Gene expression in yeast: Pichia pastoris Thomas S. Vedvick Salk Institute Biotechnology/Industrial Associates Inc., La Jolla, USA Recent studies have shown the versatility and utility of the Pichia pastoris expression system. Improvements in strains have boosted the yield of proteins and peptides to the commercially feasible range. The Pichia pastoris expression system will soon be used to manufacture proteins for human clinical trials. Current Opinion in Biotechnology 1991, 2:742-745
MUT-
Pichia pastoris, a methylotrophic yeast, is an outstanding host for high level heterologous gene expression. All expression systems require a method of transferring the DNA sequence of interest into the host cell together with a promoter capable of controlling the production of the foreign gene product. Successful promoters have a very high efficiency of transcription, are tightly regulated and are inexpensive to induce. The alcohol oxidase promoter used in the P. pastoris system was isolated and cloned by Ellis et al. [1] and transformation resulting in stable integrants of P. pastoris was first reported by Cregg et al. [2]. This promoter, for the alcohol oxidase gene (AOX1), clearly fulfills the requirements for a good heterologous gene expression system. The AOXI promoter is tightly regulated by methanol, the substrate of the enzyme. When P. pastoris strains are grown on alternative carbon sources such as glycerol or glucose, there is no induction of the alcohol oxidase promoter. Upon initiation of methanol feeding, the gene is rapidly and fully transcribed. The strength of the promoter is demonstrated by the observation that the enzyme alcohol oxidase (AOX) comprises up to 30 % of the soluble protein in extracts of P. pastoris grown on methanol in the fermentor. Another key feature of this expression system is the high cell densities that can be achieved in the fermentor using a simple feed of salts and methanol. The combination of very stable integration of foreign DNA into Pichia with this strong Picbia promoter, coupled with high cell density fermentations, produces recombinant protein products at relatively high concentrations.
When the heterologous gene vector disruptively integrates into the P. pastoris genomic AOXI sequence, the cell's ability to metabolize methanol is severely impaired. Although it is capable of slow growth resulting from expression of the less efficient AOX2 alcohol oxidase allele. Strains with a disrupted AOXI gene are described as being methanol-utilization deficient (MUT-). By integrating into the Pichia genome without disrupting the AOX1 gene, wild-type methanol metabolism is maintained. Strains with a non-disrupted AOX1 gene are described as being methanol-utilization positive (MUT+). The use of the MUT + strains of Pichia has increased the productivity of the strains and decreased total fermentation time. Brierley et al. [3"] discuss in detail the fermentation conditions for the MUT- strains, and the changes necessary for the MUT + strains. Methanol-fed batch fermentations are reduced to 50 h or less with the MUT + strains instead of the usual 140-200 h on methanol required with the MUT- strains. As the intracellular and extracellular protein levels are comparable in the two systems, the overall productivity of the MUT + strains is much higher. Clare et al. [4°°] compared the production of tetanus toxin fragment C in Pichia MUT- and MUT + strains. Their one copy MUT- and MUT + strains had similar levels of fragment C production: 6.4 mg/1 and 8.3 mg/1 respectively. However, because the MUT + material can be produced in about one quarter of the fermentor time, the cost per mg per hour is less for the MUT + strains. Brierley et al. [3"] acheived a 6.5-fold increase in the productivity of bovine lysozyme by changing from MUT- to MUT + strains.
This review covers the recent developments in the Pichia expression system. This includes the change away from the disruptive integration of vectors into the Pichia genome at the AOX1 gene, to the, now, preferred method of integration that leaves the AOXI gene intact. Also, the ability of the P. pastoris strains to process various heterologous signal and secretion sequences is described. Finally, P. pastoris strain development has produced a new generation of protease-deficient strains that improves the future outlook for the Pichia expression system.
