Highly Efficient Fructooligosaccharides Production by an Erythritol

Article pubs.acs.org/JAFC

Highly Efficient Fructooligosaccharides Production by an ErythritolProducing Yeast Yarrowia lipolytica Displaying Fructosyltransferase Lebin Zhang,†,‡ Jin An,†,‡ Lijuan Li,†,‡ Hengwei Wang,§ Dawen Liu,†,‡ Ning Li,†,‡ Hairong Cheng,*,†,‡ and Zixin Deng†,‡ †

State Key Laboratory of Microbial Metabolism and ‡School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China § Innovation & Application Institute, Zhejiang Ocean University, Zhoushan 316022, China ABSTRACT: Currently, fructooligosaccharides (FOS) are industrially transformed from sucrose by purified enzymes or fungi cells. However, these methods are expensive and time-consuming. An economical approach to producing FOS using erythritolproducing yeast cells was described in this study. Fructosyltransferase from Aspergillus oryzae was displayed on the cell surface of Yarrowia lipolytica, resulting in an engineered strain capable of transforming sucrose to FOS. An amount of 480 g/L FOS was produced within 3 h in a solution of 800 g/L sucrose and 5 g/L cells (dry cell weight, DCW) at pH 6.0 and 60 °C, with a yield of 60% of total sucrose and a productivity of 160 g/(L·h). The yeast pastes from the erythritol industry can be repeatedly used as the whole-cell catalysts at least 10 times by this newly developed approach. This efficient method is attractive for the large-scale production of FOS from sucrose. KEYWORDS: Yarrowia lipolytica, cell surface display, fructooligosaccharide, fructosyltransferase, erythritol

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INTRODUCTION Gut microbiota may serve as an important contribution to human host health, such as in the prevention of diet-related obesity.1,2 A dietary medical intervention rich in prebiotics, such as fructooligosaccharides (FOS), has been used in the treatment of human genetic obesity (Prader−Willi syndrome) and simple obesity.3 These studies showed that in the gut of obese adult human hosts, endotoxin-producing bacteria were reduced and beneficial bacterial communities were enriched, thus leading to decreased endotoxins levels in the bloodstream and significant alleviation of inflammation, adiposity, and insulin resistance.4 The mechanism of the FOS-enriched dietary intervention is to shift the metabolism in the gut microbiota from fermenting proteins and fats to carbohydrates, thus producing low-molecular-weight organic acids. The subsequent reduction of pH inhibits the growth of harmful bacteria inside the gut and corrects the dysbiosis of gut microbiota.5−7 These physicochemical properties make FOS a beneficial food ingredient in infant and elder milk formulas, baking products, and yogurts. Large-scale production of FOS involves the transformation of sucrose using free or immobilized fructosyltransferase (FTase) isolated from Aspergillus species, such as A. japonicus, A. niger, A. oryzae, and A. sydowi.8,9 However, FOS can be directly produced using whole cells of a given microorganism, such as Aureobasidium pullulans, A. japonicus, and engineered S. cerevisiae, in suspension or immobilized form.10−12 Interestingly, FOS also can also be produced by Aspergillus phoenicis biofilms on polyethylene support as reported by Aziani et al, and the maximum FOS production of total FOS (122 g/L) was achieved in a medium containing 250 g/L sucrose at 30 °C for 48 h.13 Reaction mixtures can be separated using simulated moving bed chromatography (SMB) to obtain FOS, glucose, fructose, and residual sucrose, respectively.14,15 © 2016 American Chemical Society

Currently, the production of FOS involves the synthesis of FTase by cultivating microorganisms, the purification of enzymes, and the exposure of FTase to its substrate sucrose. Current FOS production strategies are laborious and timeconsuming. FOS production by the whole-cell one-pot transformation in bioreactors is a good option;16,17 this method eliminates the need to purify FTase enzymes from cell extracts or fermentation medium. Likewise, the first step is to prepare a large number of cells using high-density cultivation, which is not only laborious and time-consuming but also expensive. Many FTase enzymes are secretory proteins, and most secreted FTases are removed when whole cells are used as biocatalysts, thus lowering the enzyme availability.18 In addition, regardless of methods used, FOS is always produced as mixtures with high concentrations of byproducts (glucose and fructose), and must be separated to obtain a high purity of FOS. However, the byproducts glucose and fructose are low-value-added. Therefore, it is important to develop an FTase-producing microorganism in which the FTase enzyme is mostly displayed on the cell surface, with the capacity of converting glucose to highvalue-added polyols, such as erythritol. Yarrowia lipolytica is one of most studied unconventional yeasts and was generally recognized as safe by the Food and Drug Administration in the United States. Y. lipolytica is widely used in the production of food ingredients such as organic acid,19 eicosapentaenoic acid (EPA),20 erythritol, and mannitol.21,22 In the erythritol fermentation industry, thousands of tons of yeast pastes (a kind of bioresource) are generated annually, which has raised much environmental concerns due to Received: Revised: Accepted: Published: 3828

January 27, 2016 April 27, 2016 April 28, 2016 April 28, 2016 DOI: 10.1021/acs.jafc.6b00115 J. Agric. Food Chem. 2016, 64, 3828−3837

Article

Journal of Agricultural and Food Chemistry

Figure 1. Primer sequences and diagram of the plasmid pINA-Pir1-A.oryFTase. (a) The sequences of primer used to amplify the YlPir1 gene. Nucleotides in the box are the FLAG-encoded sequence, and the expressed peptide can be immunorecognized by the anti-FLAG antibody. (b) The sequences of primer employed to amplify the FTase gene. (c) The schematic diagram of the recombinant plasmid.

