Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 16, Num. 2, 1999

Conventional and Non-conventional Yeasts in Modern Biotechnology
José A Cremata

Structural Analysis of Glycoproteins. Its Importance
Javier Menéndez Díaz

División de Biotecnología Industrial, Centro de Ingeniería Genética y Biotecnología. AP 6162,
CP 10600. Ciudad de La Habana, Cuba. Fax: (53-7) 21 8070. E-mail: yeastlab@cigb.edu.cu

Introduction

Yeasts are of benefit to mankind because they are widely used for the production of food, wine, beer, and a variety of biochemicals. Yeasts also cause spoilage of food and beverages and are of medical importance. At present, more than 700 yeast species are recognized, but only a few are commonly known. Relatively few natural habitats have been thoroughly investigated for yeast species, consequently, we can assume that many more species await discovery. Because yeasts are widely used in traditional and modern biotechnology, the exploration for new species should lead to additional novel technologies. Several definitions have been used to describe the yeast domain. In general, it is assumed that yeasts are unicellular fungi which reproduce by budding or fission.

At present, yeasts are widely used, not only in several industrial processes as it was aforementioned, but also as:

• models for the study of gene regulation in eukaryotic cells

• bio-factories for the expression of homologous and heterologous genes

These two aspects were extensively analyzed in the Congress Biotecnología Habana´98, held at the Center for Genetic Engineering and Biotechnology (CIGB), in Havana, Cuba.

Regulation of gene expression

The budding yeast Saccharomyces cerevisiae is a unicellular eukaryote that can exist in either haploid or diploid states. This organism has been, and will be, the focus of much research. Regarding cancer, there were two main discoveries leading to interest in yeast. First, the finding that S. cerevisiae possesses two RAS genes homologue of the human RAS oncogenes suggested that the mechanism of growth control must be similar in S. cerevisiae and in mammals. Second, about two decades ago, Leland Hartwell isolated yeast mutants defective in progression through different stages in the cell cycle. These cdc mutants have been characterized and most of the corresponding genes isolated. Perhaps not unexpectedly, many of those genes have counterparts in higher eukaryotes, which suggests that basic control of the cell cycle is universal. The fact that imbalance in this mechanism could lead to uncontrolled proliferation (i.e. cancer) brings yeast into focus again.

In S. cerevisiae, the availability of mutants in different loci, the implementation of genetic and molecular biology techniques and the easy transfer of foreign genetic material into its genome, makes this organism an excellent choice for studies on the cell cycle and the mechanism of growth control. The power of yeast genetics has contributed to the study of eukaryotic signaling pathways and transcriptional regulation as well.

The availability of nutrients in the medium results in the generation of signals from the cell surface to the nucleus. These multiple signals are mediated by intracellular signaling pathways. The yeast cell responds to the signals by changing the expression of many genes, according to the physiological status of the cell. For example, the addition of glucose to glucose-deprived cells triggers several metabolic changes, which exerts a major influence on the fermentation rate and other industrially important properties such as stress resistance. In this sense, the expression of genes involved in the utilization of carbon sources other than glucose is tightly regulated. The expression of these genes is induced by the carbon sources, and the level of enzymes for the utilization of low-carbon and low-phosphate sources is usually decreased when a better source is available.

Two of the best characterized examples of this regulation were presented by Dr. Juana María Gancedo and Dr. Cornelis Hollenberg, from the Instituto de Investigaciones Biomédicas in Spain and the Institut für Mikrobiologie, Heinrich-Heine-Universität in Düsseldorf, Germany, respectively. These works deal with the control of gluconeogenesis in yeast and the repression of genes encoding galactose-metabolizing enzymes.

Both processes are controlled by the Snf1 complex. The SNF1 gene (also called CAT1 or CCR1) is absolutely required for the derepression of genes repressed by glucose. This gene encodes a Ser/Thr protein kinase; in particular, it has a mammalian homolog, the catalytic a subunit of the AMP-activated protein kinase. A major function of the Snf1 kinase pathway is to control glucose repression by the Mig1 DNA-binding repressor protein.

Expression of foreign genes in non-conventional yeasts

For years, several biotechnological companies have used glycolytic or regulable promoters from S. cerevisiae in order to express heterologous genes. However, the interest in the study of non-conventional yeasts (yeasts other than S. cerevisiae and Schizosaccharomyces pombe) has dramatically increased in the past few years. The methylotrophic yeasts Pichia pastoris, Hansenula polymorpha, and Candida boidinii are an example within this category. They use methanol as the sole source of carbon and energy. In the particular case of P. pastoris, a very successful system for the expression of heterologous genes has been developed. Several factors have contributed to this approach:

• the use of a strong and inducible promoter from the alcohol oxidase 1 ( AOX1) gene

• P. pastoris is easy to manipulate genetically like S. cerevisiae

• there is a strong preference for aerobic metabolism

At present, more than 100 different proteins have been successfully produced in P. pastoris. In this sense, an alternative host-vector system based on a his3- mutant was developed in this yeast at the CIGB (Havana, Cuba). Using this system, several important industrial enzymes such as invertase, dextranase, and a-amylase have been expressed to a higher level.

Another important industrial yeast is Candida utilis. This microorganism is generally recognized as safe and for this reason it is allowed to be used as a food additive. For several years, the main difficulty in the employment of this yeast for expressing proteins was the lack of an efficient transformation system. However, this problem was recently overcome by two groups that developed two efficient transformation systems: one based on the use of a drug-resistance gene and the other on the use of an auxotrophic mutant defective in the URA3 gene.

Finally, all the examples described above for the use of yeast in the study of gene regulation and for the expression of foreign genes, are part of several works presented in the Congress Biotecnología Habana'98. The abstracts of these works are available in this issue of Biotecnología Aplicada, supporting the importance of conventional and non-conventional yeasts in modern biotechnology.

