Construction of Yeast Strains with High Cell Surface Lipase Activity by Using Novel Display Systems Based on the Flo1p Flocculation Functional Domain

We constructed a novel cell-surface display system, using as a new type of cell-wall anchor 3,297 or 4,341 bp of the 3′ region of the FLO1 gene (FS or FL gene, respectively), which encodes the flocculation functional domain of Flo1p. In this system, the N terminus of the target protein was fused to the FS or FL protein and the fusion proteins were expressed under the control of the inducible promoter UPR-ICL (5′ upstream region of the isocitrate lyase of Candida tropicalis). Using this new system, recombinant lipase with a pro sequence from Rhizopus oryzae (rProROL), which has its active site near the C terminus, was displayed on the cell surface. Cell-surface display of the FSProROL and FLProROL fusion proteins was confirmed by immunofluorescence microscopy and immunoblotting. Lipase activity reached 145 IU/liter (61.3 IU/g [dry cell weight]) on the surface of the yeast cells, which successfully catalyzed the methanolysis reaction. Using these whole-cell biocatalysts, methylesters synthesized from triglyceride and methanol reached 78.3% after 72 h of reaction. To our knowledge, this is the first example of cell-surface display of lipase with high activity. Interestingly, the yeast cells displaying the FLProROL protein showed strong flocculation, even though the glycosylphosphatidylinositol anchor attachment signal and cell-membrane-anchoring region of Flo1p had been deleted from this gene. The cell-surface display system based on FL thus endows the yeast strain with both novel enzyme display and strong flocculation ability.     

高效絮凝素毕赤酵母表面展示系统的构建

为了获得高效的脂肪酶毕赤酵母表面展示系统,利用来自酿酒酵母絮凝素蛋白Flo1的N端874个氨基酸残基(FS)和C端的1101个氨基酸残基(FL)作为锚定蛋白分别构建了2套载体系统.带有前肽的米黑根毛霉脂肪酶(ProRML)克隆到构建的2套展示载体中,使米黑根毛霉脂肪酶(RML)分别以N端锚定或C端锚定的方式实现在毕赤酵母细胞表面的展示.利用RMLC端的Flag标签,通过流式细胞术和激光扫描共聚焦显微镜检测2套系统中RML在酵母表面的展示情况.研究发现,N端锚定于酵母表面的展示酶FSR以pNPC为底物时,水解活力达到了105.3U/g,大约为C端锚定的展示酶FLR活力的2倍.同时FSR比FLR具有更宽的温度、pH作用范围和更好的热稳定性.与游离酶和固定化酶相比,展示酶FSR也表现出更为优良的热稳定性.结果提示,基于Flo1N端锚定的展示系统更适合展示活性中心近C端的脂肪酶,推动了展示酶的进一步研究和开发.     

Effect of cultivation conditions on cell-surface display of Flo1 fusion protein using sake yeast

The cell-surface display of the Flo1p anchor system with a flocculation functional domain was examined under various cultivation conditions. As a model system, lipase from Rhizopus oryzae with the pro sequence was genetically fused to the Flo1 short (FS) anchor (FSProROL) and displayed on the sake yeast cell-surface under the control of the SED800 promoter (pSED800). The nutrients and carbon source in the culture media affected the display of the fusion protein FSProROL on the sake yeast cell-surface. The lipase activity in whole cells cultivated in poor media, without peptone and/or yeast extracts, were higher than those cultivated in rich media. In addition, glucose and maltose were effective carbon sources for increasing the lipase activity in whole cells, and the addition of di- or tri-saccharide as the carbon source reduced the release of the lipase activity into the culture supernatants. The initial glucose concentration was found to influence the total lipase activity and it mainly affected the lipase activity in whole cells. Under the optimum condition, sake yeast was found to show high cell density and high lipase activity in short time cultivation.     

Construction of a Pichia pastoris cell-surface display system using Flo1p anchor system

A Pichia pastoris cell-surface display system was constructed using a Flo1p anchor system, which was developed in Saccharomyces cerevisiae. The lipase from Rhizopus oryzae with a pro sequence (ProROL) was used as the model protein and was genetically fused to the anchor consisting of amino acids 1-1099 of Flo1p (FS anchor). The resulting fusion protein FSProROL was expressed under the control of the alcohol oxidase 1 promoter (pAOX1). The fluorescence microscopy of immunolabeled P. pastoris cells revealed that ProROL was displayed on the cell surface, and Western blot analysis revealed that the fusion protein FSProROL was noncovalently attached to the cell wall and highly glycosylated. The lipase activity of P. pastoris cells was affected by the methanol concentration for the induction phase. Surprisingly, the activity of lipase displayed on the cells incubated at 60 degrees C was not only stable but also increased to about 6.5 times the initial value after 4 h incubation.     

