gene, together with a functional P G K g e n e , are maintained without significant loss of productivity over at least 200 generations in industrial-type media, thus providing the possibility of using pKDl-derived expression systems in continuous culture fermentations.

Systems for multi-copy integration of heterologous genes Integrative expression systems are developed for one of two reasons: either to overcome instability problems encountered with episomal vectors, or because no native plasmids were available for the construction of episomal expression vectors for the yeast species of interest (e.g. Pichia, H a n s e n u l a and Yarrowia). A disadvantage of the integrative systems used initially w a s the low gene dosage, leading to suboptimal mRNA levels and concomitantly low product yields [7"]. Therefore, a n u m b e r of strategies have been d e v e l o p e d to increase the c o p y n u m b e r of integrated expression vectors. In S. cerevisiae the gene of interest has b e e n integrated into delta elements, which are scattered throughout the S. cerevisiae g e n o m e either as solo-deltas or as part of the transposable element Ty, and these elements have b e e n used to construct a strain carrying 20 copies of the h u m a n nerve growth factor (NGF) gene [32"]. The integrant strain stably secreted three- to fourfold higher levels of h u m a n NGF than a strain transformed with a 2g-based plasmid. Integration of heterologous genes targeted to dispersed repetitive elements, such as delta

sequences, Ty elements, or tRNA genes, has b e e n the subject of a recent patent application [P4]. A similar strategy based on integration into reiterated chromosomal DNA makes use of the ribosomal DNA (rDNA) cluster, consisting of about 140 tandem repeats in S. cerevisiae and about 60 repeats in K. lactis. This technology, originally developed for S. cerevisiae (see [7"]), has n o w b e e n extended to K. lactis [35"] and Kluveromyces m a r x i a n u s (J Verbakel, personal communication). In K. lactis, c o p y - n u m b e r dependent secretion of guar a-galactosidase at levels u p to 250 mg 1-1 could be achieved with this system. Although the expression, or the presence, of the heterologous gene reduced the c o p y - n u m b e r compared to control vectors (15 versus 60 integrated copies), a-galactosidase levels obtained with the integrants were higher than those observed with strains transformed with pKDl-derived vectors. This w a s probably a result of the higher stability of the integrant strains which retained the expression cassette without loss of productivity for at least 70 generations of non-selective growth. Integration of foreign genes at the rDNA locus might be an interesting approach for yeasts like Y. lipolytica, for which only low copy-numb e r autonomously replicating vectors are available [40] and w h e r e multi-copy integration is difficult to achieve otherwise. As far as Pichia and H a n s e n u l a are concerned, stable multi-copy integration occurs without the need for specialized vectors [1",2"',33"',34"q. In Pichia, multi-copy integrants can unexpectedly be obtained b y transformation with a linear DNA fragment designed for singlec o p y gene transplacement. This has b e e n s h o w n to b e due to repeated single crossover events occurring subsequent to in vivo circularization of the fragment [2"]. Screening procedures have b e e n described based on 'dot-blot' hybridizations to facilitate the isolation of multi-copy integrants [1"',33"]. Whereas it initially a p p e a r e d that the alcohol oxidase ( A O X I ) promoter u s e d for heterologous gene expression in P. pastoris w a s strong enough to give maximal expression of a foreign gene w h e n integrated as a single copy, it is n o w clear that boosting the mRNA levels of a foreign gene further b y the use of multi-copy integrant strains can increase the final product yield significantly. This has b e e n demonstrated for the secretion of mouse epidermal growth factor giving 450 mg 1-1 [33"] and the intracellular production of tetanus toxin fragment C (yield 12 g 1-1) [2"] and B. pertussis pertactin (yield > 3 g 1-1) [1-q. With respect to pertactin, product levels varied bet w e e n 0.5% and 5% tcp as a function of copy n u m b e r (values for 1 and 30 copies, respectively). In H. polymorpha, up to 100 copies of an HBsAg expression cassette w e r e integrated using vectors originally designed for autonomous replication [34"]. These vectors, containing an autonomously replicating sequence, were found to integrate spontaneously into the g e n o m e at relatively high frequency, normally resulting in one to several integrated copies. Stable integration into H. p o l y m o r p h a chromosomal locations has also b e e n reported for vectors carrying 2~t sequences, although with lower copy numbers [3"]. An interesting aspect of