Snf1 protein kinase and transcriptional regulation in response to glucose

Marian Carlson

The Snf1 protein kinase, which has been widely conserved in eukaryotes, is essential for the regulatory response to glucose starvation in the yeast Saccharomyces cerevisiae. The kinase is found in complexes containing an activating subunit (Snf4) and a member of a family of scaffolding proteins (Sip1, Sip2, Gal83). Evidence indicates that activation of the kinase in response to glucose limitation is accompanied by conformational changes in the complex. Protein phosphorylation is implicated in the control of Snf1 kinase activity, and protein phosphatase 1 and its regulatory subunit Reg1 associate with the kinase complex. Snf1 is required for transcription of a large set of glucose-repressed genes in response to glucose limitation. A major function of the Snf1 kinase pathway is to control glucose repression by the Mig1 DNA-binding repressor protein, but Snf1 also regulates the function of transcriptional activators, including Sip4 and Cat8.

Departments of Genetics and Development and Microbiology. Columbia University, HHSC 922. Box 136 701 W. 168th Street, New York, NY 10032, USA. E-mail: mbcl@columbia.edu

Control of gluconeogenesis in yeast: a complex regulatory network

Juana M Gancedo

Growth on non-sugar carbon sources requires gluconeogenesis, which proceeds by a reversal of glycolysis except for two steps, catalyzed by phosphoenolpyruvate carboxykinase (PEPCK), and fructose-1,6-bisphosphatase (FbPase). The operation of these enzymes is not required when sugars are present in the medium and their switching off is ensured by a system of multilayered regulation. The increased concentration of fructose-2,6-bisphosphate in the presence of glucose inhibits FbPase and facilitates its phosphorylation by the cAMP-dependent protein kinases. Glucose provokes also the proteolytic degradation of FbPase and PEPCK. Finally, glucose represses strongly the transcription of the corresponding genes, FBP1 and PCK1, and decreases moderately the stability of the PCK1 mRNA. The regulation of the transcription of the gluconeogenic genes involves the interplay of positive and negative elements in their promoter under the control of Cat1, Cat8 and Mig1. In addition, cAMP downregulates the transcription of these genes. The physiological relevance of the different regulatory mechanisms will be discussed.

Instituto de Investigaciones Biomédicas. CSIC Madrid, España.

Regulation of PYC1 and PYC2 genes of the yeast Saccharomyces cerevisiae and identification of regulatory regions in their promoters

Javier Menéndez,1 Carlos Gancedo2

We investigated the regulation of the expression of PYC1 and PYC2 genes encoding isoenzymes of piruvate carboxylase from Saccharomyces cerevisiae. In order to obtain information concerning the regulation of gene expression, a fusion genes which consists of the 5' upstream region of PYC1 and PYC2 were fused to the lacZ gene from Escherichia coli and introduced into S. cerevisiae. b-galactosidase activities were measured from cells grown in glucose, ethanol and pyruvate. While b-galactosidase activity measured when the gene was expressed under the control of PYC2 promoter were similar in the conditions tested; the expression of the enzyme under the control of the PYC1 promoter seemed to be regulated by the carbon source.

Deletion analysis of PYC1 promoter revealed the presence of two elements involved in its regulation in different culture conditions. One of these two elements is a UAS located in the region between -330 and -297. This element was able to activate transcription in reporter plasmid and formed specific nuclear protein complexes in bind-shift experiments. This protein complex was abolished in rtg mutants, which suggest a role for the RTG genes in the control of PYC1 expression. On the other hand, three repressing sequences with the common central core CCGCC at positions
-457, -432 and -399 were identified.

By analyzing the expression of b-galactosidase gene from PYC2 promoter in cells grown in repression and derepression conditions, we identified a positive cis-acting element between positions -417 and -291, necessary for the expression in repression conditions. This element was able to activate transcription in the reporter plasmid. However, in our conditions we could not detect the formation of specific protein complexes when a DNA fragment corresponding to this region was used as probe in bind-shift experiments.

1Centro de Ingeniería Genética y Biotecnología, Ave. 31 e/ 158 y 190. AP 6162, CP 10600, Ciudad de La Habana, Cuba. E-mail: yeastlab@cigb.edu.cu 2Instituto de Investigaciones Biomédicas CSIC. Unidad de Bioquímica y Genética de Levaduras "Arturo Duperier" No 4, 28029 Madrid, España. E-mail: cgancedo@biomed.iib.uam.es

Interaction between Gal1p or Gal3p and Gal80p is required for induction of galactose in yeast

Ruth Engels, Verena Vollenbroich, Regina de Andrade Menezes, Cornelis P Hollenberg

When galactose is added to growing cultures of the yeast Kluyveromyces lactis, a set of genes encoding galactose-metabolizing enzymes of the so called Leloir pathway is induced. Gal1p carries out two functions in this lactose/galactose pathway. Interaction of Gal1p with the transcriptional repressor protein Gal80p in the presence of galactose leads to a rapid derepression of Gal4p-activated genes, whereas the galactokinase activity of Gal1p, as the first protein of this pathway, converts galactose into galactose-1-phosphate.

Here, we present genetic evidence obtained via a two-hybrid assay that demonstrates the interaction between Gal1p and Gal80p in vivo. The interaction depends on the presence of galactose in the medium, but not on the catalytic activity of the Gal1 protein. Mutants were isolated, in which either the regulatory or the catalytic function of Gal1p was eliminated, indicating that the two functions are separable. Mutations that lead to a loss of derepressing activity but not of galactokinase activity, were localized to two different regions that are conserved in Gal3p, ScGal1p and KlGal1p. Experiments with KlGal1p-Gal3p hybrids demonstrated that these fusions still perform regulatory activity and that both the N- and C-terminal half of Gal3p are involved in the specific interaction with Gal8p. Deletion of other N- or C-terminal regions of lexA-Gal1p leads to the loss of interaction with VP16-Gal80p, indicating that the intact structure of the galactokinase is required for protein-protein interaction.

Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf, Germany. E-mail: cornelis.hollenberg@uni-duesseldorf.de

The synthesis of trehalose: an important pathway in the life of yeasts

Carlos Gancedo, Oscar Zaragoza, Miguel A Blázquez

Trehalose (O-a-D-glucopyranosyl-a(1®1)-D-glucopyranoside) is an important reserve carbohydrate in yeast. Different results from our laboratory indicate that the sugar itself or its precursor trehalose-6-phosphate are important metabolites for a normal life of different yeasts. Evidence leading to this idea is derived from experiments with three different yeast species:

1) Evidence from Saccharomyces cerevisiae. Mutants affected in the TPS1 gene encoding trehalose-6-phosphate synthase do not grow on glucose. One type of suppressor of the glucose negative phenotype had a decreased level of hexokinase with respect to the wild type. This finding together with the metabolite profile of the mutant led to the idea that either trehalose or trehalose-6-phosphate could control the activity of hexokinase. It turned out that trehalose-6-phosphate inhibits hexokinase. We hypothesize that trehalose-6-phosphate plays a role in controlling the glycolytic flux in S. cerevisiae.

2) Evidence from Schyzosaccharomyces pombe. Hexokinase 2, the predominant hexokinase in the fission yeast, is exceptional in not being inhibited by trehalose-6-phosphate. Consistent with the hypothesis that trehalose-6-phosphate controls the glycolytic flux through its effect on hexokinase is the result that in this yeast, mutants afected in the tps1+ gene grow normally on glucose. However the spores carrying the mutation do not germinate. A mechanistic explanation for this is not evident but it suggests a role for trehalose either in germination or in the maintenance of spores.

3) Evidence from Candida albicans. Growth on glucose at 30 °C is not affected in mutants carrying disrupted copies of the TPS1 gene in both chromosomes. However the yeast-hyphae transition is blocked in the mutants in certain conditions and they are less infective than the wild type. Either disturbances produced by an altered glycolytic pathway or other effects of the lack of trehalose-6- phosphate or trehalose could be the origin of this change in behavior.

All these facts indicate the crucial role of the elements of the trehalose biosynthetic pathway for the normal life of different yeasts.

Instituto de Investigaciones Biomédicas Alberto Sols. C.S.I.C. Unidad de Bioquímica y Genética de Levaduras. C/Arturo Duperier 4. E-28029 Madrid. España. E-mail: cgancedo@iib.uam.es

Development of the methylotrophic yeast Pichia pastoris as a system for the production of heterologous proteins

James M Cregg

The methanol-utilizing yeast Pichia pastoris is a highly successful host system for the production of heterologous proteins. P. pastoris is readily grown on defined medium in continuous culture at high volume and density. The yeast does not have a strong tendency to ferment, a significant advantage since a product of fermentation, ethanol, can rapidly build to toxic levels in high-density cultures. The promoter employed to drive heterologous gene expression is derived from the methanol-regulated alcohol oxidase 1 gene (AOX1) of P. pastoris, one of the most efficient and tightly regulateds promoters known. The strength of the AOX1 promoter often results in high expression levels even in strains harboring only a single integrated copy of a foreign gene expression cassette. Levels may often be further enhanced through the integration of multiple cassette copies into the P. pastoris genome. These integrated expression cassettes are stable and not subject to problems associated with plasmid-based expression systems. The system is particularly attractive for the secretion of foreign gene products. Because P. pastoris endogenous protein secretion levels are low, foreign proteins often appear to be virtually the only proteins in the culture broth, a major advantage in purification. P. pastoris adds oligosaccharides to foreign secreted proteins that have high-mannose structures typical of lower eukaryotic organisms.

Dept. of Biochem. and Mol. Biol., Oregon Graduate Institute of Science and Technology, Portland, OR 97291, USA.

New promoters for the expression of foreign genes in Pichia pastoris

Geoffrey P Lin Cereghino, Anthony J Sunga, Joan Lin Cereghino, James M Cregg

The methylotrophic yeast Pichia pastoris has been exploited as a host system for the production of foreign proteins of commercial and academic interest. In this system, foreign proteins are typically synthesized under the transcriptional control of the AOX1 promoter obtained from the methanol-regulated alcohol oxidase 1 gene of P. pastoris. Although the AOX1 promoter has proven to be very effective, its use is not appropriate in some situations. As alternatives for the expression of certain genes, we have isolated three new P. pastoris promoters. One is from the glyceraldehyde-3-phosphate dehydrogenase gene (GAP) which strongly and constitutively expresses foreign genes, and is convenient for the synthesis of products that are not deleterious to the yeast. A second one is from the formaldehyde dehydrogenase 1 gene (FLD1), which strongly induces expression in response to either methanol as a carbon source or methylamine as a nitrogen source. This promoter is useful for regulated expression of a foreign gene under conditions where methanol is not appropriate. A third promoter is from PEX8, a gene required for peroxisome biogenesis, which, like the AOX1 promoter, is methanol regulated but is not that strong. Together, these promoters provide a variety of regulation modes and expression levels for the P. pastoris system.

Dept. of Biochem. and Mol. Biol., Oregon Graduate Institute of Science and Technology, Portland, OR 97291, USA.

Recent advances in the use of Pichia pastoris as a heterologous gene expression system

Michael Galleno

A series of six new Pichia expression vectors have been constructed that are between 3.3 and 3.5 kB in size, yet still contain all of the elements for recombinant protein expression and detection in the methylotrophic yeast, Pichia pastoris. Selection for these vectors in both Pichia and E. coli is based on a single small dominant selectable marker that confers resistance to the drug Zeocin. Expression of the zeocin-resistance gene is driven by the S. cerevisiae promoter TEF1 and is followed by the S. cerevisiae CYC1 transcription processing and polyadenylation sequence. Versions with and without the S. cerevisiae alpha factor prepro sequence for targeting expressed protein for secretion exist as well as an expanded multiple cloning site with 10 unique restriction sites. Optional C-terminal myc epitope and polyhistidine tags are included that can be used for recombinant protein detection and purification. These vectors contain a unique BglII site five prime to the AOX1 promoter sequence and a unique restriction enzyme site down stream to the Pichia transcriptional termination sequence to facilitate the generation of in vitro multimers. Data will be presented detailing the use of these vectors for transformation, cloning, and expression of tagged intracellular and secreted proteins.

