FEMS Microbiology Reviews 87 (1990) 279-284 279 Published by Elsevier

FEMSRE 00210

Emerging applications of the methylotrophie yeasts

Gene H. Wegner

Research and Development, Phillips Petroleum Company. Bartlesville. OK, U.S.A.

Key words: Pichia pastoris; Expression systems; Alcohol oxidase

1. SUMMARY

The use of methylotrophi¢ yeasts for the pro- duction of single-cell-protein (SCP), alcohol oxidase and fine chemicals has been proposed. Fermentation technology developed for the growth of these yeasts on methanol at high cell densities has been commercialized. However, it is the pro- duction of heterologous recombinant proteins by Pichia pastoris that is emerging as the most sig- nificant application of the methylotrophic yeasts.

The utilization of methanol was subsequently shown to be restricted to a limited number of species of the genera Hansenula, Pichia, Torulopsis and Candida [2].

The initial use for these yeasts was directed at producing SCP from methanol. This potential ap- plication acted as a stimulant to much of the early research into the physiology, biochemistry and genetics of these interesting organisms and to the development of other significant applications. This paper describes the emerging applications of the methylotrophic yeasts and associated technologies.

2. INTRODUCTION

Durin~ the 1960s there was considerable inter- est in using the abifity of bacteria to grow on C 1 compounds to produce SCP from methanol. The utilization of C1 compounds by bt,~teria had been known since the beginning of this century and it was relatively easy to isolate cultures that grew well on methanol. During this period several corn- parties, most notably ICI and Hoechst, developed SCP processes based on methylotrophic bacteria.

These early successes with bacteria stimulated the search in several laboratories to determine if yeasts could also be found that would grow on methanol, and by the late 1960s yeasts with this ability were discovered [1].

Correspondence to: G.H. Wegner, Research and Development, Phillips Petroleum Company, Bartlesville, OK 74004, U.S.A.

3. SINGLE-CELL-PROTEIN FERMENTA- TION TECHNOLOGY

Probably the most extensive work on methanol- yeast based SCP has been done during the past two decades at Phillips Petroleum Company. Starting in the late 1960s, yeasts isolated from natural sources and obtained from culture collec- tions were screened for growth characteristics on methanol. Eveptually a strain of Pichia pastoris was selected which exlfibited stable fermentation characteristics and high yields of biomass and protein.

Using this yeast a continuous, high-cell-density process was developed [3] which featured an ex- ceptional fermentor design with oxygen transfer rates of S00-1000 mmol 02 1 -~ h -1. Cell densities of 125-150 g / l dry cell weight were obtained. This had the advantage that the fermentor broth

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Fig. 1. Provesta/Phillips 25 000-fiter high-cell.density fermentor.

could be dried directly without further concentra- tion. Although this high-productivity fermentation technology was developed to meet the extreme mass transfer requirements for growing methylo- trophic yeasts at high cell densities, it has found applications in the production of other yeasts and in bacterial fermentations [4].

A commercial-sczde plant employing a 25000- liter fermentor was started up, using this technol- ogy in the fall of 1988, by Provesta Corporation, a subsidiary of Phillips Petroleum Company (Fig. 1). This plant has the capability of producing a number of different yeast products, employing Torula, Saccharomyces or Pichia yeasts. Initially this facility has been used to produce specialty yeast products based on Torula yeast grown on sucrose for the human and animal food markets.

4. ALCOHOL OXIDASE

Alcohol oxidase catalyzes the initial reaction of methanol metabolism in yeasts, utilizing oxygen and producing formaldehyde and hydrogen per- oxide. Under conditions of methanol limitation in continuous culture at low dilution rates, this en- zyme may constitute 35% of the soluble protein. This enzyme, readily isolated from Pichia pastoris, is commercially available from Provesta Corpora- tion.