The FTase gene of A. oryzae was obtained by RT-PCR. The total mRNA of A. oryzae CGMCC3.800 was prepared using the GenElute mRNA miniprep kit (Sigma-Aldrich). The cDNA was obtained using a reverse transcript kit (TIANScript II RT Kit, TianGen). The FTase gene of A. oryzae was amplified using synthesized cDNA as template and a pair of primers PFTase‑F and PFTase‑R (Figure 1b). The 1528 bp PCR fragment was digested with BamHI and KpnI and ligated into plasmid pINA1311-Pir1 cut with BamHI and KpnI, yielding the final plasmid pINAPir1-A.oryFTase (Figure 1c). Y. lipolytica surface-displayed vector pINA-Pir1-A.oryFTase was linearized with NotI, and the 6.0 kb linearized fragment (5′zeta-ura3d1-hp4d-Pir1-A.oryFTase-LIP2t-zeta′-3) was used to transform the erythritol-producing Y. lipolytica CGMCC7326 according to the method described by Chen et al.,25 with minor modifications. Briefly, Y. lipolytica CGMCC7326 from −80 °C was streaked on a YPD plate and incubated at 28 °C for 24 h. Then, yeast cells were collected and resuspended in 200 μL of transformation buffer (400 g/L PEG 4000, 150 mM lithium acetate (pH 6.0), 150 mM dithiothreitol, and 0.2 mg/mL single-stranded salmon sperm DNA). Samples of 3−5 μg of linearized DNA were added to the transformation buffer, vortexed thoroughly, and then incubated at 39 °C for 90 min while being shaken at regular intervals to prevent cells from submerging to the bottom of the tube. After incubation, cells were spread directly on a selective minimal medium plate (6.7 g/L yeast nitrogen base, 5 g/L ammonia sulfate, and 20 g/L sucrose, pH 5.5) and incubated at 28 °C for 3−5 days. Transformants of larger colonies were further screened on the above minimal medium plates. For the identification of Y. lipolytica transformants, the genomic DNA of the larger Y. lipolytica colonies was extracted using the method described by Cheng and Jiang (2006).26 The forward primer PFTase‑F and reverse primer PFTase‑R (as shown in Figure 1b) were used to amplify the 1.6 kb A.oryFTase gene, according to thermal cycling conditions: initial denaturation at 95 °C for 5 min, followed by 35 cycles at 94 °C for 35 s, 55 °C for 40 s, 72 °C for 90 s, and extra extension at 72 °C for 10

improper disposal. Therefore, an integrated utilization of these yeast pastes according to the concept of reducing, reusing, and recycling is receiving close attention.23 One of the alternatives is to produce prebiotics, such as FOS, using these yeast pastes as whole-cell catalysts. The idea of integrated utilization has such advantages of providing abundant, cheaper, and readily available whole-cell catalysts, meanwhile overcoming environmental problems. Therefore, the objective of this study is to develop an engineered FOS-producing yeast based on the erythritol-producing yeast Y. lipolytica CGMCC7326.

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MATERIALS AND METHODS Strains and Culture Conditions. The erythritol-producing yeast Y. lipolytica CGMCC7326 was used as the host for cellsurface-displaying fructosyltransferase and was grown at 30 °C in medium YPD (yeast extract, 10 g/L; peptone, 20 g/L; and glucose, 20 g/L).21 A. oryzae CGMCC3.800 was used to clone the fructosyltransferase gene (FTase). Yeast transformants harboring the A. oryzae FTase gene were screened using the YNS medium (6.7 g/L yeast nitrogen base without amino acids, 20 g/L sucrose, and 20 g/L agar at pH 6.0) at 30 °C for 3−5 days. The engineered Y. lipolytica was cultured in a YPS medium (yeast extract 5 g/L, peptone 5 g/L, and sucrose 20 g/ L at pH 6.0) for the synthesis of fructosyltransferase. Growth was monitored spectrophotometrically at a wavelength of 600 nm (A600). An Escherichia coli DH5α strain was used as the host for DNA manipulations. Construction of Plasmid and Transformation. The vector pINA-Pir1-A.oryFTase specific for the yeast Y. lipolytica was constructed based on the plasmid pINA1311.24 The anchor protein Pir1 DNA was amplified using genomic DNA of Y. lipolytica CGMCC7326 as the template with a pair of primers: Ppir1-F and Ppir1-R (Figure 1a). The PCR reaction began with denaturation at 94 °C for 5 min, 35 cycles of 94 °C for 30 s, 58 °C for 40 s, 72 °C for 50 s, and 72 °C for 10 min. The 890 bp PCR fragment was digested with PmlI and BamHI and ligated into plasmid pINA1311 cut with PmlI and BamHI, yielding the plasmid pINA-Pir1. 3829

DOI: 10.1021/acs.jafc.6b00115 J. Agric. Food Chem. 2016, 64, 3828−3837

Article


we performed reactions at 30, 40, 50, 55, 60, 65, 70, 75, and 80 °C at pH 5.0 and at constant agitation speed (100 rpm). To estimate the optimal pH, we carried out reactions in various buffers with different pH at the optimal temperature obtained and at constant agitation speed (100 rpm). Buffers used were citric acid−sodium citrate (100 mM, pH 3.0−5.0), acetic acid− sodium acetate buffer (100 mM, pH 5.5), Na2HPO4−NaH2PO4 (100 mM, pH 6.0−7.0), and Tris−HCl (100 mM, pH 7.5− 8.0). To determine the thermostability of A.oryFTase, we incubated the engineered Y. lipolytica cells in the absence of substrate at temperatures of 65, 70, and 75 °C for 30−180 min at the optimal pH obtained above. Samples were withdrawn at regular intervals for the assay of relative activity of A.oryFTase. To investigate the reusability of the engineered Y. lipolytica cells, we detected the FTase enzyme activity from the first reaction and the residual activity from the subsequent reactions under the optimal conditions obtained from the above experiments. Yeast cells were collected via centrifugation after reaction and then were applied for the next reaction, and so on. Each reaction took 3 h, and the FOS content was analyzed by HPLC. The first reaction activity of the engineered Y. lipolytica A.oryFTase was defined as 100%. The effects of sucrose concentration on FOS production were investigated at 100, 200, 300, 400, 500, 600, 700, and 800 g/L, respectively. The engineered Y. lipolytica cells were added to solutions of different sucrose concentrations (100 to 800 g/ L) in 5 mL of sodium citric buffer (100 mM, pH 6.0). The final cell density (OD600) in the mixture was adjusted to 20 (equivalent to 25 mg DCW in 5 mL of reaction mixture). The mixture was incubated at 60 °C in an orbital shaker at 200 rpm. Aliquots of 50 μL were taken at different times, diluted 10 times with water, and centrifuged for 15 min at 10000g. The contents of glucose, fructose, FOS, and residual sucrose were analyzed using HPLC with a TOSOH column (TSKgel Amide-80, 5 μm, 4.6 × 250 mm) and a RI101 detector with 70% acetonitrile as mobile phase at a flow rate of 1.0 mL/min at 70 °C. Production of FOS. The main purpose of this study was to produce FOS from sucrose using the A.oryFTase enzyme displaying on the surface of Y. lipolytica cells, which could also produce erythritol from glucose. First, the engineered Y. lipolytica (transformant no. 11) cells were collected from the erythritol fermentation medium and were then used as the whole-cell catalysts to synthesize FOS from sucrose. To produce erythritol, we cultured the engineered Y. lipolytica (transformant no. 11) in 50 mL of YCAG medium (5 g/L yeast extract, 3 g/L corn syrup powder, 5 g/L ammonium citrate, 10 mM MgSO4, 5 mM MnSO4, and 250 g/L glucose at pH 6.0) in a shaker at 200 rpm and 30 °C for 80 h. Cells were harvested, washed twice with PBS buffer (50 mM, pH 6.0), and then used as the whole-cell catalysts to transform 50 mL of sucrose solution (800 g/L) at 60 °C and pH 6.0 in an orbital shaker at 100 rpm. After the completion of the transformation (3 h), the reaction mixture (50 mL) was diluted by a factor of three. Yeast extract, corn syrup powder, and ammonium citrate were supplemented to the final concentrations of 3, 2, and 2 g/L, respectively, and the mixture was sterilized at 108 °C for 20 min. Then, 10 mL of parent strain Y. lipolytica CGMCC7326 cells (OD600 5.0) were inoculated into the diluted mixture to ferment glucose to erythritol in a shaker at 30 °C, initial pH of 6.0, and 200 rpm. At various time scales, aliquots were taken, and the contents of erythritol, FOS, and residual glucose were analyzed by HPLC with a sugar column SP0810 (Shodex, 8.0 ×

