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Yeast surface display is an innovative technique that can improve the stability and activity of recombinant enzymes by attaching the enzymes to the yeast cell wall. In this study, we focused on the C-terminal immobilization of recombinant proteins, as certain proteins require exclusive immobilization through their C-terminus to prevent loss of activity. C-terminal immobilization via the anchor Ccw12 protein has been a promising approach for the efficient display of various industrially relevant recombinant proteins due to the high abundance and stable attachment of the Ccw12 protein in the yeast cell wall. To further enhance the efficiency of C-terminal immobilization, we conducted a study where we examined a series of strains in which genes associated with the regulation of transcription of genes related to wall structure maintenance, mRNA processing, translation, quality control, and maintenance of ER morphology were deleted. We investigated the activity of the reporter Ccw12BLA protein in these mutants and compared it to the wild-type strain. Increased Ccw12BLA activity was observed in mutants lacking protein involved in amino acid deprivation response (gcn2), translational repressor for cell wall proteins (ssd1), a protein involved in endoplasmic reticulum-associated protein degradation pathway (doa10), and covalently linked proteins of the cell wall (scw4pir1pir2pir3pir4). Additionally, Ccw12BLA optimization was applied to the surface display of sucrose phosphorylase, an enzyme that plays a role in glucosyl glycerol synthesis and has extensive applications in various industries including cosmetics, food, and pharmaceuticals. Sucrose phosphorylase (kindly provided by Prof. B. Nidetzky, TU Graz) was successfully expressed and immobilized through the Ccw12 anchor protein on the yeast cell wall in gcn2, doa10, ssd1, and scw4pir1pir2pir3pir4 mutants, and its binding was confirmed through Western blot analysis. The activity measurement of Ccw12SP in these mutants showed a similar trend as observed for the Ccw12BLA recombinant protein, suggesting that these mutants are promising candidates for glucosyl glycerol production using yeast surface display technology. Also, we expressed methionine adenosyltransferase, a key enzyme in S-adenosylmethionine production, and confirmed its localization on the yeast cell wall.

BACKGROUND Saccharomyces cerevisiae is frequently used as a host for the production of recombinant proteins. S. cerevisiae also provides the immobilization of recombinant proteins on the cell surface as an alternative to conventional immobilization methods. Yeast surface display systems are based on genetic fusion of a gene coding for a protein of interest and a gene coding for one of yeast cell wall proteins. Since immobilization of the protein through its N- or C- terminal end can affect its activity, different surface display systems were developed. Proteins of the Pir family are standardly used for N- terminal immobilization of recombinant proteins. OBJECTIVES Pir-based display systems developed so far involve fusion of the whole Pir protein with the desired protein, resulting in a large recombinant protein whose expression cause problems in its secretion and cell wall binding. To address this problem, we introduced changes in the Pir2 sequence and the protein secretion mechanisms to increase the efficiency of the Pir2- based surface display system. METHODS Using bacterial β-lactamase as a reporter enzyme, we assessed the efficiency of surface display of non-modified and modified recombinant protein Pir2bla in different yeast mutants. Amount of protein incorporated in cell wall was assessed by measurement of β-lactamase activity and by western blot. RESULTS Results show higher β-lactamase activity in strains expressing recombinant proteins containing the modified Pir2 binding region, lacking multiple internal repeats, and an increase of activity in most of the tested strains lacking activity of certain genes involved in endoplasmic reticulum-associated degradation (ERAD) pathways, or endocytosis. ACKNOWLEDGEMENTS/REFERENCES This research was funded by The Croatian Science Foundation, grant No. IP-2019-04-2891.

After synthesizing proteins, yeast Saccharomyces cerevisiae can retain them within the cell, secrete them into the medium, or bind them to the cell surface. In the third case, yeast anchors proteins by covalently binding them to β-glucan residues, thus allowing for surface display, a cell-engineering technique that can remake the entire cell surface into catalytically active living material. To immobilize proteins of interest closer to their N-terminus, the proteins are regularly fused with one of five Pir proteins (proteins with internal repeats), which covalently bind to β-1, 3-glucan. However, beyond the requirement for glutamine in their characteristic internal repeats, the mechanism of attaching these proteins to the yeast cell wall remains unclear. Thus, the Pir-based surface display continues to rely on guesswork and intuition, which is less than efficient. To address this issue, we inserted β-lactamase at five positions in Pir2 (Hsp150) protein and followed its activity and Pir2-binding efficiency through enzymatic and immunochemical methods. Moreover, we constructed and tested additional six truncated Pir2-β-lactamase variants, thus determining the minimal portion of Pir2 required for efficient binding to the yeast cell wall. Finally, to enrich our experimental results with structural insights, we used a deep- learning Alphafold2-based algorithm to in silico predict the structure of Pir2-β-lactamase fusions. Therefore, we present and rationalize a novel set of practical guidelines for a reproducible, straightforward, and efficient Pir-based yeast surface display.

The Scw4, Scw10, and Scw11 proteins form a group of putative glucanases located in the yeast Saccharomyces cerevisiae cell wall. In the previously published study, Scw10 protein was shown to be up to 10-fold more abundant in several clinical isolates compared with laboratory yeast strain, suggesting its possible role in yeast virulence. Its paralog Scw4 undergoes complex proteolytic processing. Scw4 is processed by the Kex2 protease and subsequently by yapsin proteases, which affects the biological activity of Scw4 and its ability to bind to cell walls. The proteolytic processing or binding of Scw10 has not been thoroughly investigated. It has also been shown that both Scw4 and Scw10 can form non- covalent and covalent bonds with the wall that differ from previously known protein-wall bonds. Although these bonds resemble those of Pir proteins, both Scw4 and Scw10 lack the typical Pir binding sequence. In this study, the processing of Scw10 and its potential impact on its biological function are investigated. In addition, the role of several putative Scw4/Scw10 binding sequences in the formation of a covalent bond to cell wall structures is explored.