742
and M U T + Pichia strains
Introduction
Signal and secretion leader sequences Aprotinin, a protease inhibitor, has been produced in Pichia [5"]. The aprotinin gene was constructed using Pichia-preferred codons and fused to Saccharomyces cerevisiae m-mating factor prepro signal sequence. However, this signal sequence was not properly processed;
(~) Current Biology Ltd ISSN 0958-1669
Gene expressionin yeast: Pichia pastoris Vedvick 743 amino-terminal extensions of either 11 or four amino acids from the m-mating factor signal sequence were left on the aprotinin. This heterologous gene was reconstructed to include the sequence encoding a dipeptide (Glu-Ala), as a spacer between the cz-mating factor prepro signal sequence and the aprotinin gene product. The aprotinin analog produced depended on the number of Glu-Ala spacers - those strains with one Glu-Ala spacer had a two amino acid amino-terminal extension, and those with two Glu-Ala spacers had an amino-terminal extension of four amino acids. The Glu-Ala spacers appeared to allow for proper processing of the KEX2 (Lys--Arg) site on the factor leader sequence but no dipeptidyl amino peptidase (STE13) activity was present to finish the processing to native aprotinin. In Saccbaromyces the normal processing for the secretion of ~mating factor requires two enzyme activities. One enzyme cleaves on the carboxy-terminal side of two basic residues such as Lys-Arg or Arg-Arg and is termed KEX2. The other enzyme is a dipeptidyl amino peptidase that selectively removes dipeptides from the amino terminus of proteins. The enzyme has a high specificity for Glu-Ala and Asp-Ala sequences. It may be that aprotinin inhibited the dipeptidyi amino peptidase activity.
Ngsee and Smith [6-] have investigated the effect of modflying the bovine prolactin signal sequence fused to the mature sequence of yeast invertase on secretion of this enzyme. The wild-type prolactin signal sequence is about one-sixth as efficient at causing secretion as the wildtype yeast invertase signal peptide. However, replacing the glycine residue located between the amino terminus and the central lipophilic region of the bovine prolactin signal peptide with an alanine results in production of invertase at a level equal to the wild-type yeast invertase signal sequence. This enhanced secretion suggests that the secondary structure of the bovine prolactin signal sequence is important for recognition by the yeast secretory apparatus. This conclusion is supported by other data. Replacing the bovine prolactin signal sequence codons to those preferred by yeast did not significantly increase invertase production. Also, truncating the 5' untranslated leader sequence of the bovine prolactin signal peptide increased invertase activity only slightly. Thus, the low level of secretion seen with the bovine prolactin signal peptide-invertase fusion was caused partly by the length of the 5' untranslated leader sequence. So a very small change in the amino acid sequence of the signal sequence can result in a tremendous improvement in signal processing and secretion efficiency of recombinant proteins in Saccharomyces.
Although not all secretion signal sequences are properly processed, in general P. pastoris processes the recombinant protein to an authentic bioactive product. There is still a great deal to b e learned about the selection and modification of both signal and secretion leader sequences.
Multicopy effects Most foreign protein genes expressed in P. pastoris strains have shown a copy number effect such that the larger the number of expression cassettes, the greater the amount of protein product produced. If a strain with a single copy expression cassette produces 100mg/1, then a six-copy strain is most likely to produce up to 1 g/l of protein product. Clare et al. [4.-] have shown a dramatic increase in the production of tetanus toxin fragment C as the copy number is increased from one to nine, but little or no change when the copy number is further increased to 14. We have generally limited our maximum copy number to six for ease of strain verification. In the case of aprotinin, the nature of the final product plays some part in the level of its production. A fivecopy strain that produced aprotinin with an amino-terminal extension of 11 amino acids approached the i g/1 yield level [5"], whereas a six-copy strain with a single Glu-Ala amino-terminal amino acid extension reached a maximum output of 700-800 mg/1. A recent review article [7"] reports that multiple-copy strains have been shown to be stable throughout scale up.