Invitrogen 1600 Faraday Ave. Carlsbad,CA 92008.E-mail: mgalleno@invitrogen.com

Expression of foreign genes in the yeast Pichia pastoris

Geoffrey P Lin Cereghino, Anthony J Sunga, Joan Lin Cereghino, James M Cregg

The yeast Pichia pastoris is a highly successful system for the production of heterologous proteins. P. pastoris is readily grown in continuous culture at high volume and density. The yeast does not have a strong tendency to ferment, which is a significant advantage since a product of fermentation, ethanol, can rapidly build to toxic levels in high-density cultures. The promoter employed for expression of most heterologous genes is derived from the methanol-regulated alcohol oxidase I gene (AOX1), an exceptionally efficient and tightly regulated promoter. The system is particularly attractive for the secretion of foreign gene products. Because P. pastoris endogenous protein secretion levels are low, foreign proteins often appear to be virtually the only proteins in the culture broth, a major advantage in purification. Although the AOX1 promoter has proven to be very effective, its may not be appropriate in some situations. As alternatives, three new P. pastoris promoters have been developed and are described. Together, these promoters provide a variety of regulatory modes and expression levels for the P. pastoris system.

Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, PO Box 91000, Portland, OR 97291-1000, USA.

Ten years expressing proteins in Pichia pastoris: Our experience

Luis Rodríguez

The methylotrophic yeasts, in particular Pichia pastoris has shown to have the potential for very high level production of foreign proteins. Pichia pastoris is an industrial methylotrophic yeast initially chosen for production of single-cell protein due to its ability to grow to very high cell density in simple defined media using methanol as a sole carbon source and energy. The favorable and most advantageous characteristics of these species have resulted in an increasing number of biotechnological applications. Numerous proteins, over 100 have been expressed in Pichia pastoris with varying degrees of success.

Our expression system is based on the AOX1 promoter, which is induced with methanol, a his3- mutant as host, and HIS 3 gene from S. cerevisiae as selective marker. We have used this system to express different heterologous proteins of industrial and medical interests. Most of the constructions included secretion signals but other were designed to keep the protein intracellularly, including among others: sucrose invertase from S. cerevisiae, Alpha-amylase from
B. licheniformis, dextranase from P. miniolluteum, Neutral protease from B. amyloliquefaciens, HBAg and scFv. The expression level in all these examples reached excellent yields over 1 g/ L in single copy gene integration pattern.

Centro de Ingeniería Genética y Biotecnología, Ave. 31 e/ 158 y 190. AP 6162, CP 10600, Ciudad de La Habana, Cuba.

Preliminary comparison of dextranase expression in two different host strains of the methylotrophic yeast Pichia pastoris

Bianca García, Efraín Rodríguez, Tanilo Rivero, Yurima Hidalgo, Javier Menéndez

The expression of dextranase encoding gene (Dex) into two different host strains of the methylotrophic yeast Pichia pastoris (MP36 and GS115) was compared. The Dex gene, encodes a mature protein of 574 amino acids and it was expressed using the SUC2 signal sequence from Saccharomyces cerevisiae under the control of the AOX1 promoter. In MP36 strain (his3-) all transformants integrated the expression cassette and secreting an active enzyme to the medium after methanol induction. However, in GS115 strain (his4-), only 44% of transformants expressed the active enzyme. We also found differences in the expression level between both strains, being in the MP36 expressing transformants higher than in the GS115 transformants.

Centro de Ingeniería Genética y Biotecnología, Ave. 31 e/ 158 y 190. AP 6162, CP 10600, Ciudad de La Habana, Cuba. E-mail: yeastlab@cigb.edu.cu

The food yeast Candida utilis as a host for heterologous gene expression: Construction of a vector system for high-level expression

Keiji Kondo

The yeast Candida utilis is generally recognized as safe and is allowed to be used as a food additive. The transformation of yeast has recently become possible by using a drug resistance gene, which was constructed from C. utilis endogenous gene encoding ribosomal protein L41, as a marker. A vector system that facilitates high-level expression of a heterologous gene was also developed. The vector contains the strong promoter of the glyceraldehyde-3-phosphate decarboxylase gene from C. utilis and a promoter-deficient marker gene that allows high-copy-number integration of the vector. The vector has a unique structure that permits integration of the minimum sequences for gene expression and selection of the transformants at their target loci by single crossover recombination without the accompanying bacterial sequences. Application of this vector system to expression of a single-chain monellin gene encoding a plant sweet protein and a bacterial a-amylase gene resulted in extremely high-level expression, accounting for more than 50% of the total soluble protein. No significant decrease in the production level of both proteins was detected after 50 generations of non-selective growth, indicating that the yeast C. utilis is a promising host for protein production.

Central Laboratories for Key Technology, Kirin Brewery Co., Ltd. Fukuura, Kanazawa-ku, Yokohama-shi, Kanagawa 236, Japan. E-mail kondok@kirin.co.jp

Development of an integrative DNA transformation system for the yeast Candida utilis

Luis Rodríguez, Francisco P Chávez, Tanilo Rivero, Liliana Basabe, María E González, Julio M Delgado

We report here the development of the yeast Candida utilis as a system for gene cloning and manipulation. To facilitate molecular studies in C. utilis, we isolated auxotrophic strains for uracil biosynthesis. A genomic library from the yeast C. utilis has been constructed and employed to isolate the URA3 gene, encoding orotidine-5´-phosphate decarboxylase enzyme, by complementation in Escherichia coli pyrF and Saccharomyces cerevisiae ura3 mutations. The deduced amino-acid sequence is highly similar to that of the Ura3 proteins from other yeasts. An extensive analysis of the family of orotidine-5´-phosphate decarboxylase is shown. Integrative plasmids were constructed based on the cloned C. utilis URA3 gene and are applicable for directed insertions of genes of interest at the URA3 locus through homologous recombination.