min. A total of 25 positive transformants identified by PCR were further confirmed through assay of the fructosyltransferase activity (AT) and the hydrolase activity (AH). After determining the ratio of AT and AH (AT/AH) for A.oryFTase activity, we found that transformant no. 11 had the highest activity among all transformants tested. Therefore, the engineered Y. lipolytica transformant no. 11 was used in the subsequent experiments. A.oryFTase Enzyme Activity Assay. Transformant no. 11 was cultured in 50 mL of YPS medium at 30 °C for 48 h. The cells were then collected and washed twice using a 50 mM phosphate buffer solution (PBS buffer, pH 6.0) and centrifuged at 6000g at 4 °C for 15 min. A sample of 1 mL of cells of 1 OD600 was equivalent to 0.25 mg DCW. The 1 mL A.oryFTase reaction mixture (50 mM PBS buffer, pH 6.0) consisted of 800 mg of sucrose and 5.0 mg of yeast cells (with OD600 of 20). After incubation for 1 h at 60 °C, the reaction was stopped by heating at 95 °C for 10 min. The reaction mixture was centrifuged at 10000g for 15 min to remove yeast cells. The content of glucose, fructose, and FOS was measured using HPLC with a TOSOH column (TSKgel Amide-80, 5 μm, 4.6 × 250 mm) and a RI101 detector using 70% acetonitrile as a mobile phase at a flow rate of 1.0 mL/min at 70 °C. One unit (U) of A.oryFTase (AFTase) activity was defined as the amount of yeast cells (in DCW) required to release 1 μmole of glucose per min from sucrose under the above-mentioned conditions. The fructosyltransferase activity (AT) and the hydrolase activity (AH) were determined by measuring the content of glucose (CG) and fructose (CF) present in the reaction mixture. The content of transferred fructose (CFT) was calculated as CFT = CG − CF. One unit of fructosyltransferase activity (AT) was defined as the amount of yeast cells (in DCW) required to transfer 1 μmole of fructose per min. One unit of hydrolase activity (AH) was defined as the amount of yeast cells (in DCW) required to release 1 μmole of free fructose per min. Immunofluorescence Microscopy Assay. To examine whether the A.oryFTase enzyme was indeed displayed on the surface of Y. lipolytica, we performed immunofluorescence microscopy of recombinant Y. lipolytica cells. The control-strain Y. lipolytica CGMCC7326 and transformant no. 11 were cultured in 5 mL of YPD and YPS medium at 30 °C for 48 h, respectively. Harvested cells were then washed three times with PBS (50 mM, pH 7.4) and incubated with the mouse polyclonal antibody against FLAG tag (1:50 diluted in PBS containing 2% bovine serum albumin) on ice for 30 min. After incubation, yeast cells were washed with PBS to remove unbound antibodies before being incubated with fluorescein isothiocyanate (FITC)-conjugated goat antimouse immunoglobulin G (IgG, 1:200 diluted in PBS with 2% bovine serum albumin) on ice for 30 min in the dark. After being washed three times with PBS, fluorescent-labeled cells were observed under fluorescence microscope (Nikon Eclipse E800) using a standard fluorescein filter cube (450−490 nm excitation filter and 515 nm emission filter). Characterization of the Engineered Y. lipolytica Surface-Displaying A.oryFTase. The A.oryFTase activities of the engineered Y. lipolytica (transformant no. 11) cells were determined at different pH (3.0−8.0) and temperature (30−80 °C) values. All reactions were performed in 5 mL mixtures containing 800 mg/mL sucrose and 5 mg/mL dry cell weight unless stated otherwise. To determine the optimal temperature, 3830


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Journal of Agricultural and Food Chemistry Table 1. A.oryFTase Activity from Cells and Supernatants under Different Sucrose Concentrations YP+2% glucose AT AH AT/AH

YP+2% sucrose

YP+20% sucrose

supernatant (U/mL)

cells (U/mg DCW)

supernatant (U/mL)

cells (U/mg DCW)

supernatant (U/mL)

cells (U/mg DCW)

0.14 ± 0.02 0.01 ± 0.01 14

0.90 ± 0.05 0.06 ± 0.02 15

0.16 ± 0.02 0.01 ± 0.01 16

0.86 ± 0.05 0.06 ± 0.02 14

0.15 ± 0.02 0.01 ± 0.01 15

0.89 ± 0.05 0.06 ± 0.02 15

a

The basic YP medium contains yeast extract (5 g/L) and tryptone (5 g/L) at pH 6.0. The data represent the mean values and standard deviations of three independent experiments.