Strain improvements In all expression systems there is always room for continuous improvements and refinements. New Pichia strains have been developed at S1BIA that are deficient in some of the protease enzymes [8o]. These proteasedeficient strains display increased production of intact recombinant products and a noticeable decrease in what has been termed nicking. In the case of non-proteasedeficient strains, the protein product, insulin-like growth factor-l, is proteolysed at a particularly sensitive peptide bond. Integration of the insulin-like growth factor1 expression cassette into the protease-deficient strains reduces this nicking and results in a higher concentration of intact product. This ability to reduce the amount of proteolytic degradation of protein products has been tested by side-by-side analyses. Test peptides spiked into broth from control Picbia strains are always more susceptible to proteolysis than when spiked into proteasedeficient-strain broth.
Future work Post-translational modification of proteins in recombinant systems has always been a concern of investigators. The glycosylation of proteins in P. pastoris is similar in several respects to that observed in S. cerevisiae. In both organisms the majority of oligosaccaride chains added to proteins as they pass through the secretory pathway are the N-asparagine linked high-mannose type. However,
744
Expressionsystems there is at least one major difference. The size of the mannose side chains produced in Picbia are much smaller than the corresponding carbohydrates added by S. cerevisiae. Those in S. cerevisiae have from 50-150 mannose residues per chain, whereas in P. pastoris they are more likely to have 8-11 [9"]. In general, protein glycosylation in P. pastoris parallels the glycoprotein biosynthetic pathway in higher eukaryotes through the early stages of glycan processing, resulting in core structures that are very similar. As Pichia only adds the core structure [10], the immunogenicity of the Pichia produced recombinant protein products may not be a problem in certain therapeutic applications. Experimental testing of the immunogenicity of glycosylated proteins produced by Pichia pastoris will be critical to future work on potential human therapeutics.
3. ,.
BRIERLEYRA, BUSSINEAU C, KOSSON R, MELTON A, SIEGEL RS: Fermentation Development of Recombinant Pichta pa$toris Expressing the Heterologous Gene: Bovine Lysozyme. Biochem Engin V/1990, 589:350-362. An excellent paper, clearly describing the advantages of the MUT + Pichiapastorisstrains. Details of MUT + and M U T - fed batch protocols are given. Results showing production of Bovine lysozyme versus hours on methanol for the two types of strains show increased productivity uing MUT + strains. CLAREJJ, RAYMENTFB, B ~ SP, SREEKP,ISHNA K, ROMAN,S MA: High-level Expression of T e t a n u s Toxin Fragment C in Pichia pastoris Strains Containing Multiple T a n d e m Integrations of t h e Gene. Biotechnology 1991, 9:455-460. This paper shows both the effect of multicopy clones and MUT +, M U T - strains on production of tetanus toxin fragment C. Construction of the vectors and methods for inserting them into the Pichia starin are detailed. The mechanism for the formation of multicopy transplacement strains is also given 4. 00
5.
VEDV1CKT, BUCKHOLZ RG, ENGEL M, URCAN M, KINNEY, J, PROVOWS, SIEGEL RS, THILL GP: High-level Secretion of Biologically Active Aprotinin from t h e Yeast Pichia pastoris. J Ind Microbiol 1991, 7:197-202. Aprotinin analogs secreted at the gram per litre level are described. Problems effecting the aprotinin production were solved and are presented here. The aprotinin analogs were shown to be fully biologically active. ..
Conclusions P. pastoris has been shown to be an extremely productive recombinant protein expression system despite claims that no methylotrophs have been used for the production of heterologous proteins [11 ]. P. pastoris strains contain no antibiotic resistant genes [12] or other heterologous sequences that might be considered potential biological hazards. Secreted products from 3-90 kD have been expressed in pichia [7o,13-15]. Because of a combination of the extremely high cell densities reached by Pichia fermentation, and the stable integration of multiple copies of the expression cassette, yields of recombinant protein products in gram per litre quantities are routine. With the advent of the new protease-deficient strains, the production of proteins that have previously been difficult, if not impossible, to produce by recombinant methods, because of proteolysis, may now be possible.