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Expressionsystems this technology is the possibility of integrating different expression units at variable c o p y numbers, thus facilitating the expression of c o m p l e x composite protein structures. This has led to the construction of integrant strains coexpressing both the large S (L) and and major S (S) HBsAg u n d e r the control of the methanol-inducible methanol oxidase and formate dehydrogenase promoters [34"]. Several multimeric integrants w e r e obtained exhibiting a wide spectrum of different L to S ratios. The synthesized HBsAg assembled spontaneously into composite subviral particles containing both the S and L proteins. These particles will be useful for the production of a hepatitis B virus vaccine with the potential for i m p r o v e d immunogenicity as a result of the presence of a wider spectrum of epitopes. Whereas autoselection systems or chromosomal integration confer segregational stability on the expression vector, they cannot prevent structural instability resulting from a strong selection pressure in favor of cells harboring mutant alleles of the foreign gene, or carrying deletions of sequences required for high level expression. Enrichment of variants of a S. cerevisiae strain transformed with an HBsAg expression vector during freezing and thawing of stock cultures has b e e n described recently and the variants s h o w low levels of HBsAg expression [7"]. The variant cells exhibit greatly reduced product yields due to a mutation in the p r o m o t e r driving HBsAg expression. Similarly, w e have observed an increase in recombinant K. lactis variants carrying a deletion of the entire expression cassette b y recombination b e t w e e n direct repeats with 80% sequence h o m o l o g y (A F o u m i e r and R Fleer, unpublished data). The choice of strategy for obtaining a recombinant system intended for use on an industrial scale should be governed, at least in part, by the ease with which subtle genetic modifications, such as promoter variations or point mutations within the structural gene, can b e detected. In this respect, the use of episomal yeast-E, coli shuttle vectors, which can be rescued from the recombinant yeast and propagated in E. coli for subsequent analysis of individual clones, is clearly an advantage over m a n y of the integrative systems described above.

Influence of host strain and host species One of the factors that is often neglected w h e n optimizing yeast expression systems is the importance of the host's genetic background. W h e n screening for S. cerevisiae strains expressing Aspergillus niger glucose oxidase, a 100-fold difference w a s found b e t w e e n the lowest and the highest producing strains [41"°]. No obvious correlation b e t w e e n the expression rate and the genetic markers of the strains could b e identified. Similarly, the search for wild-type K l u y v e r o m y c e s strains secreting high levels of HSA expressed from a pKD1based vector revealed a b r o a d spectrum of productivity. Among the 50 strains tested, product yields ranged from barely detectable to 300 mg 1-1 in shake-flask cul-