Pir proteins (proteins with internal repeats) are the most prominent proteins that yeast Saccharomyces cerevisiae covalently binds to its cell wall through β-1, 3-glucan residues. As such, they are regularly used for yeast surface display when the displayed protein needs to be bound close to its N-termini. However, the role of Pir proteins in cells remains unclear, and their diversity in closely-related species unexplored. These unknowns hamper rational engineering of Pir- based surface display, a technique with the potential to convert the entire yeast cell wall into enzymatically active self-regenerating living material. To address this issue, we used a deep- learning Alphafold2-based algorithm to in silico predict the 3D protein structures of S. cerevisiae Pir proteins and compare them with structures deposited in the Protein Data Bank (PBD). Through this approach, we were able to infer proteins’ function solely through 3D homology. Moreover, we searched through the genomes of 77 species closely related to S. cerevisiae and identified over 300 Pir homologues, with which we mapped Pir evolution throughout the last 110 million years. Finally, by comparing homologous genomic loci, we show that S. cerevisiae is the only species within the Saccharomyces clade to lose one of its Pir proteins. As such, our findings point towards the role and diversity of Pir proteins in budding yeasts, thus opening novel avenues in yeast surface engineering.

Yeast surface display is a powerful and robust method for the surface immobilization of heterologous proteins with potential application in different biomedical and biotechnological processes. In biotechnology, it could be used in various bioconversion processes as enzymes of interest can be fused with anchoring proteins. This allows secretion of such protein constructs and their binding to the cell wall. The major disadvantage of this method is the low binding capacity and low overall surface dis-play efficiency of currently used systems. There are several possible approaches to resolve this problem, and in this work, we focused on the modification of the cell wall of host cells. Several mutant strains were developed and/or tested to see if it is possible to increase display efficiency by introducing changes in cell wall structures. Tested strains contained mutations in genes coding for cell wall proteins (the strain with deleted genes coding for Pir proteins, and deltacwp2 strain) or in genes coding for proteins involved in the synthesis of the GPI anchor, and in the localization of GPI anchored wall proteins (deltagpi7, deltadfg5, deltadcw1 strains). Also, the effect of deletion of the SSD1 gene coding for the translation factor involved in the translation of many cell wall proteins was tested. Effects of these mutations were investigated using reporter systems previously developed in our laboratory, composed of beta- lactamase fused with either Pir2 or Ccw12. Our preliminary results show that it is possible to obtain more than two-fold higher surface display efficiency by these modifications.

The yeast Saccharomyces cerevisiae is a prominent host for the production of recombinant proteins because it can perform protein modifications that are the same or similar to those found in other eukaryotes. At the same time, yeast provides quality control of the proteins produced, resulting in a high level of properly folded proteins. These proteins can be secreted and incorporated into the cell wall, providing their immobilization on a solid surface. Yeast surface display is an attractive alternative to classical immobilization methods because it is cost- effective, less time-consuming, and allows continuous production of immobilized protein. The major drawback is the low efficiency of surface display. There are several approaches to overcome this obstacle. One of them is the manipulation of the secretory pathway to increase the total amount of secreted protein. Overexpression of recombinant proteins increases cell stress because the increased protein load overwhelms protein production and folding mechanisms, increasing the number of misfolded proteins. Such events lead to activation of the unfolded protein response (UPR) and the endoplasmic reticulum-associated degradation (ERAD) pathways. Several published papers have indicated that activation of these pathways leads to decreased protein production. To test how mutations in genes involved in these pathways affect surface display efficiency, we have used two surface display systems in which β- lactamase is displayed using native cell wall proteins Pir2 or Ccw12 as anchors. Mutants used for the surface display contain mutations in IRE1 and YOP1 (involved in the UPR), HRD1, HRD3, DER1 and DOA10 (involved in ERAD). Additionally, we also checked the effect of a mutation in the gene SEC14, which is involved in protein secretion and regulation of UPR. Results showed increased β- lactamase activity in most of the tested strains. Results also showed that the effects of introduced mutations on surface display efficiency differed between two surface display systems.

Major disadvantages of protein surface display in yeasts as an alter-native to classical solid- surface immobilization techniques are low production, secretion, and binding capacity, or simply said low surface display efficiency of recombinant proteins. As several problems could lead to low display efficiency, we attempted to investigate how modification of different cellular processes involved in the production and secretion of proteins can affect and possibly enhance display efficiency. For this purpose, we tested the effects of mutations of genes involved in transcription and translation (deltagcn2, deltagc- n5, deltaopi1, deltarrp6) and mutations affecting properties of the endoplasmic reticulum (deltayop1). We also tested the effect of mutations of genes involved in unfolded protein response (UPR) and the endoplasmic reticulum- associated degradation (ERAD) pathways that are possibly activated due to stress conditions caused by increased protein production of overexpressed recombinant protein.To test the effect of these mutations we used two reporter systems previously developed in our laboratory. Both systems contain beta-lactamase fused with either Pir2 protein or with the GPI anchoring signal sequence of Ccw12. Results obtained by measuring the activity of surface-displayed beta-lactamase have shown that it was possible to increase surface display efficiency significantly by modifying processes involved in protein production and secretion in yeast cells.

Surface display in yeast represents a valuable alternative to enzyme immobilization on solid surfaces as it is usually less complicated, less time-consuming, and eliminates the need for expensive enzyme purification. Instead, it provides constant production of recombinant protein composed of enzyme of interest fused with fragments of native yeast cell wall proteins. This way modified cells can be used in production processes for longer time compared to the purified enzymes. However, major disadvantages of these technique are low surface display efficiency and possible hyperglycosylation of recombinant proteins. By lowering the amount of glycosylation in cells, several effects are expected. Decreased glycosylation level of enzyme of interest could result in less disturbance of its conformation. It also causes a lesser density of the outer mannan layer of the cell wall, possibly increasing the availability of the substrate. To test the effects of inactivation of genes involved in O-glycosylation (genes from PMT group) and N-glycosylation (OCH1 and genes from MMN group) on the enzyme activity and surface display efficiency, two different systems using β-lactamase as a reporter were developed. In one, bla gene coding for β-lactamase was fused with HSP150 gene coding for cell wall protein resulting in recombinant protein covalently immobilized on the wall through linkage on its N- terminal end. Other system consists of bla gene fused with a fragment of CCW12 gene coding for its signalling sequence for binding of C-terminal part of protein onto GPI anchor. Immobilization can cause differences in protein folding, possibly leading to changes in enzyme activity. To reduce interference of this effect with observed changes in enzyme activity in mutant strains, we used different immobilization approach described earlier. Protein amount in the cell wall is assessed by measuring β-lactamase activity using nitrocefin as substrate and in a semi-quantitative manner by western blot.