6. •
NGSEEJK, SMITH M: Changes in a Mammalian Signal Seq u e n c e Required for Efficient Protein Secretion by Yeasts. Gene 1990, 86:251-255. This important paper describes the effect of changing a single amino acid in the signal sequence. The boost in enzyme production is described and a rationale is clearly presented. 7. •
BUCKHOtZ RG, GLEESON MG: Alternative Yeast Systems for t h e Commercial Production of Heterologous Proteins. Biotechnology 1991, in press. This excellent review describes in some detail all of the alternative expression systems to Saccbaromyces cerevisiae. GLEESONMG, HOWARD BD: Genes W h i c h Influence Pichia Proteolytic Activity, and Uses Thereof. Patent Application Docket 50848. This patent application is an excellent piece of work that clearly describes the construction of the two protease deficient strains produced. The enzyme activities eliminated and examples of improvement in peptide stability are given. 8. .
9. •
Acknowledgments I thank my colleagues Bob Siegel, Greg Holtz, Paula Schoeneck and Martin Gleeson for critically reading this manuscript. I thank Karen Payne for her patience and help in preparing this manuscript.
References and recommended reading
TRIMBLERB, ATKINSONPH, TSCHOPPJlg, TOWNSENDRR, MALEYF: Structure of Oligosaccharides on Saccharomyces SUC2 Invertase Secreted by the Methylotrophic Yeast, Pichia pastoris. J Biol Cbem 1991, in press. This important paper is written by the authority on glycosytation patterns in Pichia pastorix 10.
WEGNERGH: Emerging Applications of the Methylotrophic Yeasts. F ~ S Microb Rev 1990, 87:279-284.
11.
LINDSTROM ME, STIRLING DI: Methylotrophs: Genetics and Commercial Applications. Annu Rev Microbiol 1990, 44:27-58.
12.
CREGGJM, DIGAN ME, TSCHOPP JF, BRIERLEY RA, CRMG WS, VELICELEBI G, SIEGEL RS, THILL GP: Expression of Foreign G e n e s in Pichia pastoris. In Genetics and Molecular Biology of Industrial Microorganisms edited by Hershberger CL, Queener SW, Hegeman G [book]. Washington IX3: American Society for Microbiology, 1989, pp 343-352.
Papers of special interest, published within the annual period of review, have been highlighted as: • of interest •. of outstanding interest 1.
ELLIS SB, BRUST PF, KOUTZ PJ, WATERS AF, HARPOLD M, GINGERAS TR: Isolation of Alcohol Oxidase and Two Other Methanol Regulatable G e n e s From t h e Yeast Pichia pastori, Mol Cell Biol 1985, 5:1111-1121.
13.
HAGENSONMJ, HOLDEN KA, PARKERKA, WOOD PJ, CRUZEJA, FUKE M, HOPKINS TR, STROMANDW: Expression of Streptoldnase in Pichia pastoris Yeast. Enzyme Microb Techno11989, 11:6504556.
2.
CREGGJM, BARRINGER KJ, HESSLER AY, MADDEN KR: Pichia pastoris as a Host System for Transformants. Mol Cell Biol 1985, 15:3376-3385.
14.
SREEKRISHNAK, NELLES L, POTENZ R, CRUZE J, MAZZAFERROP, FISH W, FUKE M, HOLDEN K, PHELPS D, WOOD P, PARKERK: High,level Expression, Purification, and Characterization of
Gene expression in yeast: Pichia pastoris Vedvick
15.
Recombinant Human Tumor Necrosis Factor Synthesized in the Methylotrophic Yeast Pichia pastorix Biochemistry 1989, 28:4117-4125.
Saccharomyces cerevisiae. Adv in Biochemical Engineering/Biotechnology 1990, 43:75-102.