tures [4"q. Veale et al. [3"'] recently reported the optimization of a H. p o l y m o r p h a strain, expressing guar 0t-galactosidase under the control of the methanol oxidase promoter, by a series of genetic crossings with wild-type strains resulting in improved growth characteristics and higher induction levels as c o m p a r e d to the initial recombinant strain. The observed differences within a given yeast species render cross-species comparisons extremely difficult. This should b e kept in mind w h e n considering the following data, which nevertheless a p p e a r to reflect s o m e general tendencies. Romanos et al. [1"] c o m p a r e d S. cerevisiae and P. p a s toris directly for their efficiency of pertactin expression. S. cerevisiae w a s transformed with a 2/.t-based vector carrying the pertactin gene under the control of the GALl promoter. In P. pastoris, the pertactin gene was driven b y the A O X 1 p r o m o t e r and integrated as single or multiple copies at the A O X 1 locus. Whereas intracellular expression of pertactin was rather low in S. cerevisiae (0.1% tcp), product levels u p to 5% tcp were obtained in P. pastoris depending on the copy number. Similarly, optimized expression of the tetanus toxin fragment C in S. cerevisiae using a synthetic gene placed under control of the GALl or ADH2 p r o m o t e r reached levels equivalent to 2-3% total soluble protein [30"q. In contrast, the same gene could be expressed in P. pastoris u n d e r the control of the A O X 1 p r o m o t e r to levels of 27% of cell protein, i.e. 12 g 1-1 in high cell density fermentations [2"]. T w o recent reports indicate that K. lactis might have a greater capacity for high level secretion than S. cerevisiae. One report describes the secretion of HSA using p K D l - b a s e d episomal vectors in wild-type, non-optimized K. lactis strains [4"]. Yields obtained in shakeflask cultures with the S. cerevisiae P G K p r o m o t e r reached a b o u t 300 mg 1-1 [4.-] and could be increased to over 400 m g 1-1 w h e n the homologous LAC4 promoter was u s e d (N Amellal and R Fleer, unpublished data). The system could be scaled up to 1000 liter fedbatch fermentations without any significant loss of productivity, yielding several grams of secreted HSA per liter. This high productivity was exploited subsequently for the production of a HSA-CD4 fusion protein, designed as a potential therapeutic agent for use in h u m a n immunodeficiency virus (HIV) infection [42"]. In contrast, attempts to secrete HSA from S. cerevisiae strains have met with variable success. Early reports indicated that HSA, although engaged in the secretory pathway, remained cell-associated [43]. Since then, S. cerevisiae strains yielding up to 55 mg 1-1 of HSA w h e n transformed with 2/.t-based expression vectors have b e e n identified [44] and Okabayashi et al. have achieved yields of 85 mg 1-1 using multiple chromosomal integration [45"]. Several rounds of mutagenesis and subsequent strain selection have led to over-secreting mutant strains exhibiting yields which are improved significantly (up to 150mg1-1) [44] but which are still lower than those obtained with nonoptimized K. lactis strains. The second e x a m p l e of a protein for which published data indicate a higher secretion capacity b y K. lactis w h e n compared to S. cere-

Engineering yeast for high level expression Fleer visiae is h u m a n IL-I~. The yields of this cytokine have b e e n estimated as 2 - 2 0 m g l - 1 in S. cerevisiae [46,47"] and 80 mg 1-1 in K. lactis [48"].

Yeasts such as Pichia, H a n s e n u l a and Yarrowia, however, also have a high potential for the secretion of foreign proteins. A recent example is the secretion of guar (x-galactosidase in H. p o l y m o r p h a , reaching 2 2 . 4 m g g - 1 dry cell weight [3"] which is equivalent to about 2 g 1-1 in a high density fermentation process. These considerations notwithstanding, parameters such as substrate range, the ease of scale-up, the specific productivity (grams of biomass and/or product per liter per hour), the availability of tightly regulated promoters, and the stability of a recombinant system, should be at least as important for industrial scale applications as minor differences in volumetric product yields.

Other approaches to optimize expression levels of heterologous genes Screening for over-producing mutants In parallel with the increasing sophistication of molecular biological methods which, as we have seen, can lead to significant increases in product yields, there is a growing awareness of the benefits of using classical techniques for strain improvement. Random mutagenesis of recombinant yeasts with chemical or physical agents, followed b y screening for over-producing strains, has led to the identification of mutants with considerably e n h a n c e d productivities [7",44]. A number of screening methods based on simple plate assays have b e e n developed, especially for the isolation of mutants showing increased levels of protein secretion [49",50]. A_rl-interesting, widely applicable screening approach to detect supersecretors has been described by Sleep et al. [44]. This plate assay is based on the precipitation of product-specific antibodies, in this case directed against HSA. The size of the halos formed around the colonies is indicative of the secretory capacity of the mutagenized strains. Several rounds of mutagenesis and selection resulted in mutants which s h o w e d a sixfold increase in HSA secretion and also p r o d u c e d higher levels of intracellularly expressed proteins such as (x-l-antitrypsin and h u m a n plasminogen activator inhibitor 2. Whereas a generalized increase in protein secretion has also b e e n observed with mutants obtained by other methods [7"], exceptions to this rule have also b e e n reported [51]. Screening procedures based on miniaturized liquid cultures have b e e n applied successfully to the identification of bovine chymosin supersecretors in filamentous fungi [52"]. We have d e v e l o p e d a similar automated screening approach to isolate HSA supersecretors involving K. lactis grown in liquid media, resulting in the identification of a strain showing increased product yields (L