The yeast cell wall is an extracellular structure with a complex role in preserving cell shape and integrity, and in interaction with different molecules and cells in its surrounding. Fact that 1200 Saccharomyces cerevisiae genes directly or indirectly affect cell wall formation and regulation (de Groot et al., 2001) clearly speaks of the importance of the cell wall. Here we present results from our recently published manuscript of in silico analysis comparing 187 S. cerevisiae proteins involved in cell wall biogenesis and function with cell wall proteomes of 92 different yeast species. The main goal of the research was to estimate the evolutionary conservation of proteins involved in protein glycosylation, cell wall synthesis and remodeling, and of cell wall proteins with still unknown functions. As expected, proteins involved in processes of great importance for cell wall integrity and its protective function were highly conserved. However, GPI anchored proteins involved in flocculation, aggregation, cell separation, and those with still unknown functions were not highly conserved. This analysis was coupled with analysis of proteins located in the cell wall by protein biotinylation and western blotting. Results of this analysis show distinctive protein patterns and significant differences in the overall amount of cell wall proteins for different yeast genera, and even for different species of the same genera. de Groot, P.W. et al. 2001. Comp. Funct. Genomics, 2(3), pp.124-142.

Surface display in yeast provides an alternative to standard protein immobilization on solid surfaces as it reduces the costs of protein purification and possibility of protein denaturation during the immobilization. Also, it provides continuous synthesis of recombinant protein composed of enzyme of interest fused with native yeast cell wall proteins. However, its major disadvantage is low surface display efficiency. Lately, it has been reported that mutations in genes encoding proteins involved in endocytosis may increase the amount of secreted heterologous proteins from the yeast cells. Glycosylation can also affect the efficiency of surface display as it could possibly have negative effect on conformation of displayed proteins and cause high density of wall outer mannan layer, limiting accessibility of the substrate. In order to test whether inactivation of genes involved in these processes would enhance surface display efficiency, two different systems using β-lactamase as a reporter were developed. In one, bla gene was fused with a fragment of CCW12 gene coding for its C-terminal GPI anchoring signal sequence. Other system consisted of bla gene coding for β-lactamase fused with PIR2 gene coding for cell wall protein that covalently binds to cell wall through the linkage on its N-terminal end. Both constructs were set under the control of an inducible PHO5 promotor. By using two different immobilization systems, the effect of different folding of the recombinant enzyme, caused by immobilization itself, on affecting enzyme activity is reduced. Therefore, the described system for expression and immobilization of recombinant reporter enzyme in the wall was examined in mutants that cannot accomplish endocytosis (end6, end3, vam4) and have lower level of glycosylation (pmt and mnn mutants). Amount of recombinant protein incorporated in cell wall was assessed by measurement of β-lactamase activity using nitrocefin as substrate and in a semi- quantitative manner by western blot.

Yeast cell wall proteins can be used for surface display of various proteins of industrial interest. Generally, yeast cell wall contains proteins that are non-covalently (Scw- proteins) or covalently (Ccw-proteins) bound to β-1, 3-glucan, the latter either through GPI- anchors and β-1, 6-glucan, or by alkali labile ester linkages between γ-carboxyl groups of glutamic acid and hydroxyl groups of glucoses (Pir-proteins). Covalently bound proteins are usually used for immobilization of different recombinant proteins. Here we explore possibility of using Scw4 and Scw10 as novel immobilization platforms. Previously it was shown that Scw4 protein can be covalently and non-covalently linked to the cell wall structures. It was also shown that proteolytic processing of Scw4 by yapsins strongly affects its covalent binding. As Scw10 shares high level of similarity with Scw4, it is interesting to investigate whether Scw10 can also form both covalent and non-covalent interactions with cell wall structures. Although protein Scw10 also contains sequence charasteristic for Kex2 processing, it is not yet clearly confirmed whether Scw10 is substrate for Kex2. Furthermore, both of these proteins have potential to be used for development of new homologous and heterologous cell surface expression system for wide range of yeast species given that genomes of some other yeast species contain SCW4/SCW10 orthologs, as it is noted before. In order to develop Scw4/Scw10 based expression systems, further characterization of this two proteins is necessary. One line of the research was focused on Scw10 proteolytic processing. Results show that Scw10 undergoes processing by Kex2 and possibly by yapsins at the same site. Results also show that this processing doesn’t affect significantly Scw10 covalent binding efficiency. Other line of the research examines the level of evolutionary preservation of these proteins among different ascomycetes yeast by analysing cell wall protein profiles. Obtained results were coupled with results of in-sillico analysis of proteomes of different yeast species and it was found that Scw4 and Scw10 show high level of evolutionary preservation, given that all of analysed ascomycetes yeast, except two Schizosaccharomyces species, contained at least one protein with high similarity to them. Further research will focus on development of Scw4/Scw10 based expression system models and usage of this models in different yeast species

Background and Objective: Xylose reductases belong to the aldo-keto reductase family of enzymes, which catalyse the conversion of xylose to xylitol. Yeast xylose reductases have been intensively studied in the last two decades due to their significance in biotechnological production of ethanol and xylitol from xylose. Due to its GRAS status and pronounced tolerance to harsh conditions, Saccharomyces cerevisiae is the ideal organism for industrial production of both xylitol and ethanol. However, Saccharomyces cerevisiae is unable to use xylose as the sole carbon source due to the lack of xylose specific transporters and insufficient activity of metabolic pathways for xylose utilisation. The aim of this paper is to give an overview of attempts in increasing biotechnological potential of xylose reductases and to highlight the prospective of this application. Results and Conclusion: In order to create strains with improved xylose utilization, different approaches were attempted including simultaneous overexpression of xylitol dehydrogenase, xylose reductase and pentose phosphate pathway enzymes, heterologous expression of putative xylose transporters or heterologous expression of genes coding for enzymes included in the xylose metabolism, respectively. Furthermore, number of attempts to genetically modify different xylose reductases is increasing. This review presents current knowledge about yeast xylose reductases and the different approaches applied in order to improve xylose metabolism in yeast.Conflict of interest: The authors declare no conflict of interest.