REISER J, GLUMOFFV, KALIN M, OCHSNER U: Transfer and Expression of Heterologous Genes in Yeasts Other Than
TS Vedvick, Salk Institute Biotechnology/Industrial Associates Inc., 505 Coast Boulevard South, La JoUa, California 92037, USA.
745
MUT-
Pichia pastoris, a methylotrophic yeast, is an outstanding host for high level heterologous gene expression. All expression systems require a method of transferring the DNA sequence of interest into the host cell together with a promoter capable of controlling the production of the foreign gene product. Successful promoters have a very high efficiency of transcription, are tightly regulated and are inexpensive to induce. The alcohol oxidase promoter used in the P. pastoris system was isolated and cloned by Ellis et al. [1] and transformation resulting in stable integrants of P. pastoris was first reported by Cregg et al. [2]. This promoter, for the alcohol oxidase gene (AOX1), clearly fulfills the requirements for a good heterologous gene expression system. The AOXI promoter is tightly regulated by methanol, the substrate of the enzyme. When P. pastoris strains are grown on alternative carbon sources such as glycerol or glucose, there is no induction of the alcohol oxidase promoter. Upon initiation of methanol feeding, the gene is rapidly and fully transcribed. The strength of the promoter is demonstrated by the observation that the enzyme alcohol oxidase (AOX) comprises up to 30 % of the soluble protein in extracts of P. pastoris grown on methanol in the fermentor. Another key feature of this expression system is the high cell densities that can be achieved in the fermentor using a simple feed of salts and methanol. The combination of very stable integration of foreign DNA into Pichia with this strong Picbia promoter, coupled with high cell density fermentations, produces recombinant protein products at relatively high concentrations.
When the heterologous gene vector disruptively integrates into the P. pastoris genomic AOXI sequence, the cell's ability to metabolize methanol is severely impaired. Although it is capable of slow growth resulting from expression of the less efficient AOX2 alcohol oxidase allele. Strains with a disrupted AOXI gene are described as being methanol-utilization deficient (MUT-). By integrating into the Pichia genome without disrupting the AOX1 gene, wild-type methanol metabolism is maintained. Strains with a non-disrupted AOX1 gene are described as being methanol-utilization positive (MUT+). The use of the MUT + strains of Pichia has increased the productivity of the strains and decreased total fermentation time. Brierley et al. [3"] discuss in detail the fermentation conditions for the MUT- strains, and the changes necessary for the MUT + strains. Methanol-fed batch fermentations are reduced to 50 h or less with the MUT + strains instead of the usual 140-200 h on methanol required with the MUT- strains. As the intracellular and extracellular protein levels are comparable in the two systems, the overall productivity of the MUT + strains is much higher. Clare et al. [4°°] compared the production of tetanus toxin fragment C in Pichia MUT- and MUT + strains. Their one copy MUT- and MUT + strains had similar levels of fragment C production: 6.4 mg/1 and 8.3 mg/1 respectively. However, because the MUT + material can be produced in about one quarter of the fermentor time, the cost per mg per hour is less for the MUT + strains. Brierley et al. [3"] acheived a 6.5-fold increase in the productivity of bovine lysozyme by changing from MUT- to MUT + strains.
This review covers the recent developments in the Pichia expression system. This includes the change away from the disruptive integration of vectors into the Pichia genome at the AOX1 gene, to the, now, preferred method of integration that leaves the AOXI gene intact. Also, the ability of the P. pastoris strains to process various heterologous signal and secretion sequences is described. Finally, P. pastoris strain development has produced a new generation of protease-deficient strains that improves the future outlook for the Pichia expression system.