fructosyltransferase activity, reaching 0.85 ± 0.04 U per mg of DCW, with a low hydrolase activity level of 0.05 ± 0.01 U per mg of DCW and a ratio of AT/AH of 17.0. We further determined whether the supernatant had enzymatic activity and whether it was induced by sucrose. Interestingly, the supernatant also had fructosyltransferase and hydrolase activity, with the similar ratio of AT/AH (Table 1). The enzymatic activity detected was at similar levels in YP media containing 20 g/L glucose, 20 g/L sucrose, or 200 g/L sucrose, suggesting that the enzyme was constitutively expressed in the transformant no. 11 and was not induced by sucrose. It could be deduced from this case that not all A.oryFTase enzyme was displayed on the cell surface of Y. lipolytica, though A.oryFTase was fused to the anchor protein Pir1. Confirmation and Characterization of the Recombinant A.oryFTase Displayed on the Cell Surface. Green fluorescence was observed on cells of transformant no. 11 under the fluorescent microscope (Figure 2, panel b2), and the

300 mm), coupled to RI101 detector using distilled water as mobile phase at a flow rate of 1.0 mL/min at 70 °C. Comparison of Transformant No. 11 and A. oryzae. The engineered Y. lipolytica transformant no. 11 and A. oryzae CGMCC3.800 were cultured in 50 mL of YPS medium at 30 °C for 48 h. Cells were harvested, washed with PBS buffer (50 mM, pH 6.5), and used as the whole-cell catalysts. Cells of transformant no. 11 and A. oryzae (25 mg in DCW) were added, respectively, to 5 mL of sodium citric buffer (100 mM and pH 5.5) containing 800 g/L sucrose. Transformations were performed at 60 °C for Y. lipolytica and 50 °C for A. oryzae in a shaker at 200 rpm. Samples were withdrawn and analyzed using HPLC with a TOSOH column (TSKgel Amide-80, 5 μm, 4.6 × 250 mm), coupled to a RI101 detector using 70% acetonitrile as the mobile phase at a flow rate of 1.0 mL/min at 70 °C.

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RESULTS Construction of Plasmid and Growth of Yeast Transformants. The plasmid pINA-Pir1-A.oryFTase for surface display of A.oryFTase was constructed as shown in Figure 1. The A.oryFTase protein was fused to the C-terminus of the YlPir1 protein, and the YlPir1 protein was anchored to glycoproteins of the cellular surface of Y. lipolytica via disulfide bonds. Between the YlPir1 and the A.oryFTase gene, a FLAG tag encoding sequence was inserted for immunofluorescent detection. A strong and constitutive promoter (hp4d) was used for the heterologous expression of the A.oryFTase gene in Y. lipolytica.27 Furthermore, when linearized with NotI, the bacterial moiety of the plasmid was removed before transformation, and only a “yeast cassette” was used for transformation of the erythritol-producing strain Y. lipolytica, thus avoiding the spread of antibiotic resistance genes into the environments. The parent-strain Y. lipolytica CGMCC7326 lacks the A.oryFTase gene and is unable to grow on the minimal medium with sucrose as the sole carbon source, whereas transformants expressing the A.oryFTase gene can grow in media with sucrose as the sole carbon source. With sucrose as a selective marker, transformants grown on the solid minimal media containing sucrose as the sole carbon source were positive transformants. The clones were purified by transferring large clones to the selective plates. Among the 25 positive transformants randomly selected, transformant no. 11 was found to have the highest fructosyltransferase activity, with 0.85 ± 0.04 U per mg of DCW, while transformant no. 2 only had 0.32 ± 0.02 U per mg of DCW. The fructosyltransferase activities of about half of transformants selected was 0.6 ± 0.1 U per mg of DCW. Then, transformant no. 11 was deposited in CGMCC with accession no. 11368 and was used in the subsequent studies. Assay of A.oryFTase Activity. High fructosyltransferase activity of selected transformant is beneficial for industrial use. Transformant no. 11 was found to possess the highest

Figure 2. Immunofluorescent labeling of A.oryFTase on the cell surface of Y. lipolytica. The microphotographs were taken at visible light (panels a1 and b1), and immunofluorescence microphotographs were taken at emission wavelength of 500 nm (panels a2 and b2). (a) The cells of Y. lipolytica parent strain as a control; (b) the cells of the transformant no. 11. Magnification: 600 ×.

control cells were not fluorescent (Figure 2, panel a2), indicating that the fusion protein was successfully displayed on the cell surface of recombinant no. 11. The highest activity of the displayed A.oryFTase was found to be at 60 °C (Figure 3a), 5−10 °C higher than that of the previously reported free enzyme.28,29 About 95% activity was lost when the reaction progressed at 80 °C for 3 h. The 3831


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Figure 3. Characterization of A.oryFTase displayed on the cell surface of Y. lipolytica. (a) Effect of temperature on the activity of surface-displayed A.oryFTase. Relative activity was calculated by assuming the activity obtained at 60 °C as being 100%. (b) Effect of pH on the activity of surfacedisplayed A.oryFTase. Relative activity was calculated by assuming the activity obtained at pH 6.0 as being 100%. (c) The thermostability of surfacedisplayed A.oryFTase. Relative activity was calculated by assuming the activity as being 100% at 0 h for each temperature tested. (d) Effect of reuse times on the activity of surface-displayed A.oryFTase. Relative activity was calculated by assuming the first round of activity as being 100%. The values that corresponded to 100% relative activity for the above four cases were 0.84−0.88 U/mg DCW. Each value represents the average for three independent measurements ± standard deviation.