Scw4p, one of noncovalently bound proteins in Saccharomyces cerevisiae cell wall, undergoes complex proteolytic processing by Kex2 and yapsins. It was suggested that this proteolytic processing can influence its cell wall incorporation, given that Scw4 can form covalent bond with βglucan, and it’s possible that processing has effect on Scw4 biological activity. Scw10 shares high level of similarity with Scw4, and also contains Kex2 processing site, but it remains unclear whether Scw10 is substrate for Kex2. Both of this proteins are considered to be glucanases, although this assumption hasn’t been proven yet. Nevertheless, importance of this two proteins is indicated through extensive phenotypic changes caused by simultaneous deletion of both SCW4 and SCW10. Their evolutionary preservation among different yeast species hasn’t been investigated, however it’s noted that genomes of some other yeast species contain SCW4/SCW10 orthologs.

Two types of genetic cassettes for the surface display of heterologous proteins in S. cerevisiae were constructed and their functionality was tested by cloning recombinant enzyme xylose reductase (XR). Cassettes contained strong and regulated host promoter GAL1 or PHO5, a signal sequence for directing the protein into the secretory pathway, an anchoring domain of native cell wall proteins for C- or N-terminal immobilisation, and genetic tags for easy detection of the recombinant protein. YEp351Pir4 plasmid was constructed for the N-terminal immobilisation of heterologous proteins, containing PIR4 under a GAL1 promoter followed by the spacer region (a stretch of eight serine residues), a region consisting of several restriction sites for the insertion of the gene of interest and, finally, followed by the -6xHis and -HA tags. The plasmid pRS425Ccw12G and pRS425Ccw12P were prepared for C-terminal immobilisation of heterologous proteins. This plasmid contains a GAL1 or PHO5 promoter, respectively, followed by the part of CCW12 coding for the signal sequence, the -HA tag, restriction sites for the insertion of the gene of interest, the part of the CCW12 coding for the GPI anchoring signal, and the downstream genetic elements of the CCW12. The S. cerevisiae gene GRE3 coding for intracellular xylose reductase was inserted into the plasmids described above using suitable restriction sites. The construct pRS425Ccw12GXR was modified by introducing the STOP codon immediately after GRE3 coding region, resulting in a secretion of recombinant xylose reductase into the growth medium. Finally, the localization, activity and characteristics of different forms of recombinant xylose reductase were determined. Both types of cassettes proved to be functional, and for this recombinant protein N-terminal immobilisation showed to be better solution for surface display.

In the past decade much effort has been devoted to the development of new expression systems and novel techniques for the surface display of heterologous proteins in yeast in order to improve their applications in biotechnology, food technology, pharmacology and medicine. Heterologous protein-encoding genes are generally fused with genes coding for yeast cell wall proteins or their fragments required for anchoring. The variety of reactions by which a protein can be displayed at the cell surface enables finding the appropriate one for each individual protein. However, it is still challenging how to improve the efficiency of display of protein complexes and increase the quantity of protein displayed on the yeast surface. Recently, synthetic protein chimeras that self-assemble into the scaffolds on the yeast surface displaying different proteins have been constructed. This review focuses on systems and techniques for display of recombinant proteins on the yeast cell surfaces and applications afforded by this technology.

Most of the vectors constructed so far for the surface display of recombinant proteins in the yeast have been created with ubiquitous plasmids. We have constructed three new plasmids for the surface display of heterologous proteins in the yeast S. cerevisiae that would enable easy insertion of the gene of interest into a prearranged genetic cassette consisting of a strong and regulated host promoter, signal sequence for directing the protein into the secretory pathway, anchoring domain of native cell wall proteins for C- or N-terminal immobilisation, and genetic tags for easy detection of the recombinant protein. YEp351Pir4 plasmid was constructed for the N-terminal immobilisation of the heterologous proteins, containing PIR4 under a GAL1 promoter followed by the spacer region (a stretch of eight serine residues), region consisting of several restriction sites and finally by the -6xHis and -HA tags. Plasmids pRS425Ccw12 and pRS425Ccw12_2 were prepared for C-terminal immobilisation of heterologous proteins. The pRS425Ccw12 plasmid contains GAL1 promoter followed by the part of CCW12 coding for the signal sequence, the -HA tag, restriction sites for the insertion of the gene of interest, the part of the CCW12 coding for the C-terminal part of Ccw12 containing the GPI anchoring signal and the downstream genetic elements of the CCW12. In the pRS425Ccw12_2 plasmid the -HA tag is placed after the heterologous gene insertion site, followed by the GPI anchoring signal and the downstream genetic elements of the CCW12. The gene GRE3 coding for xylose reductase was inserted into the plasmids described above using suitable restriction sites. Finally, the localization, abundance and the activity of the surface displayed xylose reductase were checked by Western blot method and spectrophotometric assay and the results obtained were compared to find out which type of immobilization is best for the surface display of this particular enzyme.

Yeast cell wall contains proteins that are noncovalently (Scw-proteins) or covalently (Ccw-proteins) bound to β-1, 3-glucan, the latter either through GPI-anchors and β-1, 6-glucan, or by alkali labile ester linkages between γ-carboxyl groups of glutamic acid and hydroxyl groups of glucoses (Pir-proteins, extracted from the cell wall by mild alkali). It was shown that three forms of Scw4 with different molecular weights could be isolated from the cell wall. The fact that only one site for Kex2 processing is present in the Scw4 sequence indicates existence of an additional site for processing and an additional proteolytic enzyme involved in processing. Given that group of broad specific aspartic proteases called yapsines process other cell wall proteins, it was presumed that this group of enzymes might have a role in processing of Scw4. In order to investigate the possible role of this enzymes in processing and to find the additional processing site, different mutations were introduced in HA-tagged SCW4 by which potential proteolytic sites where mutated. Native and mutated forms of HA-tagged-Scw4 were expressed in wt, kex2 yeast strain and strain lacking all yapsin genes (5yps∆) and Scw4 processing was examined by Western blot. Protein glycosilation was also examined in order to determine whether forms differ in this way. Results confirmed that the differences in the molecular weight of Scw4 forms isolated from the yeast cell wall were a consequence of its proteolytic processing with Kex2 and/or yapsin proteases using different processing sites in Scw4 sequence. Scw4 was first identified as one of the cell wall proteins non-covalently incorporated in the wall. Later, it has been found that a part of Scw4 could be extracted by mild alkalis from walls previously depleted of non-covalently attached proteins. In order to examine which forms of Scw4 where covalently linked to structures of cell wall, HA-tagged Scw4 was extracted by NaOH from cell wall preparations of w.t., kex2 and yapsin mutants previously depleted of non-covalently linked proteins. Mutants were grown either at pH 4, or at pH 7 in order to obtain all three forms of Scw4 and covalently attached part of the proteins was analysed by Western blot. Results showed that all three forms of Scw4 protein are covalently linked to wall, although smallest form of protein bounds to wall less efficiently. Additional experiments are required in order to determine which part of Scw4 is essential for covalent binding to wall. Given that Scw4 forms covalent link to cell wall, this protein is a good candidate for application in development of new homologous and heterologous expression systems. Future research will focus on identifying part of Scw4 sequence responsible for covalent binding and development of Scw4 based expression system models.