Bioindustry Division, Centro de Ingeniería Genética y Biotecnología. Ave. 31 e/ 158 y 190. AP 6162, CP 10600, Ciudad de La Habana, Cuba. E-mail: YeastLab@cigb.edu.cu

José A Cremata

División de Biotecnología Industrial, Centro de Ingeniería Genética y Biotecnología. AP 6162,
CP 10600. Ciudad de La Habana, Cuba. Fax: (53-7) 21 8070. E-mail: bioind@cigb.edu.cu

The meeting Biotecnología Habana'98 was held last November 16-21 at the Center for Genetic Engineering and Biotecnology in Havana, Cuba, focusing the attention of researchers of many countries all over the world on two fields: transgenesis and biotechnology applied to the industry. A general and exciting aspect was deeply discussed: the structural characterization of glycoproteins. Immediately, a question rises: why the interest in glycosylation analysis?

The answer could be: the importance of product glycosylation has led to a more detailed carbohydrate analysis since the beginning of the development of a new product. Improvements in glycosylation analysis have given scientists the possibility to judge how the glycan structures of recombinant glycoproteins compare to their natural human counterparts. Moreover, to keep the products' bioactivity or pharmacokinetics in vivo is in many cases a matter of maintaining unaltered the specific glycan structures present in their natural form. However, to clearly understand the importance of glycosylation in biotechnologically produced glycoproteins, it is necessary to go inside the characteristics of this phenomenon.

The biosynthesis of protein-linked carbohydrate chains involves co- and post-translational events. This implies a genetic control over the structure of the final sugar chains produced, leading to the inherent heterogeneity of the glycosylation pattern. Consequently, a glycoprotein is generally composed of a discrete family of differently-glycosylated molecules called glycoforms, which have different physico-chemical and biochemical properties. This may, in turn, lead to functional diversity.

In many cases, it is not possible to directly associate a particular biological function with a specific carbohydrate structure. Structure-function relationships must be found in the context of the glycosylation pattern. Some are trivial physico-chemical effects that may, in turn, affect the biological function of the glycoprotein in a more complex way, for example, antigenicity, stability, half-life in plasma, intracellular routing, organ targeting, and cell-glycoprotein or cell-cell recognition event.

Glycosylation can be described as an event in which a considerable number of enzymes (glycosidases and glycosyltransferases) are involved as well as a process of an enormous energy requirement, then it is easy to understand that biotechnological glycoprotein production is influenced by several factors.

A biotechnological process consists of a synthesis and an isolation step. In the first one, the glycoprotein is obtained from producing cells. In the isolation phase, the glycoprotein is isolated and purified to "homogeneity". The synthesis and recovery procedures of the product are optimized. The final goal is to manipulate metabolic processes in genetically engineered strains to obtain a desired co- or post-translational modification of the product, to increase production rates and/or to reduce the formation of side-products. Hence, the initial choice of an expression system will continue to be of crucial importance.

The perspective to direct protein glycosylation by engineering strategies in order to obtain protein-linked carbohydrate chains, which are profitable for the pharmaceutical properties of the glycoprotein, is therefore attractive. For example, Gal-specific receptors on hepatocytes and macrophages do not recognize glycoproteins exhibiting their glycan moities capped with sialic acids. The masking of the b-Gal prevents early endocytosis, and hence reduces the clearance of the glycoprotein. This implies reduced clearance, which is correlated with the administration of lower doses of a therapeutic agent. Low doses are usually associated with fewer side effects. Furthermore, the development of glycosylation engineering strategies may facilitate elimination or masking of potential carbohydrate antigens avoiding undesirable immune responses. Glycosylation engineering deals with these aspects and shows that protein glycosylation can be influenced at each stage during a biotechnological process.

Glycosyltransferases and glycosidases, which are necessary for the biosynthesis of certain carbohydrate elements, are co-expressed with recombinant glycoproteins. For example, a heterologous rat a-2,6-sialyltransferase has been expressed in Chinese hamster ovary (CHO) cells. Whereas glycoproteins from CHO cells do not contain a-2,6-linked sialic acids, the a-2,6-sialyltransferase transfectoma CHO cells show the occurrence of surface glycoproteins containing a-2,6-linked sialic acid. The glycan moitey of a glycoprotein may be also remodeled in vitro using purified glycosyltransferases and/or glycosidases.

Summarizing, the objective of glycosylation engineering is to control the aspects influencing the glycosylation pattern of the product. Proteins unappropriately glycosylated may have undesired pharmacokinetics, possibly causing side effects during and after administration. The glycosylation engineering approach is therefore important to eliminate variations in the glycosylation pattern of a given glycoprotein within batch-to-batch productions, in such a way that the pharmaceutical properties of the molecule are not influenced negatively.

The number of biotechnologically produced glycoproteins approved for therapeutic use in humans is growing. Therefore, batch-to-batch consistency analysis of glycoprotein production, in terms of protein glycosylation, is needed. Currently, methods based on oligosaccharide profiling for batch control of glycoproteins are being developed. Identification of carbohydrate chains in oligosaccharide-mapping methods is commonly based upon the migration in electrophoresis and/or the retention time in chromatographic separation compared to standard compounds. Most of the mapping methods are restricted to neutral carbohydrate chains, so sialylated glycans have to be desialylated before mapping. In these approaches, the unraveling of native sialylation patterns is ignored. This type of mapping methodologies allows a fast comparison of batch-to-batch protein glycosylation. Moreover, when these methodologies are used in conjunction with lectin affinity and exoglycosidase digestion, the structural features of the oligosaccharides are described in detail. Many of these profiling methods are based on the fluorophore derivatization of the oligosaccharide population, which remarkably increases the sensitivity of the analysis.