maximum activity was observed to be at pH 6; 21% and 62% of the activity were retained at pH 3.0 and pH 8.0, respectively (Figure 3b). About 85%, 50% and 23% of the activity were retained when cells were incubated at 65, 70, and 75 °C for 180 min, respectively (Figure 3c). With the surface-displayed expression system, we could reuse A.oryFTase as the immobilized enzyme. The enzyme activity decreased gradually with the increase of reused numbers. The enzyme activity retained 90% and 85% of the initial activity at the 9th and 11th cycle, respectively, and decreased to 55% of the initial activity at the 15th cycle (Figure 3d). From a practical perspective, cells could be reused for at least ten times. Production of FOS. The yield of FOS was increased from 43% to 60% when sucrose concentrations increased from 100 to 800 g/L. The phenomenon can be attributed to the osmotic effect imposed by higher concentration of sucrose, the lower hydrolytic activity of FTase, the lower water activity, and the greater incorporation of sucrose as the fructosyl acceptor.30 The transfructosylation activity increased as the sucrose concentration increased.28 At a sucrose concentration of 800 g/L, 480 g/L FOS was produced with a yield of 60% (Figure 4). These results showed that an initial sucrose concentration of 800 g/L was helpful to obtain a higher concentration of FOS. Sucrose concentration higher than 800 g/L (for example, 900 g/L) led to a decrease in conversion rate to 45% due to the high viscosity. The profiles of sucrose consumption, FOS production, and byproducts (glucose and fructose) formation are shown in Figure 5. During the first hour of biotransfromation, sucrose was rapidly converted to glucose and FOS. Total FOS production attained the most elevated level after 3 h of transformation. From the time point of 3 h, the total FOS concentration declined gradually (Figures 5 and 6) as a result of low sucrose content and the formation of longer-chain FOS (such as GF3, GF4, and GF5) elongated from 1-kestose (GF2).

Figure 4. Effect of sucrose concentration on the FOS formation by the displayed A.oryFTase. The mixture was incubated at pH 6.0, 60 °C, and 150 rpm for 3 h. Each value represents the average for three independent measurements ± standard deviation.

Interestingly, compared to the accentuated hydrolysis of FOS (Figure 5), which was also observed by Mussatto et al. (2009),31 Dominguez et al. (2012),10 Sheu et al. (2013),11 and ́ Marin-Navarro et al. (2015),12 the sucrose content decreased very slowly from 9.0% to 6.5% after 10 h of transformation, suggesting that the presence of a high concentration of glucose might increase the FOS hydrolysis and inactivate the transfructosylation, which led to an increase in free fructose and glucose concentrations. In fact, the glucose and fructose concentrations increased to 38.2% and 5.1%, respectively, at the time point of 10 h. Figure 6 demonstrates the chromatography profiles of the FOS production by the A.oryFTase displayed on the engineered yeasts. The highest content of GF2 (312 g/L) was obtained after 2 h of transformation, and GF4 appeared at this time point. The content of GF2 decreased to 268 g/L, along with the 3832


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Figure 5. Time course of FOS production catalyzed by the displayed A.oryFTase The mixture was incubated at pH 6.0, 60 °C, and 150 rpm for 10 h. All plots were shown as means of two independent experiments.

increase of GF3 and GF4, which reached to 189 and 24 g/L, respectively, at 3 h. The final products, GF2 (33.5%), GF3 (23.6%), and GF4 (2.9%), residual sucrose (9.0%), and the byproducts glucose (29.1%) and fructose (1.9%), were obtained after 3 h of transformation. A decrease in GF2 and GF 3 concentration and an increase in GF 4 and GF 5 concentration were observed at 10 h of transformation. It is worth noting that the longer-degree FOS, such as GF4 and GF5, has enhanced stability and better prebiotic function compared to low-degree FOS, such as GF2.32 Interestingly, the neo-FOS was produced from 1 h (indicated by the dotted arrow in Figure 6). Neo-FOS mainly comprises neo-kestose (neo-GF2, retention time of 9.0 min in Figure 6) and neo-nystose (neoGF3, retention time of 11.3 min in Figure 6), in which fructosyl units are linked to sucrose with a β-2,6-bond.33 Production of Erythritol from FOS Mixtures by the Strain Y. lipolytica CGMCC 7326. After transformation for 3 h by engineered Y. lipolytica transformant no. 11 (CGMCC11368), about 480 g/L FOS, 235 g/L glucose, and 25 g/L fructose were produced, but 63 g/L residual sucrose still remained. Figure 7 shows the profiles of carbohydrate compositions at 0, 48, and 60 h fermentation by Y. lipolytica CGMCC7326. Glucose was converted into erythritol at a yield of 50−55% and fructose was exhausted after 60 h of fermentation; meanwhile, FOS and sucrose remained constant during fermentation as expected. The final reaction mixture contained 155 g/L FOS (70.5% of total sugars), 20 g/L sucrose (9%), and 45 g/L erythritol (20.5%). Residual sucrose and produced erythritol could be separated using economic and easy-to-use nanofiltration, instead of by costly and complex SMB. Comparison of Y. lipolytica Transformant No. 11 and A. oryzae. A. oryzae was one of the microorganisms commonly used in the industrial production of FOS due to its high capacity for producing FTase via solid-state fermentation (SSF) using various agricultural byproducts.34 In this experiment, we compared the FOS-producing capacity of the yeast Y. lipolytica transformant no. 11 and the fungi A. oryzae CGMCC3.800. In the case of transformant no. 11, the maximum FOS yield reached 60% at 3 h in a solution of 800 g/L sucrose under the optimal conditions, with the highest productivity of 160 g/(L· h). In contrast, A. oryzae cells generated the maximum FOS yield of 51.4% at 15 h under similar conditions except at its optimal temperature (50 °C), with a productivity of 27.4 g/(L· h) (Figure 8). Similar results were also reported when fructofuranosidases (FTases) from various species of A. oryzae

Figure 6. Chromatography profiles of FOS production. The mixture was incubated at pH 6.0, 60 °C, and 150 rpm. Samples were taken at 0 h (a), 1 h (b), 2 h (c), 3 h (d), 5 h (e), and 10 h (f). S, sucrose; G, glucose; F, fructose.

were used.29,34 In both cases, the concentration of FOS was found to decrease after reaching the maximum values (Figure 8). 3833


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Figure 7. Chromatography profiles of carbohydrate compositions during fermentation by the erythritol-producing yeast Y. lipolytica CGMCC7326 (a) The profile at time zero of fermentation; (b) 48 h of fermentation; (c) 60 h of fermentation.

