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.     

Preparation of a whole-cell biocatalyst of mutated <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B (mCALB) by a yeast molecular display system and its practical properties

To prepare a whole-cell biocatalyst of a stable lipase at a low price, mutated Candida antarctica lipase B (mCALB) constructed on the basis of the primary sequences of CALBs from C. antarctica CBS 6678 strain and from C. antarctica LF 058 strain was displayed on a yeast cell surface by α-agglutinin as the anchor protein for easy handling and stability of the enzyme. When mCALB was displayed on the yeast cell surface, it showed a preference for short chain fatty acids, an advantage for producing flavors; although when Rhizopus oryzae lipase (ROL) was displayed, the substrate specificity was for middle chain lengths. When the thermal stability of mCALB on the cell surface was compared with that of ROL on a cell surface, T _1/2, the temperature required to give a residual activity of 50% for heat treatment of 30 min, was 60°C for mCALB and 44°C for ROL indicating that mCALB displayed on cell surface has a higher thermal stability. Furthermore, the activity of the displayed mCALB against p-nitrophenyl butyrate was 25-fold higher than that of soluble CALB, as reported previously. These findings suggest that mCALB-displaying yeast is more practical for industrial use as the whole-cell biocatalyst.     

南极假丝酵母脂肪酶B的酿酒酵母表面展示及其催化己酸乙酯的合成

从南极假丝酵母(Candida antarctica)基因组克隆得到南极假丝酵母脂肪酶B(Candida antarctica Lipase B, CALB)全基因片段, 利用连接肽celA Linker将CALB与酿酒酵母细胞表面展示蛋白a-凝集素的C端连接融合, 构建表面展示载体pICAS-celAL-CALB, 转化酵母后获得重组酵母菌Saccharomyces cerevisiae pICAS-celAL-CALB。该重组酵母菌经葡萄糖诱导表达及分析, 表明CALB已在酿酒酵母细胞表面成功展示, 水解活力达26.26 u/(g·dry cell)。重组酵母菌经冻干能有效地实现在非水相中全细胞催化己酸和乙醇酯化合成己酸乙酯。反应物己酸与乙醇的摩尔比为1:1.25, 己酸乙酯的产率为98.0%, 具有较好的操作稳定性。     

Efficient synthesis of enantiomeric ethyl lactate by <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B (CALB)-displaying yeasts

The whole-cell biocatalyst displaying Candida antarctica lipase B (CALB) on the yeast cell surface with α-agglutinin as the anchor protein was easy to handle and possessed high stability. The lyophilized CALB-displaying yeasts showed their original hydrolytic activity and were applied to an ester synthesis using ethanol and l-lactic acid as substrates. In water-saturated heptane, CALB-displaying yeasts catalyzed ethyl lactate synthesis. The synthesis efficiency increased depending on temperature and reached approximately 74% at 50°C. The amount of l-ethyl lactate increased gradually. l-Ethyl lactate synthesis stopped at 200 h and restarted after adding of l-lactic acid at 253 h. It indicated that CALB-displaying yeasts retained their synthetic activity under such reaction conditions. In addition, CALB-displaying yeasts were able to recognize l-lactic acid and d-lactic acid as substrates. l-Ethyl lactate was prepared from l-lactic acid and d-ethyl lactate was prepared from d-lactic acid using the same CALB-displaying whole-cell biocatalyst. These findings suggest that CALB-displaying yeasts can supply the enantiomeric lactic esters for preparation of useful and improved biopolymers of lactic acid.     

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.     

Development of recombinant Aspergillus oryzae whole-cell biocatalyst expressing lipase-encoding gene from Candida antarctica

To expand the industrial applications of Candida antarctica lipase B (CALB), we developed Aspergillus oryzae whole-cell biocatalyst expressing the lipase-encoding gene from C. antarctica. A. oryzae niaD300, which was derived from the wild type strain RIB40, was used as the host strain. The CALB gene was isolated from C. antarctica CBS6678 and expression plasmids were constructed with and without secretion signal peptide. The lipase gene was expressed under the control of improved glaA and pNo-8142 promoters of plasmids pNGA142 and pNAN8142, respectively. The Southern blot analysis demonstrated the successful integration of the CALB gene in the genome of A. oryzae. To determine the role of signal peptide, the expression plasmids were constructed with homologous and heterologous secretion signal sequences of triacylglycerol lipase gene (tglA) from A. oryzae and lipase B (CALB) from C. antarctica, respectively. The C-terminal FLAG tag does not alter the catalytic properties of the lipase enzyme and Western blotting analysis using anti-FLAG antibodies demonstrated the presence of cell wall and membrane bound lipase responsible for the biocatalytic activity of the whole-cell biocatalyst. The resultant recombinant A. oryzae was immobilized within biomass support particles (BSPs) made of polyurethane foam (PUF) and the BSPs were successfully used for the hydrolysis of para-nitrophenol butyrate (p-NPB) and for the optical resolution of (RS)-1-phenyl ethanol by enantioselective transesterification with vinyl acetate as acyl donor.     

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.     

Surface display of active lipase in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using Sed1 as an anchor protein

A Pichia pastoris cell-surface display system was constructed using the Sed1 anchor system that has been developed in Saccharomyces cerevisiae. Candida antarctica lipase B (CALB) was used as the model protein and was fused to an anchor that consisted of 338 amino acids of Sed1. The resulting fusion protein CALBSed1 was expressed under the control of the alcohol oxidase 1 promoter (pAOX1). Immunofluorescence microscopy of immunolabeled Pichia pastoris revealed that CALB was displayed on the cell surface. Western blot analysis showed that the fusion protein CALBSed1 was attached covalently to the cell wall and was highly glycosylated. The hydrolytic activity of the displayed CALB was more than 220 U/g dry cells after 120 h of culture. The displayed protein also exhibited a higher degree of thermostability than free CALB.     