Procedures using Mass Spectrometry as a detection system have overcome the problems associated with oligosaccharide-mapping methods where an incomplete characterization is achieved. Molecular masses and glycan fragmentation provide additional information about the structure of the carbohydrate chains. Several approaches including high performance liquid chromatography in on- or off-line configuration with Mass Spectrometry, have been described.

The aforementioned characteristics of the glycosylation process (which has persisted during evolution) were partially discussed in the symposium Structural Analysis of Glycoproteins during the last Biotecnología Habana'98 meeting, giving the attendees the possibility to be familiar with this complex but real problem when dealing with the production of genetically engineered proteins. Some of the outstanding lectures are included to facilitate the comprehension of this issue.

Mass spectrometric techniques for the rapid characterization of glycoproteins

Nimtz M, Conradt H S

The complete characterization of the heterogeneous oligosaccharide population on a natural or recombinant glycoprotein is laborious and includes enzymatic or chemical liberation of the oligosaccharides, their chromatographic purification and final structural characterization of each fraction by NMR, MS and/or enzymatic methodes. Modern mass spectrometric techniques allow a substantial reduction of this workload. The direct mass spectrometric analysis of an intact glycoprotein yields information on the mass of the protein, as well as first hints on the heterogeneity of the glycan population present. After enzymatic or chemical liberation of the oligosaccharides, the analysis of the total native or derivatized glycan pool by MALDI/TOF- or ESI-MS separates all glycans present according to their molecular weight with higher sensitivity and better resolution than that of the best HPLC techniques. Additional structural information regarding the sequence of the monosaccharide residues, the presence of isomeric structures and, to a certain extent, valuable linkage information can be obtained by the MS/MS analysis of selected molecular ion species by collision induced decomposition from picomolar amounts of material. Thus, the terminal substitution pattern of complex type glycans typical for natural or recombinant glycoproteins like sialylated or non sialylated antennae, modification by peripheral or proximal fucose, the Gal 1-3Gal-R motif, lactosamine repeats, the Gal versus GalNAc exchange and the presence of sulfate or phosphate groups can readily be determined from individual oligosaccharides of an unseparated glycan pool. Furthermore, parent ion scans can be used for the selective detection of the structural motifs mentioned above from very complex matrices. Complementary information about the glycosylation sites and the peptide backbone can be acquired by MALDI/TOF- or HPLC/ESI-MS analysis of the proteolytic peptide mixture obtained from the respective glycoprotein.

Dept. of Structure Research, GBF - Gesellschaft für Biotechnologische. Forschung mbH, Mascheroder Weg 1, D-38124 Braunschweig, Germany.

Using a new profiling methodology for the characterization of N-glycosylation in natural and recombinant glycoproteins

José A Cremata, Raquel Montesino, Omar Quintero, Rossana García

Recently we described a simple and sensitive two-dimensional (2D) sugar-mapping technique of 8-amine-1,3,6-naphthalene trisulfonic acid derivatives (ANTS-derivatives) of neutral and sialyloligosaccharides for structure analysis and characterization of N-linked oligosaccharides using picomoles of samples. The method includes: 1) reductive amination with ANTS of enzymaticaly released oligosaccharides; 2) simultaneous separation of oligosaccharide derivatives in FACE and NH2-HPLC column under ion suppression conditions; 3) plotting of the relative migration indexes (RMI) (X-axis) and relative retention times (trMan7) (Y-axis) in graph; 4) when necessary, additional exoglycosidase digestion. The principal advantage of the new methodology here discussed is the finding of linear dependence within each group of compounds, i.e., oligomannosides, asialo complex oligosaccharides, sialylated diantennary, triantennary and tetraantennary structures.

The suitability of this methodology will be illustrated by the glycosylation profiling and structural analysis of oligosaccharide population of a series of glycoproteins of different origen which includes: recombinant proteins expressed in yeast, native fungal Endoglucanase 1, monoclonal IgG antibodies, and human a1 anti-trypsin.

GlycoLab, División de Bioindustria. Centro de Ingeniería Genética y Biotecnología, Ave. 31 e/ 158 y 190. AP 6162, CP 10600, Ciudad de La Habana, Cuba.

Glycosylation of cellobiohydrolase I from Trichoderma reesei

Marleen Maras1, Kathleen Piens2, Marc Claeyssens2, André De Bruyn,3 Roland Contreras1

By a combination of different techniques N-glycans from different strains of Trichoderma reesei have been analysed. The N-linked oligosaccharides have been released from CBH I using N-glycanase F and the complexity of the N-glycan mixture was evaluated by PAGE analysis after labeling the non reducing sugar ends with ANTS or AMAC. Biogel-P4 gelfiltration has been used to fractionate neutral oligosaccharides. These glycans were further analysed by HPAE-PAD chromatography and finally by NMR spectroscopy. With the RUTC 30 strain of Trichoderma reesei elucidation of certain N-glycan structures led to the conclusion that oligosaccharides were of limited sizes, that they were of the high-mannose type, and that substituent groups such as phosphates or glucoses were abundantly present. Characterizations of the N-glycan structures of Trichoderma reesei strain VTT D-80133 are in progress. PAGE and HPAEC.PAD analyses allready indicate that the latter strain synthesizes several different charged N-glycans (probably phosphorylated) not found with the RUTC 30 strain. A small amount of Man5GlcNAc2 was detected and NMR analysis gave proof that its structure was the same as that synthesized by mammalian cells. Indeed, this oligosaccharide was found to be acceptor substrate for human N-acetylglucosaminyltransferase I. With a third, uncharacterized, strain of Trichoderma reesei, the combination of PAGE and HPAEC.PAD analyses showed yet another N-glycan pattern on CBH I. Here, the presence of different glucosylated compounds could be suggested.

The conclusion is that fungal N-glycosylation shows strain dependent differences and that this can play a decisive role for future conversions of these glycans to a mammalian type.