purpose, a plasmid pINA-Pir1-A.oryFTase was first constructed (Figure 1). The plasmid contained the Pir1 gene fused with the FTase gene from A. oryzae. The A.oryFTase gene-expression cassette harbored the homologous recombinant arm zeta sequence, a promoter hp4d, an anchor protein Pir1, a terminator LIP2t, and the target gene A.oryFTase from A. oryzae, which was widely used and had a long history in food fermentation. All other DNA elements except for the A.oryFTase gene were from the Y. lipolytica yeast itself. The erythritol-producing Y. lipolytica was then transformed with this expression cassette, and A.oryFTase gene expression was used as a dominant selective marker because the parent yeast was unable to use sucrose as carbon source. Transformants were observed to be able to grow on this sucrose-containing medium, indicating that sucrose could be an effective selective marker. Y. lipolytica is sensitive to antibiotics hygromycin B and bleomycin, and the heterologous expression of these genes conferring such antibiotics has been popularly used in laboratories. However, antibiotic resistance markers in food microorganisms has raised concerns in the European Union regarding the risk of propagating antibiotic resistance genes to

Figure 8. Time courses of FOS production by the engineered Y. lipolytica transformant no. 11 and A. oryzae. The transformation was performed using the transformant no. 11 and A. oryzae in the reaction mixture containing 800 g/L sucrose at their optimal temperatures of 60 and 50 °C, respectively. All plots were shown as means of two independent experiments.

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DISCUSSION In this work, we aimed to develop an engineered FOSproducing yeast, Y. lipolytica CGMCC11368, based on the erythritol-producing yeast Y. lipolytica CGMCC7326. For this 3834


Article

Journal of Agricultural and Food Chemistry Table 2. FOS Yields and Productivity Levels from Different Processes Using Various FTases FTase sources A. oryzae CFR 202 A. oryzae CFR 202 A. japonicus ATCC 20236 A. japonicus ATCC 20236 A. japonicus ATCC 20236 A. pullulans Aspergillus sp. N74 engineered Y. lipolytica a

processes two-stage two-stage two-stage one-stage one-stage one-stage one-stage one-stage

process process process process process process process process

using using using using using using using using

reaction time (h)

extracellular enzyme FTase immobilized on corn germ FTase produced by SSF immobilized cells on corncobs immobilized cells on coffee silverskin free cells free cells free cells

18 8 20 21 16 48 4 3

yield (%)a productivity (g/L·h) 53 60 87 66 61−70 64.1 70 60

17.6 45 10.44 6.61 8.05 2.6 122.5 160

references 34 45 43 31 44 10 17 this study

The yield is the ratio of FOS (g/L) to total initial sucrose (g/L).

fructosidase from A. oryzae FS4 in P. pastoris and found that 10 of 13 putative glycosylation sites were N-glycosylated,40 and such glycosylation made the recombinant enzyme more thermostable at higher temperatures. In our study, the higher optimal temperature for FTase displayed on the cell surface of Y. lipolytica could decrease the viscosity of high-concentration sucrose and improve the reaction speed and product yield, which are beneficial for industrial FOS production. In the fructosyl-transferring reaction, along with FOS production, large amounts of low-valued glucose were produced (Figure 6). The presence of glucose and fructose made it difficult to purify FOS. Thus, the product was low in terms of purity (60% FOS, 30% glucose, 8−10% sucrose, and 2% fructose). High-purity FOS can be obtained by removing glucose via nanofiltration, SMB, or using glucose oxidase coupled to hydrogen peroxide, S. cerevisiae, and P. heimii cofermentation.11,32 In our study, glucose was converted to a higher-value-added erythritol using the strain Y. lipolytica CGMGG7326 at a yield of 50−55%, and a small amount of fructose was also exhausted by this yeast (Figure 7). The final reaction mixture contained 155 g/L FOS (70.5% of total sugars), 20 g/L sucrose (9%), and 45 g/L erythritol (20.5%). Nanofiltration membranes have nominal molecular weight cut-offs (MWCO, molecular weight of solute that was 90% rejected by the membrane) from 200 to 1000 Da in the rejection of neutral solutes by their porous active layer with estimated pore sizes of 1−2 nm. It is one of the new membrane separation technologies and is widely used in the fields of water treatment, manufacturing of pharmaceuticals, food production, and so on.41,42 Sucrose and erythritol can be separated from the fermentation medium simply using nanofiltration membrane owed to their lower molecular weight (342 and 122.1 Da, respectively) than that of FOS (more than 504 Da). Therefore, it is expected to be easy to obtain high-purity FOS by the nanofiltration technology instead of by complex and costly SMB technology. The yield and productivity of a FOS production process are the two most important factors in FOS industrial application. Table 2 presents a comparison between the FOS production yields and productivity levels in the literatures. Though the maximum FOS yield of 87% was obtained by the two-stage process using A. japonicus FTase produced by SSF, the FOS productivity was only 10.44 g/(L·h).43 However, in our study, the maximum FOS productivity reached 160 g/(L·h) at a yield of 60%. Production processes that allow the obtaining FOS with elevated productivity levels have potential industrial applications because they contribute to the lower capital and operating costs. Taken together, this new FOS-synthesis strategy had several advantages: (i) the full utilization of the waste yeast paste from

previously insusceptible microbes in the environment or human gut, making them more resistant to the available antibiotics.35 Therefore, the use of antibiotic resistance markers in yeast for the production of food additives is not allowed in many countries. Instead, the use of safe and sustainable food-grade selection markers is strongly recommended. In this study, we showed that the fructosyltranferase gene (A.oryFTase) could also be used as a safe selection marker for the production of FOS. At present, four different methods of FOS production have been developed. These methods included the use of whole cells (A. japonicus, A. niger, A. oryzae, A. pullulans, etc.) capable of producing intracellular FTase, purified FTase from culture media, immobilized whole cells, and immobilized FTase in supporting materials such as calcium alginate and ligonocellulosic materials.30,31,36 Among them, immobilization technology was extensively employed to enhance enzyme stability and successive recycling capacity. However, from the view of industrial applications, the high immobilized carrier cost and multistep operations are limited factors. The yeast cell-surface display system has been used successfully for the development of microbial enzymes production.37,38 The cell itself could act as a carrier because the production of the immobilized enzyme was simply reduced to cell separation by filtration just after cultivation, thus eliminating the cost of a support carrier. In our experiment, the optimal reaction temperature of surface-displayed FTase was increased to 60 °C (Figure 3a), 5− 10 °C higher than that of the free enzyme reported previously.28 Due to the improved FTase stability immobilized on the cell wall, the recycling number was increased to at least 10 times, and only 10% of the activity of FTase was lost (Figure 3d). In contrast, Menéndez et al. reported that 10% of its original activity was lost after only five cycles of continuous operation.39 It is well-known that N-glycosylation is essential for the thermal stability of enzymes. As described above, the Y. lipolytica strain has been shown to be an excellent host for secreted expression of recombinant proteins with post-translational modifications, including moderate glycosylation, which contains only short oligosaccharide chains of about 8−10 mannose residues, compared to the glycosylation pattern of P. pastoris and H. polymorpha, which produce glycosylations with chains of 8−14 mannose residues.27 It has been found that there are nine putative glycosylation sequons (Asn-Xaa-Ser/ Thr stretch, where Xaa is amino acids other than proline) on A. oryzae FTase (GenBank no. GU477633), six of which (Asn59, Asn107, Asn204, Asn261, Asn399, and Asn437) have the potential to be N-glycosylated with a score above 0.5, analyzed using the online NetNGlyc 1.0 (http://www.cbs.dtu.dk/services/ NetNGlyc). Generally, N-glycosylation occurs at or above the score value. Xu et al. (2014) expressed FOS-producing β3835