So far, the only commercial application of this enzyme has been as a diagnostic tool for determin- ing alcohol concentrations. Alcohol oxidase gener- ates hydrogen peroxide and has thus been consid- ered for use in detergents as a bleaching agent, especially by Unilever [5]. This oxidase has con-

siderable potential for generating hydrogen per- oxide and recently the enzyme from Pichia pasto- ris has been shown to produce hydrogen peroxide in excess of 6~ concentrations under the proper conditions (L. Nelles, personal communication).

Alcohol oxidase may be used for the produc- tion of aldehydes from alcohols. The oxidation of benzyl alcohol to benzaldehyde using two phase systems has been demonstrated recently [6]. This enzyme also has potential use as an oxygen scavenger.

5. FINE CHEMICALS PRODUCTION

The use of the methylotrophic yeast for the biosynthesis of fine chemicals has emerged as another potential application of these organisms. Production of several different amino acids has been reported including alanine, glutamic acid, lysine, threonine, valine and tryptophan [7,8]. Methyl ketones were found to be produced by a secondary alcohol dehydrogenase from methylo- trophic yeasts by EXXON scientists [9]. Tani and co-workers have reported on a process to produce ATP using Candida boidinii, and they have also studied the formation of formaldehyde [10]. Dihy- droxyacetone and glycerol were found to be pro- duced by a dihydroxyaeetone kinase-deficient mutant of Hansenula polymorpha, an example of over-production of metabolites directly related to methanol assimilation [111.

6. HETEROLOGOUS GENE EXPRESSION IN PICHIA PASTORIS

6.1. Development of express-on systems Recent research has demonstrated the high-level

expression of heterologous proteins in the methylotrophic yeast Pichia pastoris [12,131. This emerging use of the methylotrophic yeasts may be their most important application. The develop- ment of expression systems utilizing this yeast is the culmination of research initiated in the early 1980s, which, in turn, was catalyzed by earlier observations and research with P. pastoris in SCP production.

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Although alcohol oxidase (AOX), dihydroxy- acetone synthase and other methanol regulated genes have been isolated [14], it is the regulatory sequences from the highly expressed and tightly controlled AOX gene that have been most used to drive expression of heterologous protei~;~ ,~n P. pastoris.

In the Pichia system a histidinol dehydrogenase mutant GSl l5 has been used as the host strain, and the HIS4 gene isolated from w~dd-type P. pastoris has been incorporated into expression vectors as the selectable marker for transforma- tions of this mutant [15]. Two basic types of expression vectors have been developed, autono- mous replication vectors and integration vectors. Integrat, ions can be site-directed at the HIS4 gene or at the primary alcohol oxidase locus (AOX1). This yeast strain also contains a secondary alcohol oxidase gene (AOX2) which allows reduced growth rate on methanol even when the AOX1 gene i:- displaced by integration of the vector [16].

Taking advantage of these characteristics, a second-generation integrative expression vector specifically designed for site-directed integration of an expression cassette at the AOXI chro-- mosomal locus was developed [17]. The expression cassette contains the 5'-controlling sequences, the foreign gene, the HIS4 gene and a 3'-flanking AOXI region. This linear cassette is released from the circular vector by Bglll digestion, is readily intJodueed into the host strain and is stably in- tegrated. Transformants that exhibit the His + phenotype and slow growth on methanol are indi- cative of having the A OX1 gene replaced by the expression cassette.

In batch fermentations, strains with the metha- nol-slow-growth phenotype are usually grown up on 5-10% glyce:ol and after exhaustion of the glycerol, expression is turned on by feeding methanol. For continuous fermentations, glycerol- methanol mixtures have been used. Several mod- ifications of these basic procedures have been successfully employed with different constructs [18,191.

6.2. Production of intracellular proteins The Pichia system has been used by scientists

at Phillips Petroleum Company and Salk Institute

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Table 1 Proteins expressed in Piclffa pastoris

Intracellular proteins Secreted proteins Tumor necrosis factor Invertase TNF analogs Human serum albumin Streptokinase Tissue plasminogen activator Hepatitis B surface antigen Bovine lysozyme fl-Galactosidase Salmon growth hormone Mature IL-2 Gamma interferon Human serum albumin HIV antigens

Biotechnology and Industrial Associates (SIBIA) to express a variety of proteins (Table 1). These proteins were from such diverse sources as hu- mans, fish, bacteria and viruses, an indication of the versatility of this system. Hagenson [20] has recently reviewed production of recombinant pro- reins in P. pastoris.