1Department of Molecular Biology, K.L.Ledeganckstraat 35, 9000 Ghent, Belgium. 2Department of Biochemistry,Fysioology and Microbiology, K.L.Ledeganckstraat 35, 9000 Ghent, Belgium. 3Department of Organic chemistry, Krijgslaan 241 (S4), 9000 Ghent, Belgium.

Deglycosilation of fungal enzymes for structure determination

Jerry Ståhlberg1, Rossana García3, José A Cremata,3 T Alwyn Jones2

Although it is not the primary goal of our research, the problems we have encountered in the crystallisation of fungal cellulases, have forced us to acquire at least a rudimentary insight into how these enzymes are glycosylated. The glycosylation alters the surface properties of a protein and thereby how it may crystallise. Therefore heterogeneity, which is common in the carbohydrate portion, may have a detrimental effect. Some examples will be shown from studies of extracellular cellulose degrading enzymes from the filamentous fungus Trichoderma reesei (Tr herein) illustrating that glycosylation may vary not only between proteins, but also with the strain and the cultivation conditions. In the case of Cellobiohydrolase 1 (CBH1), which apparently requires a single GlcNAc at one site for crystallisation, it was possible to obtain a homogenuous preparation of the protein by choosing the right strain and proper cultivation conditions. The amount of sugar present on the catalytic domain was surprisingly small, only 3 single GlcNAc. The N-glycans are probably cleaved by an enzyme with similar specificity as Endo F and Endo H and the enzyme must be present in relatively large amounts in Trichoderma to cope with the high expression levels of CBH1. An easier way to obtain homogenuous protein preparations is to use enzymatic deglycosylation. We have developed a method by which we can deglycosylate several mg of protein for crystal structure determination at an affordable cost. A solution containing 10 mg protein and 0.2 mg Jack bean alpha-mannosidase (Boehringer-Mannheim) in 5 ml 0.1 M sodium acetate, pH 5.0, 2 mM zinc acetate, was sterile filtered and incubated overnight. Beta-mercaptoethanol and EDTA were aseptically added to 2 mM and 10 mM concentration, respectively, as well as 20 DGU (deglycosylation units) of a crude mixture of Endo F and PNGase F (Oxford Glycosystems). After overnight incubation, another portion of 20 DGU Endo F/PNGase F was added and the reaction left to proceed for two more days. All the incubations were done at 37 degrees C. We have applied this procedure on several of the fungal cellulases under study. Only in the case of the catalytic domain of Tr Endoglucanase 1 (EG1) we have made a proper characterization of the glycosylation before and after treatment. In the original preparation more than 10 glycoforms were present, the dominating one containing only a single GlcNAc and one Man. Some of the longer N-glycans were charged due to the presence of fosfate groups linked between the outermost mannose residues. After deglycosylation we could obtain a seemingly homogeneous preparation of the protein. Unfortunately we have not yet been able to obtain any crystals of the deglycosylated EG1. With the other enzymes the evaluation has been limited to wether the protein crystallises or not. Deglycosylation of CBH1 gave reproducible preparations that crystallised readily. Tr Cellobiohydrolase 2 (CBH2) on the other hand crystallises without deglycosylation although it has 2-3 N-glycans and 6 O-glycans attached and is heterogenuous. After deglycosylation, however, it does not crystallise under the same conditions. With CBH 58 from another fungus, Phanerochaete chrysosporium, it was only with the deglycosylated protein that we could obtain crystals and solve the structure. It should be remembered that this study only includes enzymes of fungal origin. Glycosylation is relatively simple, the only monosaccharides found being GlcNAc, Man and Glc. N-glycosylation is only of the high-mannose type and the O-glycosylation seems to be direct O-mannosylation and we havent seen any signs of the hyper-glycosylation commonly found in yeast. Although the method has proven useful for our studies, it may not work on proteins from other organisms and with more complex glycosylation.

1Swedish University of Agricultural Sciences, 2Uppsala University, Dept. Molecular Biology, PO Box 590, SE-75124 Uppsala, Sweden. 3Centro de Ingeniería Genética y Biotecnología, Ave. 31 e/ 158 y 190. AP 6162, CP 10600, Ciudad de La Habana, Cuba.

Bacterial 1,3-1,4-b-glucanases: mechanism, specificity and applications through functional redesign by protein engineering

Antoni Planas

The 1,3-1,4-b-D-glucan 4-glucanohydrolases ( EC 3.2.1.73, 1,3-1,4-b-glucanases [1,2]) hydrolyze beta-glucans from the cell walls of Gramineae, which are particularly abundant in the starchy endosperm cells of cereals such as barley, sorghum, wheat and rice. 1,3-1,4-b-glucanase is an endoglycosidase acting with retention of configuration [3] and with a cleavage specificity for b-1,4 glycosidic bonds in 3-O-substituted glucopyranose units. The bacterial enzymes (family 16 of glycosyl hydrolases) are more active and thermally stable than the plant isozymes (family 17), and they are a target in our group [3-9] as an endoglycosidase model for studies on the mechanism of action and protein-carbohydrate molecular recognition, as well as for the redesign of the enzyme properties for biotechnological applications (brewing and animal foodstuff). This lecture summarizes recent studies focused on the details of the catalytic mechanism and the redesign of the enzymatic activity, and the structural/functional analysis of protein-carbohydrate interactions defining substrate specificity.

a) Catalytic mechanism. After identifying the essential catalytic residues by site-directed mutagenesis [4] (E138 as a general acid/base and E134 as a nucleophile in the Bacillus licheniformis enzyme), their functional role has been analyzed by a chemical rescue methodology on inactive mutants [4]. Addition of exogenous nucleophiles to the E134A and E138A mutants restores the enzymatic activity. Kinetic and structural analysis of the 'chemical rescue' indicates that the reactions proceed through different mechanisms and provides functional information on the catalytic pathway. The detection of a novel alfa-glycosyl formate intermediate will be discussed. It mimics the proposed glycosyl-enzyme intermediate in retaining glycosidases, and it is used to develop a new concept for rebuilding the active site towards new enzymatic activities.

