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.     

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.     

Screening for Glycosylphosphatidylinositol-Modified Cell Wall Proteins in Pichia pastoris and Their Recombinant Expression on the Cell Surface

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro. In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by β-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface.     

Endogenous signal peptides efficiently mediate the secretion of recombinant proteins in Pichia pastoris

By predicting the potential signal peptides from proteins that are naturally secreted by Pichia pastoris, we identified three possible endogenous signal peptides: Scw, Dse and Exg. We compared their capability to mediate the secretion of enhanced green fluorescent protein (EGFP) and Candida antarctica lipase B (CALB) with that of the Saccharomyces cerevisiae α-factor prepro-signal. EGFP entered the secretory pathway of P. pastoris and was efficiently secreted into the culture medium by all three endogenous peptides. Further, these three putative endogenous signal peptides were also effective in secreting CALB. These endogenous signal peptides thus have the potential to mediate the efficient secretion of heterologous proteins in P. pastoris.     

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.     

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.     

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.     

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.     

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.     

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.     

Construction of a Novel <Emphasis Type="Italic">Pichia pastoris</Emphasis> Cell-Surface Display System Based on the Cell Wall Protein Pir1

A novel system based on Pir1 from Saccharomyces cerevisiae was developed for cell-surface display of heterologous proteins in Pichia pastoris with the alpha-factor secretion signal sequence. As a model protein, enhanced green fluorescence protein (EGFP) was fused to the N-terminal of the mature peptide of Pir1 (Pir1-a). The expression of fusion protein EGFP-Pir1-a was irregular throughout the P. pastoris cell surface per detection by confocal laser scanning microscopy. A truncated sequence containing only the internal repetitive sequences of Pir1-a (Pir1-b) was used as a new anchor protein in further study. The fusion protein EGFP-Pir1-b was expressed uniformly on the cell surface. The fluorescence intensity of the whole yeast was measured by spectrofluorometer. Western blot confirmed that the fusion proteins were released from cell walls after mild alkaline treatment. The results indicate that a Pir1-based system can express proteins on the surface of P. pastoris and that the fusion proteins do not affect the manner in which Pir1 attaches to the cell wall. The repetitive sequences of Pir1 are required for cell wall retention, and the C-terminal sequence contributes to the irregular distribution of fusion proteins in P. pastoris.     

Accurate analysis of fusion expression of <Emphasis Type="Italic">Pichia pastoris</Emphasis> glycosylphosphatidylinositol-modified cell wall proteins

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have diverse intrinsic functions in yeasts, and they also have different uses in vitro. The GPI-modified cell wall proteins GCW21, GCW51, and GCW61 of Pichia pastoris were chosen as anchoring proteins to construct co-expression strains in P. pastoris GS115. The hydrolytic activity and the amount of Candida antarctica lipase B (CALB) displayed on cell surface increased significantly following optimization of the fusion gene dosage and combination of the homogeneous or heterogeneous cell wall proteins. Maximum CALB hydrolytic activity was achieved at 4920 U/g dry cell weight in strain GS115/CALB-GCW (51 + 51 + 61 + 61) after 120 h of methanol induction. Changes in structural morphology and the properties of the cell surfaces caused by co-expression of fusion proteins were observed by transmission electron microscopy (TEM) and on plates containing cell-wall-destabilizing reagent. Our results suggested that both the outer and inner cell layers were significantly altered by overexpression of GPI-modified cell wall proteins. Interestingly, quantitative analysis of the inner layer components showed an increase in β-1,3-glucan, but no obvious changes in chitin in the strains overexpressing GPI-modified cell wall proteins.     

Double Candida antarctica lipase B co-display on Pichia pastoris cell surface based on a self-processing foot-and-mouth disease virus 2A peptide

To develop a high efficiency Candida antarctica lipase B (CALB) yeast display system, we linked two CALB genes fused with Sacchromyces cerevisiae cell wall protein genes, the Sed1 and the 3′-terminal half of Sag1, separately by a 2A peptide of foot-and-mouth disease virus (FMDV) in a single open reading frame. The CALB copy number of recombinant strain KCSe2ACSa that harbored the ORF was identified, and the quantity of CALB displayed on the cell surface and the enzyme activity of the strain were measured. The results showed that the fusion of multiple genes linked by 2A peptide was translated into two independent proteins displayed on the cell surface of stain KCSe2ACSa. Judging from the data of immunolabeling assay, stain KCSe2ACSa displayed 94?% CALB-Sed1p compared with stain KCSe1 that harbored a single copy CALB-Sed1 and 64?% CALB-Sag1p compared with stain KCSa that harbored a single copy CALB-Sag1 on its surface. Besides, strain KCSe2ACSa possessed 170?% hydrolytic activity and 155?% synthetic activity compared with strain KCSe1 as well as 144?% hydrolytic activity and 121?% synthetic activity compared with strain KCSa. Strain KCSe2ACSa even owned 124?% hydrolytic activity compared with strain KCSe2 that harbored two copies CALB-Sed1. The heterogeneous glycosylphosphatidylinositol-anchored proteins co-displaying yeast system mediated by FMDV 2A peptide was shown to be an effective method for improving the efficiency of enzyme-displaying yeast biocatalysts.     

Temperature limited fed-batch technique for control of proteolysis in <Emphasis Type="Italic">Pichia pastoris</Emphasis> bioreactor cultures

Background  

A temperature limited fed-batch (TLFB) technique is described and used for Pichia pastoris Mut⁺ strain cultures and compared with the traditional methanol limited fed-batch (MLFB) technique. A recombinant fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced and secreted by this strain.     

