Cell surface display of organophosphorus hydrolase using ice nucleation protein

A new anchor system based on the ice nucleation protein (InaV) from Pseudomonas syringae INA5 was developed for cell surface display of functional organophosphorus hydrolase (OPH). The activity and stability of cells expressing the truncated InaV (INPNC)-OPH fusions were compared to cells with surface-expressed OPH using two other fusion anchors based on Lpp-OmpA and the truncated InaK protein. Whole cell activity was as much as 5-fold higher using the InaV anchor. Majority of the OPH activity was located on the cell surface as determined by protease accessibility and cell fractionation experiments. The surface localization of OPH was further verified by immunofluorescence microscopy. Constitutive expression of OPH on the surface using the InaV anchor resulted in no cell lysis or growth inhibition, in contrast to the Lpp-OmpA anchor. Suspended cultures also exhibited good stability, retaining almost 100% activity over a period of 3 weeks. Therefore, the InaV anchor system offers an attractive alternative to the currently available surface anchors, providing high-level expression and superior stability.     

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 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.     

Organophosphorus compound detection on a cell chip with yeast coexpressing hydrolase and eGFP

Organophosphorus compounds (OPs) such as pesticides, fungicides, and herbicides are highly toxic but are nevertheless extensively used worldwide. To detect OPs, we constructed a yeast strain that co-displays organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (EGFP) on the cell surface using a Flo1p anchor system. OP degradation releases protons and causes a change in pH. This pH change results in structural deformation of EGFP, which triggers quenching of its fluorescence, thereby making this cell useful for visual detection of OPs. Fluorescence microscopy confirmed the high-intensity fluorescence displayed by EGFP on the cell surface. The yeast strain possessed sufficient OPH hydrolytic activities for degrading OPs, as measured by incubation with 1 mM paraoxon for 24 h at 30°C. In addition, with 20 mM paraoxon at 30°C, fluorescence quenching of EGFP on the single yeast cell was observed within 40 s in a microchamber chip. These observations suggest that engineered yeast cells are suitable for simultaneous degradation and visual detection of OPs.     

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.     

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.     

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.     

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.     

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.     

High level recombinant protein expression in Ralstonia eutropha using T7 RNA polymerase based amplification

We report further development of a novel recombinant protein expression system based on the Gram-negative bacterium, Ralstonia eutropha. In this study, we were able to express soluble, active, organophosphohydrolase (OPH), a protein that is prone to inclusion body formation in Escherichia coli, at titers greater than 10 g/L in high cell density fermentation. This represents a titer that is approximately 100-fold greater than titers previously reported in E. coli for this enzyme. R. eutropha strains expressing OPH were generated in two cloning steps. First, the T7 RNA polymerase gene was placed under the control of the strong, inducible phaP promoter and integrated into the phaP locus of R. eutropha NCIMB 40124. Second, a single copy of the oph gene under control of the T7 promoter was randomly integrated into the chromosome using a transposon cloning vector.     

Surface display of organophosphorus hydrolase on Saccharomyces cerevisiae

The gene encoding organophosphorus hydrolase (OPH) from Flavobacterium species was expressed on the cell surface of Saccharomyces cerevisiae MT8-1 using a glycosylphosphatidylinositol (GPI) anchor linked to the C-terminal region of OPH. Immunofluorescence microscopy confirmed the localization of OPH on the cell surface, and fluorescence intensity measurement of cells revealed that 1.4 x 10(4) molecules of OPH per cell were displayed. Seventy percent of OPH whole-cell activity was detected on the cell surface by protease accessibility assay. The activity of OPH was highly dependent on cell growth conditions. The maximum activity was obtained when cells were grown in a synthetic dextrose medium lacking tryptophan (SD-W) buffered by 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES, 200 mM, pH 7.0) at 20 degrees C, and cobalt chloride was added at 0.1 mM. S. cerevisiae MT8-1 displaying OPH which exhibited a higher activity than Escherichia coli displaying OPH using the ice nucleation protein (INP) anchor. The use of S. cerevisiae MT8-1, which has a "generally regarded as safe (GRAS)" status, as a host for the easy expression of the OPH gene provides a new biocatalyst useful for simultaneous detoxification and detection of organophosphorus pesticides.     