b) Protein-carbohydrate interactions. 1,3-1,4-b-glucanases from Bacillus have an extended binding cleft containing 6 subsites, four on the non-reducing end from the scissile glycosidic bond (-IV to -I) and two on the reducing end (+I and +II) [6]. By means of kinetic analyses with a series of oligosaccharide substrates with variable degrees of polymerization (obtained by chemical as well as enzymatic synthesis [7]), the contribution of each subsite to enzyme-substrate binding has been evaluated [8]. The amino acid residues involved in substrate binding belong to two well defined structural motifs: a surface loop that partially covers subsites -IV and -III, an a beta-sheet composed of 6 antiparallel beta-strands on the concave face of the protein molecule (jellyroll beta-sandwich structure as determined by X-ray crystallography of the free enzyme) [9]. Most of these residues have been mutated by site-directed mutagenesis. The structural/functional interpretation of protein-carbohydrate recognition based on the mutational analysis in combination with the molecular modeling will be discussed.

1. Parrish FW, Perlin AS, Reese ET. Can J Chem 1960;38:2094-2104.

2. Anderson MA, Stone BA. FEBS Lett 1975;52: 202-7.

3. Malet C, Jiménez-Barbero J, Bernabé M, Brosa C, Planas A. Biochem J 1993;296:735-758.

4. Juncosa M, Pons J, Dot T, Querol E, Planas A. J Biol Chem 1994;269:14530-5.

5. Viladot JLl, de Ramón E, Durany O, Planas A. Biochemistry 1998;37:11332- 42.

6. Malet C, Planas A. Biochemistry 1997;36:13838-48.

7. Viladot JL, Moreau V, Planas A, Driguez H. J Chem Soc Perkin Trans 1997;I:2383-7.

8. Planas A. Mutational analysis of specificity and catalysis in Bacillus 1,3-1,4-b-glucanases. In: Claeyssens M, Piens K, Nerinckx W, editors. Carbohydrases from Trichoderma reesei and other microorganisms. Royal Society of Chemistry, London 1998. p.21-38.

9. Hahn M, Pons J, Planas A, Querol E, Heinemann U. FEBS Lett 1995;374:221-4.

Laboratori de Bioquímica, Institut Químic de Sarrià, Universitat Ramon. Llull, Via Augusta, 390, 08017 Barcelona, España. E-mail: aplan@iqs.url.es

Glycosylation profiling of high level heterologous proteins expression in the methylotrophic yeast Pichia pastoris

Raquel Montesino, Omar Quintero, Rossana García, José A Cremata

N-linked oligosaccharides from heterologous proteins secreted by the methylotrophic yeast Pichia pastoris under the control of alcohol oxidase (AOX1) promoter were characterized by a combination of Fluorophore-Assisted Carbohydrate Electrophoresis (FACE), using 8-amino-1,3,6-naphthalene trisulfonic acid (ANTS) as the fluorophore, and the separation of the same ANTS-oligosaccharide derivatives on NH2-HPLC column. These proteins included: A bacterial enzyme, Bacillus licheniformis a-amylase; three fungal enzymes, Saccharomyces cerevisiae invertase, Penicillium minioluteum dextranase and Mucor pusillus aspartic protease; and two higher eukaryotic proteins, Boophilus microplus (tick) gut antigen and bovine enterokinase catalytic subunit.

Man8GlcNAc2 and Man9GlcNAc2 (Man8, Man9) are the structures most frequently found in most of the characterized glycoproteins despite oligomannosides of lower degree of polymerization (Man6) were also found on invertase as well as higher structures (up to Man18) were common on aspartic protease and enterokinase. Phosphorylated oligosaccharides were observed on one protein, aspartic protease. In any case, oligosaccharides do not undergo terminal addition of a1,3-linked mannose. From these results, it is apparent that most foreign proteins secreted from P. pastoris are not subjected to the extensive mannosylation (hyperglycosylation) that commonly occurs to proteins secreted from S. cerevisiae. Additionally, slight changes in oligosaccharide profiles were observed when culture conditions were modified.

GlycoLab, División de Bioindustria. Centro de Ingeniería Genética y Biotecnología, Ave. 31 e/ 158 y 190. AP 6162, CP 10600, Ciudad de La Habana, Cuba.

The major exoglucanase secreted by Saccharomyces cerevisiae as a model to study protein glycosylation

Germán Larriba, Rosario Cueva, Luis Burgos

The major yeast exoglucanase (ExgIb) consists of a 408 aminoacid polypeptide carrying two short N-linked oligosaccharides attached to asparagines 165 (Asn165) and 325 (Asn325). These oligosaccharides are very similar, in both length and composition, to those present in the vacuolar protease carboxypeptidase Y. Minor glycoforms of exoglucanase arise by underglycosylation of the protein precursor (Exg165 and Exg325) or by elongation of the second oligosaccharide (ExgIa). The fact that these glycoforms can be readily separated and identified by HPLC and/or Western blots converts ExgI in an excellent model to study the role of the several components or branches of the precursor oligosaccharide in the eficiency and selectivity of the oligosaccharidyl transferase in vivo. We have found that the presence of a single glucose attached to Dol-PP-GlcGlcNAc2-Man9 increases the efficiency of transfer of that oligosaccharide to the protein acceptor. Also, the glucotriose unit appears to be involved in the selection of the sequons to be occupied, in such a way that its absence results in a bias towards the glycosylation of a particular sequon. Finally, we have shown the transfer of GlcNAc2 from Dol-PP-GlcNAc2 to exoglucanase, an indication that this intermediate is able to translocate from the cytoplasmic to the lumenal face of the endoplasmic reticulum membrane.

Departamento de Microbiología, F. de Ciencias, Universidad de Extremadura, 06071 Badajoz, España.

Copyright 1999 Elfos Scientiae