Sreekrishna and co-workers [12,18] at Phillips obtained expression levels exceeding 30% of the soluble protein with tumor necrosis factor (TNF) in P. pastor&. TNF expression was stably main- rained during high-cell-density fermentation (100 g dry cell weight/l) and resulted in a TNF pro- duction level of 6-10 g/ l .

Exceptional results were also obtained with hepatitis B surface antigen (HBsAg) [21]~ Expres- sion levels of 3-4~ of the soluble protein were observed in a strain in which the AOX1 gene was deleted and replaced by the expression cassette. Although the concentration of HBsAg protein was comparable in S. cerevisiae and P. pastoris, the concentration of antigenically active HBsAg 22 nm particles in P. pastoris (2-3% of soluble pro- rein) was at least 10-times higher.

6.3. Secretion of heterologous proteins Secretion of beterologous proteins by P. pasto-

ris has been demonstrated by SIBIA and Phillips scientists using this system (Table 1). Both yeast and mammalian secretion-signal sequences have been found to function with this yeast [13,19].

Using the SUC2 gene from S. cerevisiae behind the AOX1 promoter in Pichia, Tschopp and co-

workers [13] at SIBIA found that invertase secre- tion into the medium reached levels of up to 2.5 g/1. In S. cerevisiae this enzyme is secreted into the periplasmic space. The invertase produced by P. pastoris was found to contain much less carbohydrate than that produced by S. cerevisiae and the giycosylation pattern resembled that of higher animals, which indicates that P. pastoris may be a superior host for production of glyco- proteins for human therapeutic use [22].

Efficient secretion of human serum albumin in excess of 1 g / l also has been demonstrated (J. Tschopp and K. Sreekrishna, personal communi- cation).

7. CONCLUSIONS

The initial .application of the methylotrophic yeast was during the early 1970s when there was considerable interest in using them to produce SCP. This application has not yet been com- mercialized because of economic constraints, al- though the high-cell-density fermentation technol- ogy developed for the methanol-yeast process has been put into commercial use by Provesta Corpo- ration to produce higher-value-speciality yeast products.

Alcohol oxidase continues to enjoy a small market as a diagnostic enzyme. However, poten- tially large volume applications, such as to pro- duce aldehydes or hydrogen peroxide, are yet to emerge.

Perhaps the most significant application of the methylotrophic yeast will be as a host organism for the production of heterologous proteins. High-level expression systems have been devel- oped, employing the tightly regulated and highly expressed AOX1 regulatory sequences in P. pasto- ris. Moreover, this yeast possesses genetic and physiological features that makes it an excellent host for production of recombinant proteins. In the future it may even be possible to target foreign proteins to the peroxisomes in these yeasts to protect them from proteolysis.

Philfips Petroleum Company has been licensing the P. pastoris high-level expression system since 1988, and with this tool now in the hands of a

growing n u m b e r o f scientists, this use of the metho ylo t rophic yeas ts is emerg ing as their m o s t note.- wor thy appl icat ion.

A C K N O W L E D G E M E N T S

T h e au th o r is indebted to the persona l a n d profess iona l associa t ion a n d effor ts o f the 150 or so pe r sons at Phill ips a n d SIBIA, who, in the last two decades, have t aken initial observa t ions and research wi th P. pastoris as a SCP o r g a n i s m a n d used t hem in the deve lopmen t o f commerc ia l processes for the p roduc t ion o f yeas t p roduc ts , a lcohol oxidase a n d the express ion o f he te ro logous prote ins . W i t h o u t t h e m an d their con t r ibu t ions th is paper could n o t have been wri t ten,

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