Most of the vectors constructed so far for the surface display of recombinant proteins in the yeast have been created with ubiquitous plasmids. We have constructed three new plasmids for the surface display of heterologous proteins in the yeast S. cerevisiae that would enable easy insertion of the gene of interest into a prearranged genetic cassette consisting of a strong and regulated host promoter, signal sequence for directing the protein into the secretory pathway, anchoring domain of native cell wall proteins for C- or N-terminal immobilisation, and genetic tags for easy detection of the recombinant protein. YEp351Pir4 plasmid was constructed for the N-terminal immobilisation of the heterologous proteins, containing PIR4 under a GAL1 promoter followed by the spacer region (a stretch of eight serine residues), region consisting of several restriction sites and finally by the -6xHis and -HA tags. Plasmids pRS425Ccw12 and pRS425Ccw12_2 were prepared for C-terminal immobilisation of heterologous proteins. The pRS425Ccw12 plasmid contains GAL1 promoter followed by the part of CCW12 coding for the signal sequence, the -HA tag, restriction sites for the insertion of the gene of interest, the part of the CCW12 coding for the C-terminal part of Ccw12 containing the GPI anchoring signal and the downstream genetic elements of the CCW12. In the pRS425Ccw12_2 plasmid the -HA tag is placed after the heterologous gene insertion site, followed by the GPI anchoring signal and the downstream genetic elements of the CCW12. The gene GRE3 coding for xylose reductase was inserted into the plasmids described above using suitable restriction sites. Finally, the localization, abundance and the activity of the surface displayed xylose reductase were checked by Western blot method and spectrophotometric assay and the results obtained were compared to find out which type of immobilization is best for the surface display of this particular enzyme.

It was previously shown that S. cerevisiae cell wall protein Scw4p undergoes proteolytic processing with Kex2p. However, we observed that three forms of Scw4p with different molecular weights could be isolated from the cell wall. The fact that only one site for Kex2p processing is present in the Scw4p sequence indicates existence of an additional site for processing and an additional proteolytic enzyme involved in processing. According to the potential proteolytic processing sites found in the Scw4p sequence we presumed that aspartic proteases called yapsins might have a role in processing of Scw4p. In order to investigate the possible role of this enzymes in processing and to find the additional processing site, different mutations were introduced in SCW4 by which potential proteolytic sites where mutated. Native and mutated forms of Scw4p were expressed in wt, kex2 yeast strain and strain lacking all yapsin genes (5yps∆) and Scw4p processing was examined. Protein glycosilation was also examined in order to determine whether forms differ in this way. Results confirmed that the differences in the molecular weight of Scw4p forms isolated from the yeast cell wall were a consequence of its proteolytic processing with Kex2p and/or yapsin proteases using different processing sites in Scw4p sequence. In order to determine if there is a link between proteolytic processing and protein activity, mortality rate of yeast strains expressing different forms of Scw4p was estimated by methylene blue staining and MTT test.

Studies of microbial cell envelopes and particularly cell surface proteins and mechanisms of their localization brought about new biotechnological applications of gained knowledge in surface display of homologous and heterologous proteins. By fusing surface proteins, or their anchoring domains with different proteins of interest their so called genetic immobilization is achieved. Hybrid proteins are engineered in a way that they are expressed in the host cells, secreted to the cell surface and incorporated into the wall/envelope moiety. In this way laborious and often detrimental procedure of chemical immobilization of the protein is avoided by letting the cells do the whole procedure. Both bacterial and yeast cells have been used for this purpose and a number of potential biotechnological applications of surface displayed proteins have been reported. Examples range from microbial whole cell biocatalysts, biosorbents, biosensors and biostimulants development to design and screening of protein and peptide libraries. When surface immobilized enzymes are used, substrates do not need to cross membrane barriers, i.e. enzymes are free to access any externally added substrate. Thus, often complex and expensive purification of the enzymes used on an industrial scale is bypassed. In addition, the multi-step transformation can be performed using microbial cells displaying different enzymes that catalyze cascade reactions. In recent years particular attention has been paid to yeast systems for surface display of proteins since most yeasts are generally regarded as safe (GRAS) microorganisms, yeast cell walls are capable of binding more proteins, and the cells are bigger. Besides, yeasts are generally more suitable for expression of proteins originating from higher eukaryotes. In this talk our current knowledge on mechanisms and molecular systems for surface display of proteins on bacterial and yeast cell surfaces will be discussed.

Studies of microbial cell envelopes and particularly cell surface proteins and mechanisms of their localization brought about new biotechnological applications of gained knowledge in surface display of homologous and heterologous proteins. By fusing surface proteins, or their anchoring domains with different proteins of interest their so called genetic immobilization is achieved. Hybrid proteins are engineered in a way that they are expressed in the host cells, secreted to the cell surface and incorporated into the wall/envelope moiety. In this way laborious and often detrimental procedure of chemical immobilization of the protein is avoided by letting the cells do the whole procedure. Both bacterial and yeast cells have been used for this purpose and a number of potential biotechnological applications of surface displayed proteins have been reported. Examples range from microbial whole cell biocatalysts, biosorbents, biosensors and biostimulants development to design and screening of protein and peptide libraries. When surface immobilized enzymes are used, substrates do not need to cross membrane barriers, i.e. enzymes are free to access any externally added substrate. Thus, often complex and expensive purification of the enzymes used on an industrial scale is bypassed. In addition, the multi-step transformation can be performed using microbial cells displaying different enzymes that catalyze cascade reactions. In recent years particular attention has been paid to yeast systems for surface display of proteins since most yeasts are generally regarded as safe (GRAS) microorganisms, yeast cell walls are capable of binding more proteins, and the cells are bigger. Besides, yeasts are generally more suitable for expression of proteins originating from higher eukaryotes. In this talk our current knowledge on mechanisms and molecular systems for surface display of proteins on bacterial and yeast cell surfaces will be discussed.