Louvel and R Fleer, unpublished data). Finally, using a screening method which involved the scoring of clearing zones on a lawn of sensitive cells resulting from killer toxin secretion, Chow et al. [53"'] have identified a S. cerevisiae gene conferring fourfold enhanced levels of total protein secreted per cell w h e n re-introduced into the parental strain on a multi-copy vector. This gene, termed PSEI (protein secretion enhancer), does not seem to be related to any of the SEC genes the sequences of which have so far b e e n established. The repertoire of proteins that are found to be secreted in greater quantities include killer toxin, (x-factor and acid phosphatase. Preliminary experiments involving the secretion of (x-amylase suggest that overexpression of PSE1 also increases the yield of a heterologous secretory protein. This is the first example showing that protein secretion can be increased by overexpressing a single gene.

Optimization of fermentation and scale-up parameters Maximizing product yields in yeasts, as in any recombinant system, is not limited to strain improvements brought about by the techniques of classical or molecular genetics. The control of growth and induction parameters offered b y modern fermenter technology contributes considerably to high level gene expression. Recent examples include the increase in tetanus toxin fragment C yields, from 10% tcp in shake-flask cultures to 27% tcp (12 g l - 1 ) in fed-batch fermenters, b y optimizing the fermentation conditions for a recombinant P. pastoris strain [2"]. The optimization of a fermentation process for the secretory production of a human pro-urokinase variant in S. cerevisiae is another example [54"]. A supersecreting yeast strain ( p m r l 1), harboring multiple copies of integrated expression units under the control of a glycolytic promoter, was grown batchwise and at a constant respiratory quotient (RQ). Despite lower cell densities, higher product yields could be achieved in a pulsed batch fermentation (1863 International Units per ml at 45 g 1-1 dry cell weight) than in an RQ-controlled fed-batch culture (1108International Units per ml at 77g1-1 dry cell weight). Other parameters contributing to optimal product yields were phosphate concentration (twofold increase of secreted single-chain urinary plasminogen activator at 40 g 1-1 instead of 10 g 1-1), p H (fivefold higher yields b y buffering the medium at p H 6.0), and temperature (45-50% higher yields were obtained at 25 °C as c o m p a r e d to 22.5 °C, 27.5 °C or 30 °C). Finally, optimization of a fermentation process for the secretory production of A. n i g e r glucose oxidase in S. cerevisiae resulted in the highest expression levels ever reported for baker's yeast, demonstrating the capability of the latter to secrete a large protein efficiently (the molecular weight of the yeast-derived enzyme was estimated to be 350 000-400000, primarily due to extensive Nlinked glycosylation) [41-'].