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(8) Bali, V.; Panesar, P. S.; Bera, M. B.; Panesar, R. Fructooligosaccharides: production, purification and potential applications. Crit. Rev. Food Sci. Nutr. 2015, 55, 1475−1490. (9) Nobre, C.; Teixeira, J. A.; Rodrigues, L. R. New trends and technological challenges in the industrial production and purification of fructo-oligosaccharides. Crit. Rev. Food Sci. Nutr. 2015, 55, 1444− 1455. (10) Dominguez, A.; Nobre, C.; Rodrigues, L. R.; Peres, A. M.; Torres, D.; Rocha, I.; Lima, N.; Teixeira, J. New improved method for fructooligosaccharides production by Aureobasidium pullulans. Carbohydr. Polym. 2012, 89, 1174−1179. (11) Sheu, D. C.; Chang, J. Y.; Wang, C. Y.; Wu, C. T.; Huang, C. J. Continuous production of high-purity fructooligosaccharides and ethanol by immobilized Aspergillus japonicus and Pichia heimii. Bioprocess Biosyst. Eng. 2013, 36, 1745−1751. (12) Marín-Navarro, J.; Talens-Perales, D.; Polaina, J. One-pot production of fructooligosaccharides by a Saccharomyces cerevisiae strain expressing an engineered invertase. Appl. Microbiol. Biotechnol. 2015, 99, 2549−2555. (13) Aziani, G.; Terenzi, H. F.; Jorge, J. A.; Guimarães, L. H. S. Production of fructooligosaccharides by Aspergillus phoenicis biofilm on polyethylene as inert support. Food Technol. Biotechnol. 2012, 50, 40− 45. (14) Gomes, P. S.; Minceva, M.; Rodrigues, A. E. Simulated moving bed technology: Old and new. Adsorption 2006, 12, 375−392. (15) Tiihonen, J.; Markkanen, I.; Paatero, E. Complex stability of sugars and sugar alcohols with Na+, Ca2+, and La3+ in chromatographic separations using poly(styrene-co-divinylbenzene) resins and aqueous organic eluents. Chem. Eng. Commun. 2002, 189, 995−1008. (16) Jung, K. H.; Bang, S. H.; Oh, T. K.; Park, H. J. Industrial production of fructooligosaccharides by immobilized cells of Aureobasidium pullulans in a packed bed reactor. Biotechnol. Lett. 2011, 33, 1621−1624. (17) Sánchez, O.; Guio, F.; Garcia, D.; Silva, E.; Caicedo, L. Fructooligosaccharides production by Aspergillus sp. N74 in a mechanically agitated airlift reactor. Food Bioprod. Process. 2008, 86, 109−115. (18) Rodríguez, M. A.; Sánchez, O. F.; Alméciga-Díaz, C. J. Gene cloning and enzyme structure modeling of the Aspergillus oryzae N74 fructosyltransferase. Mol. Biol. Rep. 2011, 38, 1151−1161. (19) Finogenova, T.; Morgunov, I.; Kamzolova, S.; Chernyavskaya, O. Organic acid production by the yeast Yarrowia lipolytica: a review of prospects. Appl. Biochem. Microbiol. 2005, 41, 418−425. (20) Xue, Z.; Sharpe, P. L.; Hong, S.-P.; Yadav, N. S.; Xie, D.; Short, D. R.; Damude, H. G.; Rupert, R. A.; Seip, J. E.; Wang, J. Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica. Nat. Biotechnol. 2013, 31, 734−740. (21) Cheng, H. R.; Lv, J. Y.; Wang, B.; Li, D. C.; Deng, Z. X. Yarrowia lipolytica strain and method thereof for synthesizing erythritol. Patent CN201310282059, 2013. (22) Tomaszewska, L.; Rywińska, A.; Gładkowski, W. Production of erythritol and mannitol by Yarrowia lipolytica yeast in media containing glycerol. J. Ind. Microbiol. Biotechnol. 2012, 39, 1333−1343. (23) Makkar, R.; Cameotra, S. An update on the use of unconventional substrates for biosurfactant production and their new applications. Appl. Microbiol. Biotechnol. 2002, 58, 428−434. (24) Nicaud, J. M.; Madzak, C.; Broek, P.; Gysler, C.; Duboc, P.; Niederberger, P.; Gaillardin, C. Protein expression and secretion in the yeast Yarrowia lipolytica. FEMS Yeast Res. 2002, 2, 371−379. (25) Chen, D.-C.; Beckerich, J.-M.; Gaillardin, C. One-step transformation of the dimorphic yeast Yarrowia lipolytica. Appl. Microbiol. Biotechnol. 1997, 48, 232−235. (26) Cheng, H.-R.; Jiang, N. Extremely rapid extraction of DNA from bacteria and yeasts. Biotechnol. Lett. 2006, 28, 55−59. (27) Madzak, C.; Gaillardin, C.; Beckerich, J.-M. Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review. J. Biotechnol. 2004, 109, 63−81.

the erythritol industry; (ii) the high production and yield (480 g/L and 160 g/(L·h), respectively); (iii) the increased optimal reaction temperature that led to a higher reaction speed and higher productivity; and (iv) the efficient conversion of glucose to erythritol, a value-added product. With these advantages, it is expected to be a promising method to produce FOS in industrial scale.