742
and M U T + Pichia strains
Introduction
Signal and secretion leader sequences Aprotinin, a protease inhibitor, has been produced in Pichia [5"]. The aprotinin gene was constructed using Pichia-preferred codons and fused to Saccharomyces cerevisiae m-mating factor prepro signal sequence. However, this signal sequence was not properly processed;
(~) Current Biology Ltd ISSN 0958-1669
Gene expressionin yeast: Pichia pastoris Vedvick 743 amino-terminal extensions of either 11 or four amino acids from the m-mating factor signal sequence were left on the aprotinin. This heterologous gene was reconstructed to include the sequence encoding a dipeptide (Glu-Ala), as a spacer between the cz-mating factor prepro signal sequence and the aprotinin gene product. The aprotinin analog produced depended on the number of Glu-Ala spacers - those strains with one Glu-Ala spacer had a two amino acid amino-terminal extension, and those with two Glu-Ala spacers had an amino-terminal extension of four amino acids. The Glu-Ala spacers appeared to allow for proper processing of the KEX2 (Lys--Arg) site on the factor leader sequence but no dipeptidyl amino peptidase (STE13) activity was present to finish the processing to native aprotinin. In Saccbaromyces the normal processing for the secretion of ~mating factor requires two enzyme activities. One enzyme cleaves on the carboxy-terminal side of two basic residues such as Lys-Arg or Arg-Arg and is termed KEX2. The other enzyme is a dipeptidyl amino peptidase that selectively removes dipeptides from the amino terminus of proteins. The enzyme has a high specificity for Glu-Ala and Asp-Ala sequences. It may be that aprotinin inhibited the dipeptidyi amino peptidase activity.
Ngsee and Smith [6-] have investigated the effect of modflying the bovine prolactin signal sequence fused to the mature sequence of yeast invertase on secretion of this enzyme. The wild-type prolactin signal sequence is about one-sixth as efficient at causing secretion as the wildtype yeast invertase signal peptide. However, replacing the glycine residue located between the amino terminus and the central lipophilic region of the bovine prolactin signal peptide with an alanine results in production of invertase at a level equal to the wild-type yeast invertase signal sequence. This enhanced secretion suggests that the secondary structure of the bovine prolactin signal sequence is important for recognition by the yeast secretory apparatus. This conclusion is supported by other data. Replacing the bovine prolactin signal sequence codons to those preferred by yeast did not significantly increase invertase production. Also, truncating the 5' untranslated leader sequence of the bovine prolactin signal peptide increased invertase activity only slightly. Thus, the low level of secretion seen with the bovine prolactin signal peptide-invertase fusion was caused partly by the length of the 5' untranslated leader sequence. So a very small change in the amino acid sequence of the signal sequence can result in a tremendous improvement in signal processing and secretion efficiency of recombinant proteins in Saccharomyces.
Although not all secretion signal sequences are properly processed, in general P. pastoris processes the recombinant protein to an authentic bioactive product. There is still a great deal to b e learned about the selection and modification of both signal and secretion leader sequences.
Multicopy effects Most foreign protein genes expressed in P. pastoris strains have shown a copy number effect such that the larger the number of expression cassettes, the greater the amount of protein product produced. If a strain with a single copy expression cassette produces 100mg/1, then a six-copy strain is most likely to produce up to 1 g/l of protein product. Clare et al. [4.-] have shown a dramatic increase in the production of tetanus toxin fragment C as the copy number is increased from one to nine, but little or no change when the copy number is further increased to 14. We have generally limited our maximum copy number to six for ease of strain verification. In the case of aprotinin, the nature of the final product plays some part in the level of its production. A fivecopy strain that produced aprotinin with an amino-terminal extension of 11 amino acids approached the i g/1 yield level [5"], whereas a six-copy strain with a single Glu-Ala amino-terminal amino acid extension reached a maximum output of 700-800 mg/1. A recent review article [7"] reports that multiple-copy strains have been shown to be stable throughout scale up.