Long anchor using Flo1 protein enhances reactivity of cell surface-displayed glucoamylase to polymer substrates

We investigated the influence of anchor length on the reactivity to polymer substrate of enzyme displayed on yeast cell surfaces. Using various lengths [42, 102, 146, 318, 428, and 1,326 amino acids (aa)] of the C-terminal region of the Saccharomyces cerevisiae Flo1 protein (Flo1p), which plays a major role in yeast flocculation, six display systems with various anchor lengths were constructed. In these systems, the target protein was displayed on the yeast cell surface under the control of the 5'-upstream region of the isocitrate lyase gene of Candida tropicalis ( UPR-ICL). Cell-surface display of Rhizopus oryzae glucoamylase by these systems was induced and confirmed in all systems by immunofluorescence microscopy and immunoblotting. Flow-cytometer measurement of the fluorescence intensity of immunofluorescence-labeled yeast cells displaying glucoamylase indicated that glucoamylase displayed with longer anchors, especially those of 428 and 1,326 aa in length, had higher reactivity to antibodies. The reactivity of starch to displayed glucoamylase, which was evaluated by plate assay, increased with anchor length, as did the cell growth-rate in starch-containing medium. These results indicate that cell-surface display systems using 428- and 1,326-aa length anchors of Flo1p are effective for the display of enzymes on the outer surface of yeast cells.     

The contribution of Pir protein family to yeast cell surface display

Proteins with internal repeats (Pir) in the Baker’s yeast are located on the cell wall and include four highly homologous members. Recently, Pir proteins have become increasingly used as anchor proteins in yeast cell surface display systems. These display systems are classified into three types: N-terminal fusion, C-terminal fusion, and inserted fusion. In addition to the GPI (glycosylphosphatidyl inositol) and the FL/FS anchor proteins, these three Pir-based systems significantly increase the choices for target proteins to be displayed. Furthermore, Pir proteins can also be used as a fusion partner for target proteins to be effectively secreted into culture medium. Here, we summarize the development and application of Pir proteins as anchor proteins.     

Development of yeast cells displaying <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B and their application to ester synthesis reaction

We isolated the lipase B from Candida antarctica CBS 6678 (CALB CBS6678) and successfully constructed CALB-displaying yeast whole-cell biocatalysts using the Flo1p short (FS) anchor system. For the display of CALB on a yeast cell surface, the newly isolated CALB CBS6678 exhibited higher hydrolytic and ester synthesis activities than the well-known CALB, which is registered in GenBank (Z30645). A protease accessibility assay using papain as a protease showed that a large part of CALB, approximately 75%, was localized on an easily accessible part of the yeast cell surface. A comparison of the lipase hydrolytic activities of yeast whole cells displaying only mature CALB (CALB) and those displaying mature CALB with a Pro region (ProCALB) revealed that mature CALB is preferable for yeast cell surface display using the Flo1p anchor system. Lyophilized yeast whole cells displaying CALB were applied to an ester synthesis reaction at 60°C using adipic acid and n-butanol as substrates. The amount of dibutyl adipate (DBA) produced increased with the reaction time until 144 h. This indicated that CALB displayed on the yeast cell surface retained activity under the reaction conditions.     

Construction of system for localization of target protein in yeast periplasm using invertase

We constructed a novel system for periplasmic localization of target proteins, using yeast external invertase (INV) as anchor protein, in which the C- or N-terminal of the target protein was fused to the invertase and the fusion proteins expressed under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (GAPDH). Unlike in conventional cell-surface display, the system enables the target fusion protein to localize in yeast periplasm in a free state. As a model, enhanced green fluorescence protein (EGFP) was localized in yeast periplasm using the new system. Yeast-periplasm localization of INV-EGFP and EGFP-INV fusion proteins was confirmed by fluorescence microscopy and immunoblotting: green fluorescence was observed at the cell outline and, in western blot analysis, most fusion proteins were detected in the cell-surface fraction, indicating that the fusion proteins had been transported to the cell-surface layer. In addition, in both C- and N-terminal fusion, invertase showed activity, indicating dimer formation. These results demonstrate that invertase is a useful anchor for localizing target protein in the yeast periplasm.     

Combination of site-directed mutagenesis and yeast surface display enhances <Emphasis Type="Italic">Rhizomucor miehei</Emphasis> lipase esterification activity in organic solvent

To increase the activity of Rhizomucor miehei lipase (RML) in organic solvent, multiple sequence alignments and rational site-directed mutagenesis were used to create RML variants. The obtained proteins were surface-displayed on Pichia pastoris by fusion to Flo1p as an anchor protein. The synthetic activity of four variants showed from 1.1- to 5-fold the activity of native lipase in an esterification reaction in heptane with alcohol and caproic acid as substrates. The increase in esterification activity may be attributed to the four mutations changing the flexibility of RML or facilitating the reaction. In conclusion, this method demonstrated that multiple sequence alignments and rational site-directed mutagenesis combined with yeast display technology is a faster and more effective means of obtaining high-efficiency esterification lipase variants compared with previous similar methods.     