Expression and characterization of the flocculin Flo11/Muc1, a Saccharomyces cerevisiae mannoprotein with homotypic properties of adhesion

The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticus strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.     

Enhanced biodegradation of toxic organophosphate compounds using recombinant Escherichia coli with sec pathway-driven periplasmic secretion of organophosphorus hydrolase

Although Escherichia coli can be genetically engineered to degrade environmental toxic organophosphate compounds (OPs) to nontoxic materials, a critical problem in such whole cell systems is limited substrate diffusion. The present work examined whether periplasmic expression of organophosphorus hydrolase (OPH) resulted in better whole cell enzymatic activity compared to standard cytosolic expression. Recombinant OPH periplasmic expression was achieved using the general secretory (sec) pathway with the pelB signal sequence. We found that while total OPH activity in periplasmic-expressing cell lysates was lower compared to that in cytosolic-expressing cell lysates whole cell OPH activity was 1.8-fold greater at 12 h post-induction in the periplasmic-expressing cells as a result of OPH translocation into the periplasmic space ( approximately 67% of whole cell OPH activity was found in the periplasmic fraction). These data suggest that E. coli engineered to periplasmically secrete OPH via the sec pathway may provide an improved whole cell biodegradation system for destruction of environmental toxic OPs.     

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.     

Displaying Lipase B from Candida antarctica in Pichia pastoris Using the Yeast Surface Display Approach: Prospection of a New Anchor and Characterization of the Whole Cell Biocatalyst

Yeast Surface Display (YSD) is a strategy to anchor proteins on the yeast cell wall which has been employed to increase enzyme stability thus decreasing production costs. Lipase B from Candida antarctica (LipB) is one of the most studied enzymes in the context of industrial biotechnology. This study aimed to assess the biochemical features of this important biocatalyst when immobilized on the cell surface of the methylotrophic yeast Pichia pastoris using the YSD approach. For that purpose, two anchors were tested. The first (Flo9) was identified after a prospection of the P. pastoris genome being related to the family of flocculins similar to Flo1 but significantly smaller. The second is the Protein with Internal Repeats (Pir1) from P. pastoris. An immunolocalization assay showed that both anchor proteins were able to display the reporter protein EGFP in the yeast outer cell wall. LipB was expressed in P. pastoris fused either to Flo9 (FLOLIPB) or Pir1 (PIRLIPB). Both constructions showed hydrolytic activity towards tributyrin (>100 U/mg_dcw and >80 U/mg_dcw, respectively), optimal hydrolytic activity around 45°C and pH 7.0, higher thermostability at 45°C and stability in organic solvents when compared to a free lipase.     

Capacity of N- and C-domains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> flo1p cell wall protein homologue to expose lip2 lipase on cell surface of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> yeast

The closest homologue of the Saccharomyces cerevisiae Flo1p cell wall protein was detected in Yarrowia lipolytica yeast (YALI0C09031p) by the method of genomic analysis, and capacity of its N- and C-domains to expose the Lip2 lipase on the cell surface was studied. The efficient fixation of the enzyme on the Y. lipolytica cell wall surface was demonstrated. The activity of the cell-bound lipase was 9170 and 3200 units per 1 g of dry solid matter when using N- and C-domains of the cell wall protein, respectively. At the same time, in the case of immobilization using the N-domain, approximately 30% of the total lipase activity was detected in the culture medium, whereas when using C-domain of the cell wall protein YALI0C09031p, practically all lipase was in the immobilized state. Obtained values of the level of the cell-bound lipase activity considerably exceed previously published data opening a prospect for new technological solutions which meet industrial needs.     