Strain improvements In all expression systems there is always room for continuous improvements and refinements. New Pichia strains have been developed at S1BIA that are deficient in some of the protease enzymes [8o]. These proteasedeficient strains display increased production of intact recombinant products and a noticeable decrease in what has been termed nicking. In the case of non-proteasedeficient strains, the protein product, insulin-like growth factor-l, is proteolysed at a particularly sensitive peptide bond. Integration of the insulin-like growth factor1 expression cassette into the protease-deficient strains reduces this nicking and results in a higher concentration of intact product. This ability to reduce the amount of proteolytic degradation of protein products has been tested by side-by-side analyses. Test peptides spiked into broth from control Picbia strains are always more susceptible to proteolysis than when spiked into proteasedeficient-strain broth.
Future work Post-translational modification of proteins in recombinant systems has always been a concern of investigators. The glycosylation of proteins in P. pastoris is similar in several respects to that observed in S. cerevisiae. In both organisms the majority of oligosaccaride chains added to proteins as they pass through the secretory pathway are the N-asparagine linked high-mannose type. However,
744
Expressionsystems there is at least one major difference. The size of the mannose side chains produced in Picbia are much smaller than the corresponding carbohydrates added by S. cerevisiae. Those in S. cerevisiae have from 50-150 mannose residues per chain, whereas in P. pastoris they are more likely to have 8-11 [9"]. In general, protein glycosylation in P. pastoris parallels the glycoprotein biosynthetic pathway in higher eukaryotes through the early stages of glycan processing, resulting in core structures that are very similar. As Pichia only adds the core structure [10], the immunogenicity of the Pichia produced recombinant protein products may not be a problem in certain therapeutic applications. Experimental testing of the immunogenicity of glycosylated proteins produced by Pichia pastoris will be critical to future work on potential human therapeutics.
3. ,.
BRIERLEYRA, BUSSINEAU C, KOSSON R, MELTON A, SIEGEL RS: Fermentation Development of Recombinant Pichta pa$toris Expressing the Heterologous Gene: Bovine Lysozyme. Biochem Engin V/1990, 589:350-362. An excellent paper, clearly describing the advantages of the MUT + Pichiapastorisstrains. Details of MUT + and M U T - fed batch protocols are given. Results showing production of Bovine lysozyme versus hours on methanol for the two types of strains show increased productivity uing MUT + strains. CLAREJJ, RAYMENTFB, B ~ SP, SREEKP,ISHNA K, ROMAN,S MA: High-level Expression of T e t a n u s Toxin Fragment C in Pichia pastoris Strains Containing Multiple T a n d e m Integrations of t h e Gene. Biotechnology 1991, 9:455-460. This paper shows both the effect of multicopy clones and MUT +, M U T - strains on production of tetanus toxin fragment C. Construction of the vectors and methods for inserting them into the Pichia starin are detailed. The mechanism for the formation of multicopy transplacement strains is also given 4. 00
5.
VEDV1CKT, BUCKHOLZ RG, ENGEL M, URCAN M, KINNEY, J, PROVOWS, SIEGEL RS, THILL GP: High-level Secretion of Biologically Active Aprotinin from t h e Yeast Pichia pastoris. J Ind Microbiol 1991, 7:197-202. Aprotinin analogs secreted at the gram per litre level are described. Problems effecting the aprotinin production were solved and are presented here. The aprotinin analogs were shown to be fully biologically active. ..
Conclusions P. pastoris has been shown to be an extremely productive recombinant protein expression system despite claims that no methylotrophs have been used for the production of heterologous proteins [11 ]. P. pastoris strains contain no antibiotic resistant genes [12] or other heterologous sequences that might be considered potential biological hazards. Secreted products from 3-90 kD have been expressed in pichia [7o,13-15]. Because of a combination of the extremely high cell densities reached by Pichia fermentation, and the stable integration of multiple copies of the expression cassette, yields of recombinant protein products in gram per litre quantities are routine. With the advent of the new protease-deficient strains, the production of proteins that have previously been difficult, if not impossible, to produce by recombinant methods, because of proteolysis, may now be possible.