Display of Candida antarctica lipase B on Pichia pastoris and its application to flavor ester synthesis

Two alternative cell-surface display systems were developed in Pichia pastoris using the α-agglutinin and Flo1p (FS) anchor systems, respectively. Both the anchor cell wall proteins were obtained originally from Saccharomyces cerevisiae. Candida antarctica lipase B (CALB) was displayed functionally on the cell surface of P. pastoris using the anchor proteins α-agglutinin and FS. The activity of CALB displayed on P. pastoris was tenfold higher than that of S. cerevisiae. The hydrolytic and synthetic activities of CALB fused with α-agglutinin and FS anchored on P. pastoris were investigated. The hydrolytic activities of both lipases displayed on yeast cells surface were more than 200 U/g dry cell after 120 h of culture (200 and 270 U/g dry cell, respectively). However, the synthetic activity of CALB fused with α-agglutinin on P. pastoris was threefold higher than that of the FS fusion protein when applied to the synthesis of ethyl caproate. Similarly, the CALB displayed on P. pastoris using α-agglutinin had a higher catalytic efficiency with respect to the synthesis of other short-chain flavor esters than that displayed using the FS anchor. Interestingly, for some short-chain esters, the synthetic activity of displaying CALB fused with α-agglutinin on P. pastoris was even higher than that of the commercial CALB Novozyme 435.     

Improvement in organophosphorus hydrolase activity of cell surface-engineered yeast strain using Flo1p anchor system

Organophosphorus hydrolase (OPH) hydrolyzes organophosphorus esters. We constructed the yeast-displayed OPH using Flo1p anchor system. In this system, the N-terminal region of the protein was fused to Flo1p and the fusion protein was displayed on the cell surface. Hydrolytic reactions with paraoxon were carried out during 24 h of incubation of OPH-displaying cells at 30°C. p-Nitrophenol produced in the reaction mixture was detected by HPLC. The strain with highest activity showed 8-fold greater OPH activity compared with cells engineered using glycosylphosphatidylinositol anchor system, and showed 20-fold greater activity than Escherichia coli using the ice nucleation protein anchor system. These results indicate that Flo1p anchor system is suitable for display of OPH in the cell surface-expression systems.     

Direct production of ethanol from raw corn starch via fermentation by use of a novel surface-engineered yeast strain codisplaying glucoamylase and alpha-amylase

Direct and efficient production of ethanol by fermentation from raw corn starch was achieved by using the yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis alpha-amylase by using the C-terminal-half region of alpha-agglutinin and the flocculation functional domain of Flo1p as the respective anchor proteins. In 72-h fermentation, this strain produced 61.8 g of ethanol/liter, with 86.5% of theoretical yield from raw corn starch.     

酿酒酵母表面展示表达系统及应用

酵母细胞表面展示表达系统是一种固定化表达异源蛋白质的真核展示系统,即把异源靶蛋白基因序列与特定的载体基因序列融合后导入酵母细胞,利用酿酒酵母细胞内蛋白转运到膜表面的机制（GPI锚定）使靶蛋白定位于酵母细胞表面并进行表达。它利用细胞表面展示技术使外源蛋白固定化于细胞表面,从而生产微生物细胞表面蛋白,可应用于生物催化剂、细胞吸附剂、活疫苗、环境治理、蛋白质文库筛选、高亲和抗体、生物传感器、抗原／抗体库构建、免疫检测及亲和纯化、癌症诊断等领域。国内对这一方面研究较少,本文主要介绍了该技术的基本原理、研究现状、应用及其发展前景。     

Display of both N- and C-terminal target fusion proteins on the Aspergillus oryzae cell surface using a chitin-binding module

A novel cell surface display system in Aspergillus oryzae was established by using a chitin-binding module (CBM) from Saccharomyces cerevisiae as an anchor protein. CBM was fused to the N or C terminus of green fluorescent protein (GFP) and the fusion proteins (GFP-CBM and CBM-GFP) were expressed using A. oryzae as a host. Western blotting and fluorescence microscopy analysis showed that both GFP-CBM and CBM-GFP were successfully expressed on the cell surface. In addition, cell surface display of triacylglycerol lipase from A. oryzae (tglA), while retaining its activity, was also successfully demonstrated using CBM as an anchor protein. The activity of tglA was significantly higher when tglA was fused to the C terminus than N terminus of CBM. Together, these results show that CBM used as a first anchor protein enables the fusion of both the N and/or C terminus of a target protein.     