Cold-active lipases have attracted attention in recent years due to their potential applications in reactions requiring lower temperatures. Both bacterial and fungal lipases have been investigated, each having distinct advantages for particular applications. Among yeasts, cold- active lipases from the genera Candida, Yarrowia, Rhodotorula , and Pichia have been reported. In this paper, biosynthesis and properties of a novel cold-active lipase from Candida zeylanoides isolated from refrigerated poultry meat are described. Heat-sterilized olive oil was found to be the best lipase biosynthesis inducer, while nonionic detergents were not effective. The enzyme was purified to homogeneity using hydrophobic chromatography and its enzymatic properties were tested. Pure enzyme activity at 7 ° C was about 60% of the maximal activity at 27°C. The enzyme had rather good activity at higher temperatures, as well. Optimal pH of pure lipase was between 7.3 and 8.2, while the enzyme from the crude extract had an optimum pH of about 9.0. The enzyme was sensitive to high ionic strength and lost most of its activity at high salt concentrations. Due to the described properties, cold-active C. zeylanoides lipase has comparative advantages to most similar enzymes with technological applications and may have potential to become an industrially important enzyme.

The aim of the project "Potential of microencapsulation in cheese production" was to investigate the possibility of microencapsulation of natural rennet obtained from the rennet of lambs (which are mostly not further used) and the possibility of simultaneous microencapsulation of rennet and isolated and identified microbial culture (lactic acid bacteria). This research has revitalised the production technology of rennet for the production of cheese “Paški sir” by introducing a new and more modern method of microencapsulation of rennet and microbial culture. The applied method of rennet and microbial culture production eliminates the use of imported industrial rennet and milk cultures, which is of particular importance for protecting the originality of the cheese according to EU criteria. The result of the research refers to the application of a traditional technological process modified in the laboratory in the production technology of different types of cheese, regardless of the type of milk. The isolation of autochthonous microbial cultures and their use in cheese production contribute to the preservation of biodiversity and to a greater diversity of cheese varieties on a global and regional level. The technology developed allows the product to be adapted to the needs of customers in the production of traditional and "boutique" cheeses.

Preserving genome stability is of critical importance to living organisms. Lack of genome stability results in severe conditions such as cancer and premature aging in mammals. Stability of E. coli genome is determined by metabolism of 3´-ending tails at DNA double-strand breaks, which are faithfully mended by homologous recombination catalyzed by RecBCD enzyme. The rare cases of aberrant DNA transactions that lead to imprecise recombination events in wild-type E. coli are unwound by prototypic genome guardian, RecQ helicase, thus preventing the appearance of illegitimate recombination. We used ssb-1 conditional mutation and measured the frequency of aberrant λ-prophage excision from the E. coli genome (Spi- assay) for quantifying illegitimate recombination in the bacteria of wild-type as well as of several other E. coli genetic backgrounds. We reveal that SSB protein prevents illegitimate recombination, which depends on SSB binding to ssDNA. Since for illegitimate recombination SSB overproduction complements RecQ deficiency, our results suggest that SSB prevents appearance of aberrant recombination events, whereas RecQ dismantles those that do occur in the E. coli genome.

In addition to being a rich source of nutrients, autochthonous fermented foods are a reservoir of beneficial lactic acid bacteria (LAB) starter cultures and potential probiotics. The bacteriocin-based antibacterial capacity of the autochthonous LAB is investigated as a strategy for biopreservation. Therefore, the aim here was to screen the antibacterial activity in a selected group of Lactiplantibacillus plantarum strains belonging to different food microbiomes, autochthonous cheese or sauerkraut. Since Listeria monocytogenes and Staphylococcus aureus are frequently detected as foodborne pathogens, they have been applied as test microorganisms to demonstrate antibacterial activities. All tested L. plantarum strains exhibited antilisterial and antistaphylococcal activities when grown on a solid medium, and when the cell-free supernatant (CFS) was analysed, where D13 and SF15C showed the greatest inhibition. Antibacterial activity in broth cultures was re-established or enhanced by coculturing with sensitive bacterial strains S. aureus 3048 and L. monocytogenes ATCC®19111™ which can be associated with the induction of bacteriocin production typically present among LAB. Additionally, the proteinase nature of CFSs has been confirmed as another feature of plantaricins. PCR using total DNA from L. plantarum strains resulted in the amplification of plantaricin-related genes plnA, plnEF and plnJ of expected amplicon size, suggesting that the genome could harbour a pln gene clusters. Whole genome sequencing (WGS) revealed the existence of five plantaricin specific operons regulated by a three- component regulatory system, involved in a quorum sensing (QS) mechanism, of L. plantarum D13 and SF15C strain, with high similarity to L. plantarum WCFS1. The 3D structures of PlnEF and PlnJK, were predicted using AlphaFold Protein Structure Database, while physicochemical properties and amino acid sequence of α-helixes of Pln were determined by HeliQuest analysis server. Taken together, these findings contribute to the characterisation of plantaricins of L. plantarum D13 and SF15C, and their implementation as either a functional starter cultures or even probiotics. Their application could improve the quality of fermented foods, which could be achieved by controlling contamination by L. monocytogenes, which would extend the shelf-life of the final product.

Foods for special medical purposes (FSMPs) are unique therapeutic products formulated for the specific dietary management of disease-related conditions or specific medical disorders, for which distinctive nutritional requirements, based on recognized evidence-based scientific principles, are established. The human microbiome is composed of microbes that have a tremendous potential to impact our physiology, both in health and in disease. Since high-fiber diet impacts the entire gut microbe community and improves blood- glucose regulation, they can be used as FSMPs, in which they pose a beneficial effect in maintaining the gastrointestinal health.Therefore the aim of this research was to establish the lactogenic and bifidogenic effects of three different innovative fibers (K1, D1, D2), in order to develop innovative formulations of foods for diabetic medical purposes, using a diabetic mouse model. Standard diabetic fiber mixures and food without added fibers, were used as controls. All tested innovative dietary fibers showed in vivo lactogenic and in vitro bifidogenic and lactogenic effect, where the best effects were achieved with the addition of D2 innovative fiber mixture, respectively. Additionally, after feeding the mice, all innovative dietary fibers showed a positive impact on the growth of Lactococcus spp., with the best results upon the addition of D2 innovative fiber mixture. Innovative fiber mixture (D1) reduced the number of L. monocytogenes to undetectable cell count level, significantly decreased the number of S. aureus and Salmonella spp., while innovative fiber mixture (D2) significantly decreased the number of L. monocytogenes and S. aureus, in diabetic mice faeces samples in vivo. The reduction of L. monocytogenes was also achieved with innovative (K1) and standard (K0) fiber mixtures, to undetectable cell count level. Finally, innovative fiber mixtures (D1 and D2), along with the standard fiber mixtures (K0) showed the impact on the incresement of the total bacteria number in vivo. In conclusion, two innovative mixtures of dietary fibers, D1 and D2, can be used as nutritional supplementation for foods for diabetic medical purposes.