6. •
NGSEEJK, SMITH M: Changes in a Mammalian Signal Seq u e n c e Required for Efficient Protein Secretion by Yeasts. Gene 1990, 86:251-255. This important paper describes the effect of changing a single amino acid in the signal sequence. The boost in enzyme production is described and a rationale is clearly presented. 7. •
BUCKHOtZ RG, GLEESON MG: Alternative Yeast Systems for t h e Commercial Production of Heterologous Proteins. Biotechnology 1991, in press. This excellent review describes in some detail all of the alternative expression systems to Saccbaromyces cerevisiae. GLEESONMG, HOWARD BD: Genes W h i c h Influence Pichia Proteolytic Activity, and Uses Thereof. Patent Application Docket 50848. This patent application is an excellent piece of work that clearly describes the construction of the two protease deficient strains produced. The enzyme activities eliminated and examples of improvement in peptide stability are given. 8. .
9. •
Acknowledgments I thank my colleagues Bob Siegel, Greg Holtz, Paula Schoeneck and Martin Gleeson for critically reading this manuscript. I thank Karen Payne for her patience and help in preparing this manuscript.
References and recommended reading
TRIMBLERB, ATKINSONPH, TSCHOPPJlg, TOWNSENDRR, MALEYF: Structure of Oligosaccharides on Saccharomyces SUC2 Invertase Secreted by the Methylotrophic Yeast, Pichia pastoris. J Biol Cbem 1991, in press. This important paper is written by the authority on glycosytation patterns in Pichia pastorix 10.
WEGNERGH: Emerging Applications of the Methylotrophic Yeasts. F ~ S Microb Rev 1990, 87:279-284.
11.
LINDSTROM ME, STIRLING DI: Methylotrophs: Genetics and Commercial Applications. Annu Rev Microbiol 1990, 44:27-58.
12.
CREGGJM, DIGAN ME, TSCHOPP JF, BRIERLEY RA, CRMG WS, VELICELEBI G, SIEGEL RS, THILL GP: Expression of Foreign G e n e s in Pichia pastoris. In Genetics and Molecular Biology of Industrial Microorganisms edited by Hershberger CL, Queener SW, Hegeman G [book]. Washington IX3: American Society for Microbiology, 1989, pp 343-352.
Papers of special interest, published within the annual period of review, have been highlighted as: • of interest •. of outstanding interest 1.
ELLIS SB, BRUST PF, KOUTZ PJ, WATERS AF, HARPOLD M, GINGERAS TR: Isolation of Alcohol Oxidase and Two Other Methanol Regulatable G e n e s From t h e Yeast Pichia pastori, Mol Cell Biol 1985, 5:1111-1121.
13.
HAGENSONMJ, HOLDEN KA, PARKERKA, WOOD PJ, CRUZEJA, FUKE M, HOPKINS TR, STROMANDW: Expression of Streptoldnase in Pichia pastoris Yeast. Enzyme Microb Techno11989, 11:6504556.
2.
CREGGJM, BARRINGER KJ, HESSLER AY, MADDEN KR: Pichia pastoris as a Host System for Transformants. Mol Cell Biol 1985, 15:3376-3385.
14.
SREEKRISHNAK, NELLES L, POTENZ R, CRUZE J, MAZZAFERROP, FISH W, FUKE M, HOLDEN K, PHELPS D, WOOD P, PARKERK: High,level Expression, Purification, and Characterization of
Gene expression in yeast: Pichia pastoris Vedvick
15.
Recombinant Human Tumor Necrosis Factor Synthesized in the Methylotrophic Yeast Pichia pastorix Biochemistry 1989, 28:4117-4125.
Saccharomyces cerevisiae. Adv in Biochemical Engineering/Biotechnology 1990, 43:75-102.
REISER J, GLUMOFFV, KALIN M, OCHSNER U: Transfer and Expression of Heterologous Genes in Yeasts Other Than
TS Vedvick, Salk Institute Biotechnology/Industrial Associates Inc., 505 Coast Boulevard South, La JoUa, California 92037, USA.
745