Surface Display and Bioactivity of Bombyx mori Acetylcholinesterase on Pichia pastoris

A Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE) was constructed and its bioactivity was studied. The modified Bombyx mori acetylcholinesterase gene (bmace) was fused with the anchor protein (AGα1) from Saccharomyces cerevisiae and transformed into P. pastoris strain GS115. The recombinant strain harboring the fusion gene bmace-AGα1 was induced to display BmAChE on the P. pastoris cell surface. Fluorescence microscopy and flow cytometry assays revealed that the BmAChE was successfully displayed on the cell surface of P. pastoris GS115. The enzyme activity of the displayed BmAChE was detected by the Ellman method at 787.7 U/g (wet cell weight). In addition, bioactivity of the displayed BmAChE was verified by inhibition tests conducted with eserine, and with carbamate and organophosphorus pesticides. The displayed BmAChE had an IC₅₀ of 4.17×10⁻⁸ M and was highly sensitive to eserine and five carbamate pesticides, as well as seven organophosphorus pesticides. Results suggest that the displayed BmAChE had good bioactivity.     

Buffer-mediated activation of Candida antarctica lipase B dissolved in hydroxyl-functionalized ionic liquids

Ionic-liquid buffer having phosphate anion was synthesized for the development of buffered enzymatic ionic liquid systems. Both the conformation and transesterification activity of Candida antarctica lipase B (CALB) dissolved in the hydroxyl-functionalized ionic liquids were buffer dependent. Intrinsic fluorescence studies indicated that the CALB possessed a more compact conformation in the medium consisted of ionic liquid buffer having phosphate anion and hydroxyl-functionalized ionic liquids like 1-(1-hydroxyethyl)-3-methyl-imidazolium tetrafluoroborate and 1-(1-hydroxyethyl)-3-methyl-imidazolium nitrate. High activity and outstanding stability could be obtained with the CALB enzyme in the buffered ionic liquids for the transesterification.     

de novo Design and Synthesis of Candida antarctica Lipase B Gene and α-Factor Leads to High-Level Expression in Pichia pastoris

Candida antarctica lipase B (CALB) is one of the most widely used and studied enzymes in the world. In order to achieve the high-level expression of CALB in Pichia, we optimized the codons of CALB gene and α-factor by using a de novo design and synthesis strategy. Through comparative analysis of a series of recombinants with different expression components, we found that the methanol-inducible expression recombinant carrying the codon-optimized α-factor and mature CALB gene (pPIC9KαM-CalBM) has the highest lipase production capacity. After fermentation parameters optimization, the lipase activity and protein content of the recombinant pPIC9KαM-CalBM reached 6,100 U/mL and 3.0 g/L, respectively, in a 5-L fermentor. We believe this strategy could be of special interest due to its capacity to improve the expression level of target gene, and the Pichia transformants carrying the codon-optimized gene had great potential for the industrial-scale production of CALB lipase.     

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.     

Influence of cosolvents on the hydrophobic surface immobilization topography of Candida antarctica lipase B

The presence of cosolvents and co-solutes during the immobilization of lipases on hydrophobic supports may influence the extent of lipase immobilization and the long-term catalytic stability of the biocatalyst. Candida antarctica B lipase immobilization was examined on a hydrophobic surface, i.e., gold modified with a methyl-terminated, self-assembled alkylthiol layer. Lipase adsorption was monitored gravimetrically using a quartz crystal microbalance (QCM). Lipase activity was determined colorimetrically by following p-nitrophenol propionate hydrolysis. Adsorbed lipase topography was examined by atomic force microscopy (AFM). Lipase adsorption from low ionic strength aqueous buffer produced a uniform confluent protein monolayer. Inclusion of 10% (vol) ethanol in the buffer during immobilization resulted in a 33% adsorbed mass increase. Chemically similar cosolvents, all at 10% by volume in buffer, were also individually examined for their influence on CALB adsorption. Glycerol or 1-propanol increased mass adsorption by 10%, while 2-propanol increased mass adsorption by 33%. QCM dissipation values increased threefold with the inclusion of either ethanol or 2-propanol in the medium during lipase adsorption, indicating formation of multilayers of CALB. Partial multilayer formation using 10% ethanol was confirmed by AFM. Inclusion of 10% ethanol in the CALB immobilization buffer decreased the specific activity of the immobilized lipase by 37%. The formation of lipase multilayers in the presence of certain cosolvents thus results in lower specific activity, which might be due to either influences on lipase conformation or substrate active site accessibility.     

Analysis of the Conformational Stability and Activity of Candida antarctica Lipase B in Organic Solvents: INSIGHT FROM MOLECULAR DYNAMICS AND QUANTUM MECHANICS/SIMULATIONS*

The conformational stability and activity of Candida antarctica lipase B (CALB) in the polar and nonpolar organic solvents were investigated by molecular dynamics and quantum mechanics/molecular mechanics simulations. The conformation change of CALB in the polar and nonpolar solvents was examined in two aspects: the overall conformation change of CALB and the conformation change of the active site. The simulation results show that the overall conformation of CALB is stable in the organic solvents. In the nonpolar solvents, the conformation of the active site keeps stable, whereas in the polar solvents, the solvent molecules reach into the active site and interact intensively with the active site. This interaction destroys the hydrogen bonding between Ser¹⁰⁵ and His²²⁴. In the solvents, the activation energy of CALB and that of the active site region were further simulated by quantum mechanics/molecular mechanics simulation. The results indicate that the conformation change in the region of active sites is the main factor that influences the activity of CALB.