In Escherichia coli, double-strand DNA breaks (DSBs) are repaired by the RecBCD pathway of homologous recombination. In the initiation (presynaptic) stage of this process, the RecBCD enzyme resects double-strand DNA ends and loads RecA recombinase to the emerging single-strand DNA tails. RecA catalyzes the central reactions of homologous DNA pairing and strand exchange (synapsis), while RuvABC complex and RecG helicase process recombination intermediates (post- synapsis). In this work we have studied chromosome morphology and segregation after introduction of DSBs by I-SceI endonuclease in E. coli strains carrying mutations in presynaptic (recB, recO) and postsynaptic (ruvABC, recG) recombination functions. Induction of DSBs by I-SceI caused severe chromosome segregation defects and DNA-less cell formation in the ruvABC, recG, and ruvABC recG mutants. This phenotype was efficiently suppressed by the recB mutation, while the recO mutation had no suppressive effect. These findings indicate that attempted DSB repair via the RecBCD recombination pathway influences DNA distribution within the cell as well as its transmission upon cell division. We assume that the DNA strand exchange between sister chromosomes during homologous recombination causes a temporary delay in chromosome segregation and disruption of cell division. In the absence of the postsynaptic RuvABC and/or RecG functions, the RecBCD-mediated DSB repair leads to accumulation of recombination intermediates that interfere with chromosome partition and cell division.

Metoda izlaganja heterolognih proteina na površini stanice kvasca Saccharomyces cerevisiae alternativa je klasičnim metodama imobilizacije proteina, a temelji se na fuziji gena koji kodira za protein od interesa sa genom koji kodira za neki od proteina stanične stijenke. Optimizacija sustava za izlaganje proteina na površini stanice kvasca u ovome je radu provedena uvođenjem promjena u sekvencu proteina Pir2 te promjenom pozicije proteina od interesa unutar sekvence proteina Pir2. U promatranom sustavu mjerenjem aktivnosti ˝reporter˝ enzima β-laktamaze te metodom imunoblota proteina stanične stijenke procijenjena je količina rekombinantnog proteina Pir2bla u staničnoj stijenki. Navedenim metodama promatran je i utjecaj mutacija u endocitozi, korištenjem mutanata end3 i end6, na efikasnost izlaganja rekombinantnog proteina na površini stanice u svrhu daljnje optimizacije sustava za imobilizaciju. Surface display of enzymes at the cell wall of yeast Saccharomyces cerevisiae presents an alternative to chemical immobilization methods. Methods for yeast surface display are based on the fusion of a gene encoding a protein of interest with a gene encoding one of the cell wall proteins. Optimization of the protein surface display system in this paper was performed by introducing changes in the Pir2 protein sequence and changing the position of the reporter protein within the Pir2 protein sequence. In the observed system, the amount of recombinant Pir2bla protein in the cell wall was estimated by measuring the activity of the reporter enzyme β-lactamase and the immunoblot of yeast cell wall proteins. Before mentioned methods were also used to investigate the impact of mutations in endocytosis, by using mutants end3 and end6, on efficiency of surface display system in order to optimize immobilization method.

„Yeast surface display“ sustav predstavlja metodu imobilizacije rekombinantnih proteina na površinu stanice kvasca pri čemu se gen koji kodira za željeni protein fuzionira sa čitavim genom ili fragmentom gena koji kodira za jedan od proteina stanične stijenke. Ova genetička fuzija omogućuje ekspresiju i ugradnju rekombinantnog proteina u staničnu stijenku kvasca. Osnovni nedostatci ove metode su relativno mala količina na površinu stanice ugrađenih rekombinantnih proteina, potencijalno smanjenje aktivnost rekombinantnih proteina uslijed fuzije sa proteinom stijenke te ponekad njihova hiperglikozilacija. Glikozilacija je postsintetska preinaka proteina koja može utjecati na smatanje, aktivnost i stabilnost proteina. Cilj ovog rada jest ispitati utjecaj O-glikozilacije na efikasnost ugradnje i aktivnost rekombinantnog enzima β-laktamaze u staničnu stijenku. Gen koji kodira za ovaj enzim je u jednom slučaju fuzioniran sa genom koji kodira za Pir2, a u drugom slučaju sa dijelom gena koji kodira za Ccw12 protein stijenke. Rekombinantni enzim eksprimiran je u stanicama kvasca divljeg tipa i u stanicama koji imaju mutaciju u O-glikozilaciji proteina (pmt mutanti). Količina enzima koja se ugradila u stijenku analizirana je mjerenjem aktivnosti enzima korištenjem nitrocefina kao supstrata i western blot analizom. Yeast „surface display“ system represents an alternative method of immobilization of recombinant proteins on the yeast cell wall surface in which the gene encoding for the protein of interest is fused with whole or with fragment of a gene encoding for a native yeast cell wall proteins. This genetic fusion provides constant expression and incorporation of the recombinant protein into the cell wall. However, one of the major disadvantages of this method are relatively low yield of the recombinant protein in the cell wall, potential low activity of incorporated recombinant protein due to their fusion with native cell wall protein and their hypermannosylation. Glycosylation is one of major posttranslational modifications which effects protein folding, their activity and stability. Theme of this thesis is to study the effect of O-glycosylation on incorporation of the recombinant enzyme β-lactamase into the cell wall. In one case, the gene encoding for this enzyme is fused with gene encoding for Pir2 protein, and in the other the gene is fused with the gene encoding for Ccw12 protein. The recombinant enzyme was expressed in the wild type yeast cells and in O-glycosylation mutants (pmt mutants). The amount of enzyme in the cell wall is assessed by measuring β-lactamase activity using nitrocefine as substrate and by western blot.