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Expressionsystems

Conclusion The past y e a r has w i t n e s s e d a growing interest in y e a s t s p e c i e s o t h e r t h a n S. cerevisiae, e s p e c i a l l y w i t h respect to the large-scale production of proteins of p h a r m a c e u t i c a l o r a g r o - a l i m e n t a r y i n t e r e s t . W h e r e a s P. p a s t o r i s h a s a c o n f i r m e d r o l e as a n e x c e l l e n t h o s t f o r the production of heterologous proteins [l"',2",P5,P6], H. p o l y m o r p h a a n d K. l a c t i s s h o w p r o m i s e o f a c a p a c ity f o r h i g h l e v e l s e c r e t i o n , y i e l d i n g g r a m q u a n t i t i e s o f p l a n t a n d m a m m a l i a n p r o t e i n s p e r liter o f c u l t u r e s u p e r n a t a n t [3",4"']. T h e d e v e l o p m e n t o f a n u m b e r o f n o vel tools has resulted in higher product yields, mostly d u e t o a n i n c r e a s e i n g e n e d o s a g e , h i g h e r s t r a i n stability, o r b e t t e r r e g u l a t i o n o f t r a n s c r i p t i o n , t n a d d i t i o n , t h e e l i m i n a t i o n o f f o r t u i t o u s t r a n s c r i p t i o n t e r m i n a t i o n signals by using synthetic genes with a higher GC-content h a s b e e n s h o w n to a f f e c t p r o d u c t i v i t y d r a m a t i c a l l y i n certain cases. Despite the growing importance of nonc o n v e n t i o n a l y e a s t s , S. cerevisiae, w h i c h h a s t r a d i t i o n ally b e e n t h e s y s t e m o f c h o i c e f o r m o l e c u l a r e n g i n e e r ing due to the extensive knowledge accumulated for this species, remains an attractive organism especially f o r t h e f u n c t i o n a l e x p r e s s i o n o f m a m m a l i a n g e n e s [55"]. As a c o n s e q u e n c e , S. c e r e v i s i a e g a i n s m o r e a n d m o r e importance as a eukaryotic model organism which can b e e n g i n e e r e d to s e r v e i n r o b u s t a n d s i m p l e a s s a y s f o r n o v e l d r u g s c r e e n i n g [P7-,P8"]. I n a d d i t i o n , c a r e ful s e l e c t i o n o f t h e r i g h t h o s t s t r a i n a n d o p t i m i z a t i o n o f t h e f e r m e n t a t i o n c o n d i t i o n s c a n m a k e S. c e r e v i s i a e an excelleiat host for the production of industrially important proteins, which has been demonstrated by the e n g i n e e r i n g o f s t r a i n s s e c r e t i n g o v e r 3 g 1-1 o f a f u n g a l g l u c o s e o x i d a s e [41"].

Acknowledgements I would like to thank my colleagues A Foumier, E Conseiller, P Yeh and J Crouzet for reading this manuscript critically, L Ferrero for linguistic improvements, and H Bouras for valuable secretarial help.

2. •.

CLAREJJ, RAYMENT FB, BALLANTINE SP, SREEKRISHNA K, ROMANOSMA: High-level E x p r e s s i o n of Tetanus Toxin Fragment C i n Pichia pastoris Strains C o n t a i n i n g Multiple T a n d e m Integrations of the Gene. Biotechnology 1991, 9:455-460. Optimization of a number of parameters, including the site and type of integration, copy number and scale-up, resulted in a host/vector system producing the highest levels of a foreign protein ever reported for yeast (12gl-1). The mechanism leading to multi-copy gene transpIacements is analyzed and a screening method for the rapid identification of multi-copy integrants is described. 3. •o

VEALERA, GUISEPPIN MLF, VAN EIJK HMJ, SUDBERY PE, VERRIPSCT: D e v e l o p m e n t o f a Strain o f Hansenula polymorpha for the Efficient E x p r e s s i o n o f Guar czGalactosidase. Yeast 1992, 8:361-372. Initial low yields of 0t-galactosidase could be increased significantly, mainly by changing the host cell background subsequent to a series of genetic crossings, by stably integrating the expression unit into the genome, and by optimizing the culture conditions using a chemostat. 4. •.

ble Multi¢opy Vectors for High-level Secretion of Rec o m b i n a n t H u m a n S e r u t n Albnmlrt b y Kluyveromyces Yeasts. BiotechnoIogy 1991, 9:968-975.

This is the first report describing the use of pKDl-based expression vectors for the production of mammalian proteins in K. lactis. Optimization of the host/vector system included parameters such as host strains, promoters, secretion signals, role of the propeptide, culture medium and scale-up conditions. Several grams per liter of correctly folded and processed HSA could be obtained at the 1000 liter scale. 5.

BUCKHOLZRG, GLEESONMA: Yeast Systems for t h e Commercial Production o f H e t e r o l o g o u s Proteins. Biotechnology 1991, 9:1067-1072. An excellent review focusing on the performance of alternative yeast expression systems relative to S. cerevisiae. ROMANOSMA, SCORERCA, CLAREJJ: Foreign G e n e Expression i n Yeast: A Review. Yeast 1992, 8:423-488. This outstanding, very up