Simultaneous degradation of organophosphate and organochlorine pesticides by Sphingobium japonicum UT26 with surface-displayed organophosphorus hydrolase

A genetically engineered microorganism (GEM) capable of simultaneously degrading organophosphate and organochlorine pesticides was constructed for the first time by display of organophosphorus hydrolase (OPH) on the cell surface of a hexachlorocyclohexane (HCH)-degrading Sphingobium japonicum UT26. The GEM could potentially be used for removing the two classes of pesticides that may be present in mixtures at contaminated sites. A surface anchor system derived from the truncated ice nucleation protein (INPNC) from Pseudomonas syringae was used to target OPH onto the cell surface of UT26, reducing the potential substrate uptake limitation. The surface localization of INPNC–OPH fusion was verified by cell fractionation, western blot, proteinase accessibility, and immunofluorescence microscopy. Furthermore, the functionality of the surface-exposed OPH was demonstrated by OPH activity assays. Surface display of INPNC–OPH fusion (82 kDa) neither inhibited cell growth nor affected cell viability. The engineered UT26 could degrade parathion as well as γ-HCH rapidly in minimal salt medium. The removal of parathion and γ-HCH by engineered UT26 in sterile and non-sterile soil was also studied. In both soil samples, a mixture of parathion (100 mg kg^?1) and γ-HCH (10 mg kg^?1) could be degraded completely within 15 days. Soil treatment results indicated that the engineered UT26 is a promising multifunctional bacterium that could be used for the bioremediation of multiple pesticide-contaminated environments.     

Effect of culture conditions on the whole cell activity of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing periplasmic organophosphorus hydrolase and cytosolic GroEL/ES chaperone

Specific whole cell activity strongly affects sensitivity and detection limit of whole cell-based biosensors. Previously, we developed recombinant Escherichia coli coexpressing periplasmic organophosphorus hydrolase (OPH) and cytosolic chaperone GroEL-GroES (GroEL/ES). In present work, we investigated the effect of culture conditions on whole cell OPH activity. Especially, the whole cell OPH activity was significantly affected by the concentration of tetracycline that is an inducer for chaperone GroEL/ES. When cultured at 20°C for 31 h in M9 medium containing 1 mM IPTG, 50 ng/mL tetracycline, and 500 µM CoCl₂, the recombinant E. coli exhibited a specific whole cell OPH activity (U/OD₆₀₀) of ~0.55, which is 2.6-fold higher than that of recombinant E. coli cultured as previously described conditions. In addition, recombinant cells showed adequate storage stability for 1 week with 100% of original response. Finally, the improved activity and adequate stability in the whole cell biocatalyst will contribute to sensitivity, detection time, and stability of a whole cell-based biosensor for the detection of toxic organophosphates.     

Coexpression of molecular chaperone enhances activity and export of organophosphorus hydrolase in Escherichia coli

Periplasmic secretion has been used in attempts to construct an efficient whole‐cell biocatalyst with greatly reduced diffusion limitations. Previously, we developed recombinant Escherichia coli that express organophosphorus hydrolase (OPH) in the periplasmic space using the twin‐arginine translocation (Tat) pathway to degrade environmental toxic organophosphate compounds. This system has the advantage of secreting protein into the periplasm after folding in the cytoplasm. However, when OPH was expressed with a Tat signal sequence in E. coli, we found that the predominant OPH was an insoluble premature form in the cytoplasm, and thus, the whole‐cell OPH activity was significantly lower than its cell lysate activity. In this work, we, for the first time, used a molecular chaperone coexpression strategy to enhance whole‐cell OPH activity by improving the periplasmic translocation of soluble OPH. We found that the effect of GroEL‐GroES (GroEL/ES) assistance on the periplasmic localization of OPH was secretory pathway dependent. We observed a significant increase in the amount of soluble mature OPH when cytoplasmic GroEL/ES was expressed; this increase in the amount of mature OPH might be due to enhanced OPH folding in the cytoplasm. Importantly, the whole‐cell OPH activity of the chaperone–coexpressing cells was ～5.5‐fold greater at 12 h after induction than that of cells that did not express the chaperone as a result of significant Tat‐based periplasmic translocation of OPH in the chaperone–coexpressing cells. Collectively, these data suggest that molecular chaperones significantly enhance the whole‐cell activity of periplasmic OPH‐secreting cells, yielding an effective whole‐cell biocatalyst system with highly reduced diffusion limitations. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 925–930, 2012