Izlaganje proteina na staničnoj površini (˝surface display˝) predstavlja poboljšanu tehniku imobilizacije proteina te omogućuje kontinuiranu sintezu rekombinantnih proteina zahvaljujući fuziji gena koji kodira za ispitivani enzim i gena koji kodiraju za nativne proteine stanične stijenke. U ovom radu rekombinantni protein β-laktamaza (gen bla) fuzioniran je sa proteinom Pir2, odnosno sa dijelom proteina Ccw12. Pir2 veže se za stijenku svojim Nterminalnim dijelom. Dio gena CCW12, korišten u ovom radu, kodira za C-terminalnu signalnu sekvencu na koju se veže GPI-sidro, preko kojeg se protein veže u stijenku. Najveći je nedostatak ovakvog pristupa mali prinos imobiliziranih proteina u stijenku. S pretpostavkom da mutacije u N-glikozilaciji i endocitozi povećavaju učinkovitost imobilizacije ispitivan je utjecaj mutacija u N-glikozilaciji (och1 mutant i mnn mutanti) te mutacija u endocitozi (vam4 mutant i end mutanti) na efikasnost ugradnje rekombinantnog proteina u stijenku mjerenjem aktivnost β-laktamaze, što omogućuje procjenu količine imobiliziranog rekombinantnog proteina u staničnoj stijenci. „Surface display“ system provides enhanced immobilization technique that enables countinuous synthesis of recombinant protein. This is possible because of the genetic fusion between genes encoding for native cell wall proteins and enzyme that is reasearched. In this study the enzyme β-lactamase (gene bla) and Pir2 or a fragment of Ccw12 are fuzed. Pir2 is linked to the cell wall on its N-terminal end, while Ccw12 is linked to the cell wall by GPI anchor on C- terminal region. One of the major disadvantages of this approach is low yield in protein immobilization. With the assumption that mutation in N-glycosylation and endocytosis enhances surface display system efficiency, this system was examined in mutants in Nglycosylation (och1 mutant and mnn mutants) and in mutants in endocytosis (vam4 mutant and end mutants). By measuring β-lactamase activity, it is possible to estimate the amount of recombinant protein immobilized in cell wall.

Scw4 protein u stijenku kvasca Saccharomyces cerevisiae veže se kovalentnim i neokovalentnim vezama, međutim, točan mehanizam kovalentnog povezivanja i dalje je nepoznat. Polazišna je pretpostavka da je način vezanja sličan onome PIR proteina, s obzirom na jednak način ekstrakcije kovalentno vezane frakcije ovog proteina iz stijenke. Cilj ovog istraživanja bio je odrediti koja je aminokiselina odgovorna za kovalentno vezanje Scw4 u stijenku kvasca. U ciljanu regiju gena SCW4 uvedene su mutacije, a uzgoj mutanata proveden je u nepuferiranom mediju, te pri pH=4 i pH=7. Nakon uzgoja nekovalentno vezani proteini su izolirani iz stijenki pomoću vrućeg SDS-a, a kovaletno vezani pomoću 30 mM NaOH. Dobiveni proteinski ekstrakti analizirani su Western blot metodom. Iz dobivenih rezultata možemo zaključiti kako je za kovalentno vezanje Scw4 u stijenku S. cerevisiae odgovoran ostatak glutamina Q₂₄₁. The Scw4 protein of Saccharomyces cerevisiae yeast can be bound both covalently and non-covalently to the cell wall, however, the exact mechanism of covalent binding is still unknown. The initial assumption is that the binding mechanism is similar to that of PIR proteins, given the same method of extraction of covalently bound fraction of these proteins from the yeast cell wall. The aim of this study was to determine which amino acid is responsible for covalent binding of Scw4 to the yeast wall. Mutations were introduced into the target region of the SCW4 gene, followed by the cultivation in an unbuffered medium, as well as at pH=4 and pH=7. After cultivation, non-covalently bound proteins were isolated by hot SDS, while covalently bound proteins were extracted by 30 mM NaOH. Protein extracts were analyzed by the Western blot method. The obtained results showed that glutamine Q₂₄₁ is responsible for covalent binding of Scw4 to the cell wall of S. cerevisiae.

U novije vrijeme, veliki interes za heterologne proteine dovodi do potrebe za pronalaskom efikasnih tehnika za njihovu proizvodnju. Tehnika izlaganja heterologih proteina na površini stanične stijenke kvasca je značajan predmet istraživanja u znanosti i industriji. Njezinom primjenom omogućava se istovremena proizvodnja i sekrecija heterolognih proteina te njihova imobilizacija na površini stanice i primijena kvasaca kao staničnih biokatalizatora. Osnovni nedostatak metode je relativno niska koncentracija imobiliziranih proteina na površini stanica. Ta je činjenica glavni razlog sve većeg broja istraživanja u svrhu poboljšanja metode. Nastoje se pronaći optimalni sustavi za izlaganje heterolognih proteina te pogodni domaćini. Uz do sada najviše istraženi kvasac S. cerevisiae, sve veća važnost se pridaje metilotrofnim kvascima. Cilj ovog rada je prikazati nekonvencionalne metilotrofne kvasce, P. pastoris i H. polymorpha, kao pogodne domaćine za proizvodnju heterolognih proteina te njihovo izlaganje na površini stanica. In recent times, the great interest in heterologous proteins has led to the need of finding efficient techniques for their production. Yeast surface display of heterologous proteins has become a significant subject of research in science and industry. Its application enables the simultaneous production and secretion of heterologous proteins, as well as their immobilization on the cell surface and the use of yeast as whole-cell biocatalysts. The main disadvantage of the method is the relatively low concentration of immobilized proteins on the cell surface. This fact is the main reason for the progress of research in order to improve the method. Great effort has been put into finding the optimal systems for surface display of heterologous proteins and finding the suitable host. In addition to the most researched yeast S. cerevisiae so far, increasing importance is given to methylotrophic yeast. The aim of this study is to present non-conventional methylotrophic yeasts, P. pastoris and H. polymorpha, as suitable hosts for the production of heterologous proteins and their display on the cell surface.