Deletion of tandem repeats causes flocculation phenotype conversion from Flo1 to NewFlo in Saccharomyces cerevisiae

A gene, FLONS, conferring NewFlo-type flocculation ability in yeast was cloned. The 3,396-bp ORF encoded a peptide of 1,132 amino acids with high identity to Flo1 protein. Aligned with the FLO1 gene, two repeated regions (675 and 540 bp) were lost in the middle of FLONS, revealing that this gene was a derived form of the FLO1 gene. The missing repeated sequence contained three highly homologous repeat units. Although the flocculation phenotype of the transformant YTS-S with the FLONS gene was inhibited by both mannose and glucose, it exhibited some distinguished physiological characteristics from the reported typical NewFlo-type flocculation during detailed investigation. The deletion of repeats was suspected to cause conversion of the flocculation phenotype from Flo1 to NewFlo, suggesting that intragenic tandem repeats generated functional variability in Flo1 protein.     

Direct Production of Ethanol from Raw Corn Starch via Fermentation by Use of a Novel Surface-Engineered Yeast Strain Codisplaying Glucoamylase and α-Amylase

Direct and efficient production of ethanol by fermentation from raw corn starch was achieved by using the yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis α-amylase by using the C-terminal-half region of α-agglutinin and the flocculation functional domain of Flo1p as the respective anchor proteins. In 72-h fermentation, this strain produced 61.8 g of ethanol/liter, with 86.5% of theoretical yield from raw corn starch.     

Flocculation protein structure and cell–cell adhesion mechanism in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Cell–cell adhesion occurs in a broad spectrum of biological processes, of which yeast flocculation is an area of interest for evolutionary scientists to brewers and winemakers. The flocculation mechanism is based on a lectin-carbohydrate interaction but is not yet fully understood, although the first model dates back to the 1950s. This review will update the current understanding of the complex mechanism behind yeast flocculation. Moreover, modern technologies to measure the forces involved in single carbohydrate-lectin interactions, are discussed. The Flo1 protein has been extensively described as the protein responsible for strong flocculation. Recently, more research has been directed to the detailed analysis of this flocculin. Due to the advances in the field of bioinformatics, more information about Flo1p could be obtained via structurally or functionally related proteins. Here, we review the current knowledge of the Flo1 protein, with a strong emphasis towards its structure.     

Region of Flo1 Proteins Responsible for Sugar Recognition

Yeast flocculation is a phenomenon which is believed to result from an interaction between a lectin-like protein and a mannose chain located on the yeast cell surface. The FLO1 gene, which encodes a cell wall protein, is considered to play an important role in yeast flocculation, which is inhibited by mannose but not by glucose (mannose-specific flocculation). A new homologue of FLO1, named Lg-FLO1, was isolated from a flocculent bottom-fermenting yeast strain in which flocculation is inhibited by both mannose and glucose (mannose/glucose-specific flocculation). In order to confirm that both FLO1 and Lg-FLO1 are involved in the yeast flocculation phenomenon, the FLO1 gene in the mannose-specific flocculation strain was replaced by the Lg-FLO1 gene. The transformant in which the Lg-FLO1 gene was incorporated showed the same flocculation phenotype as the mannose/glucose-specific flocculation strain, suggesting that the FLO1 and Lg-FLO1 genes encode mannose-specific and mannose/glucose-specific lectin-like proteins, respectively. Moreover, the sugar recognition sites for these sugars were identified by expressing chimeric FLO1 and Lg-FLO1 genes. It was found that the region from amino acid 196 to amino acid 240 of both gene products is important for flocculation phenotypes. Further mutational analysis of this region suggested that Thr-202 in the Lg-Flo1 protein and Trp-228 in the Flo1 protein are involved in sugar recognition.     

Quantitative evaluation of Candia antarctica lipase B displayed on the cell surface of a Pichia pastoris based on an FS anchor system

A new approach is described to quantify the number of enzyme molecules, such as Candia antarctica lipase B, that are displayed on the cell surface of Pichia pastoris. Enhanced green fluorescent protein (EGFP) and Candida antarctica lipase B (CALB) were fused and displayed on the surface of P. pastoris by linking to the anchor flocculation functional domain of FLO1p from Saccharomyces cerevisiae. Confocal laser scanning microscopy, flow cytometry, and fluorescence spectrophotometry were used to monitor the fluorescence intensity of fused EGFP. Combined with the corresponding protein concentration detected in the medium, a standard curve describing the relationship between the fusion protein concentration and fluorescence intensity were obtained and could be used to number CALB displayed on the cell surface. The results showed that approx. 10⁴ molecules of CALB molecules were immobilized on the single P. pastoris cell wall based on FS anchor system.