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.     

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.     

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.     

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.     

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.     

Production of glucoamylase in pyruvate decarboxylase deletion mutants of the yeast Kluyveromyces lactis

We have developed a novel Escherichia coli cell surface display system by employing PgsA as an anchoring motif. In our display system, C-terminal fusion to PgsA anchor protein from Bacillus subtilis was used. The enzymes selected for display were α-amylase (AmyA) from Streptococcus bovis 148 and lipase B (CALB) from Candida antarctica. The molecular mass values of AmyA and CALB are approximately 77 and 34 kDa, respectively. The enzymes were displayed on the surface as a fusion protein with a FLAG peptide tag at the C terminus. Both the PgsA-AmyA-FLAG and PgsA-CALB-FLAG fusion proteins were shown to be displayed by immunofluorescence labeling using anti-FLAG antibody. The displayed enzymes were active forms, and AmyA and CALB activities reached 990 U/g (dry cell weight) and 4.6 U/g (dry cell weight), respectively. AmyA-displaying E. coli cells grew utilizing cornstarch as the sole carbon source, while CALB-displaying E. coli cells catalyzed enantioselective transesterification, indicating that they are effective whole-cell biocatalysts. Since a target enzyme with a size of 77 kDa and an industrially useful lipase have been successfully displayed on the cell surface of E. coli for the first time, PgsA protein is probably a useful anchoring motif to display various enzymes.     

Displaying Candida antarctica lipase B on the cell surface of Aspergillus niger as a potential food-grade whole-cell catalyst

Aspergillus niger is a recognized workhorse used to produce food processing enzymes because of its extraordinarily high protein-producing capacity. We have developed a new cell surface display system de novo in A. niger using expression elements from generally recognized as safe certified microorganisms. Candida antarctica lipase B (CALB), a widely used hydrolase, was fused to an endogenous cell wall mannoprotein, CwpA, and functionally displayed on the cell surface. Localization of CALB was confirmed by enzymatic assay and immunofluorescence analysis using laser scanning confocal microscopy. After induction by maltose for 45 h, the hydrolytic activity and synthesis activity of A. niger mycelium-surface displayed CALB (AN-CALB) reached 400 and 240 U/g dry cell, respectively. AN-CALB was successfully used as a whole-cell catalyst for the enzymatic production of ethyl esters from a series of fatty acids of different chain lengths and ethanol. In a solvent-free system, AN-CALB showed great synthetic activity and afforded high substrate mole conversions, which amounted to 87 % for ethyl hexanoate after 2 h, 89 % for ethyl laurate after 2 h, and 84 % for ethyl stearate after 3 h. These results suggested that CwpA can act as an efficient anchoring motif for displaying enzyme on A. niger, and AN-CALB is a robust, green, and cost-effective alternative food-grade whole-cell catalyst to commercial lipase.     

Synthesis of functional dipeptide carnosine from nonprotected amino acids using carnosinase-displaying yeast cells

Carnosine (β-alanyl-l-histidine) is one of the bioactive dipeptides and has antioxidant, antiglycation, and cytoplasmic buffering properties. In this study, to synthesize carnosine from nonprotected amino acids as substrates, we cloned the carnosinase (CN1) gene and constructed a whole-cell biocatalyst displaying CN1 on the yeast cell surface with α-agglutinin as the anchor protein. The display of CN1 was confirmed by immunofluorescent labeling, and CN1-displaying yeast cells showed hydrolytic activity for carnosine. When carnosine was synthesized by the reverse reaction of CN1, organic solvents were added to the reaction mixture to reduce the water content. The CN1-displaying yeast cells were lyophilized and examined for organic solvent tolerance. Results showed that the CN1-displaying yeast cells retained their original hydrolytic activity in hydrophobic organic solvents. In the hydrophobic organic solvents and hydrophobic ionic liquids, the CN1-displaying yeast cells catalyzed carnosine synthesis, and carnosine was synthesized from nonprotected amino acids in only one step. The results of this research suggest that the whole-cell biocatalyst displaying CN1 on the yeast cell surface can be used to synthesize carnosine with ease and convenience.     

Expression in Pichia pastoris of Candida antarctica Lipase B and Lipase B Fused to a Cellulose-Binding Domain

Candida antarctica lipase B (CALB) and C. antarctica lipase B fused to a cellulose-binding domain (CBD-CALB) were expressed functionally in the methylotrophic yeast Pichia pastoris. The cellulose-binding domain originates from cellulase A of the anaerobic rumen fungus Neocallimastix patriciarum. The genes were fused to the α-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter. The recombinant proteins were secreted into the culture medium reaching levels of approximately 25 mg/L. The proteins were purified using hydrophobic interaction chromatography and gel filtration with an overall yield of 69%. Results from endoglycosidase H digestion of the proteins showed that CALB and CBD-CALB were N-glycosylated. The specific hydrolytic activities of recombinant CALB and CBD-CALB were identical to that reported for CALB isolated from its native source. The fusion of the CBD to the lipase resulted in a greatly enhanced binding toward cellulose for CBD-CALB compared with that for CALB.     

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.     

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.