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AUTHOR INFORMATION

Corresponding Author

*H.C. tel.: +86 2134206722; fax: +86 2134206722; e-mail: [email protected]. Author Contributions

L.Z. and J.A. contributed equally to this work. L.Z, J.A., L.L., D.L., and N.L. performed this research; H.W. and H.C. analyzed the data; H.C. designed the study and wrote the paper; Z.D. discussed the results. Funding

We acknowledge financial support through grants from the National Basic Research Program of China (no. 2013CB733903) and the National High-Tech R&D Program (no. 2012AA021503). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

We thank Dr. Muhammad Zohaib Nawaz for his kind revision of this manuscript.

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ABBREVIATIONS USED A.oryFTase, fructosyltransferase gene of A.oryzae; A.oryFTase, fructosyltransferase enzyme of A. oryzae; CGMCC, China General Microbiological Culture Collection Center; DCW, dry cell weight; FOS, fructooligosaccharide; FTase, fructosyltransferase enzyme; FTase, fructosyltransferase gene; HPLC, highperformance liquid chromatography; rpm, rotation per min; SMB, simulated moving bed; SSF, solid-state fermentation

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REFERENCES

(1) Fei, N.; Zhao, L. An opportunistic pathogen isolated from the gut of an obese human causes obesity in germfree mice. ISME J. 2013, 7, 880−884. (2) Zhao, L. The gut microbiota and obesity: from correlation to causality. Nat. Rev. Microbiol. 2013, 11, 639−647. (3) Xiao, S.; Fei, N.; Pang, X.; Shen, J.; Wang, L.; Zhang, B.; Zhang, M.; Zhang, X.; Zhang, C.; Li, M. A gut microbiota-targeted dietary intervention for amelioration of chronic inflammation underlying metabolic syndrome. FEMS. Microbiol. Ecol. 2014, 87, 357−367. (4) Pokusaeva, K.; Fitzgerald, G. F.; van Sinderen, D. Carbohydrate metabolism in Bif idobacteria. Genes Nutr. 2011, 6, 285−306. (5) Russell, W. R.; Gratz, S. W.; Duncan, S. H.; Holtrop, G.; Ince, J.; Scobbie, L.; Duncan, G.; Johnstone, A. M.; Lobley, G. E.; Wallace, R. J.; Duthie, G. G.; Flint, H. J. High-protein, reduced-carbohydrate weight-loss diets promote metabolite profiles likely to be detrimental to colonic health. Am. J. Clin. Nutr. 2011, 93, 1062−1072. (6) Flint, H. J.; Scott, K. P.; Duncan, S. H.; Louis, P.; Forano, E. Microbial degradation of complex carbohydrates in the gut. Gut Microbes 2012, 3, 289−306. (7) Zhang, C.; Yin, A.; Li, H.; Wang, R.; Wu, G.; Shen, J.; Zhang, M.; Wang, L.; Hou, Y.; Ouyang, H. Dietary modulation of gut microbiota contributes to alleviation of both genetic and simple obesity in children. EbioMed. 2015, 2, 966−982. 3836


Article


fermentation using agricultural by-products. Appl. Microbiol. Biotechnol. 2004, 65, 530−537.

(28) Chang, C.-T.; Lin, Y.; Tang, M.; Lin, C. Purification and properties of beta-fructofuranosidase from Aspergillus oryzae ATCC 76080. Biochem. Mol. Biol. Int. 1994, 32, 269−277. (29) Kurakake, M.; Masumoto, R.; Maguma, K.; Kamata, A.; Saito, E.; Ukita, N.; Komaki, T. Production of fructooligosaccharides by βfructofuranosidases from Aspergillus oryzae KB. J. Agric. Food Chem. 2010, 58, 488−492. (30) Cruz, R.; Cruz, V. D.; Belini, M. Z.; Belote, J. G.; Vieira, C. R. Production of fructooligosaccharides by the mycelia of Aspergillus japonicus immobilized in calcium alginate. Bioresour. Technol. 1998, 65, 139−143. (31) Mussatto, S. I.; Aguilar, C. N.; Rodrigues, L. R.; Teixeira, J. A. Fructooligosaccharides and β-fructofuranosidase production by Aspergillus japonicus immobilized on lignocellulosic materials. J. Mol. Catal. B: Enzym. 2009, 59, 76−81. (32) Mutanda, T.; Mokoena, M.; Olaniran, A.; Wilhelmi, B.; Whiteley, C. Microbial enzymatic production and applications of short-chain fructooligosaccharides and inulooligosaccharides: recent advances and current perspectives. J. Ind. Microbiol. Biotechnol. 2014, 41, 893−906. (33) Kritzinger, S. M.; Kilian, S. G.; Potgieter, M. A.; du Preez, J. C. The effect of production parameters on the synthesis of the prebiotic trisaccharide, neokestose, by Xanthophyllomyces dendrorhous (Phaf f ia rhodozyma). Enzyme Microb. Technol. 2003, 32, 728−737. (34) Sangeetha, P. T.; Ramesh, M. N.; Prapulla, S. G. Fructooligosaccharide production using fructosyl transferase obtained from recycling culture of Aspergillus oryzae CFR 202. Process Biochem. 2005, 40, 1085−1088. (35) Schjørring, S.; Krogfelt, K. A. Assessment of bacterial antibiotic resistance transfer in the gut. Int. J. Microbiol. 2011, 2011, 312956. (36) Markosyan, A.; Abelyan, L.; Adamyan, M.; Ekazhev, Z.; Akopyan, Z.; Abelyan, V. Production of fructooligosaccharide syrup from sucrose in combination with palatinose and trehalose. Appl. Biochem. Microbiol. 2007, 43, 383−389. (37) Gai, S. A.; Wittrup, K. D. Yeast surface display for protein engineering and characterization. Curr. Opin. Struct. Biol. 2007, 17, 467−473. (38) Yamakawa, S. I.; Yamada, R.; Tanaka, T.; Ogino, C.; Kondo, A. Repeated fermentation from raw starch using Saccharomyces cerevisiae displaying both glucoamylase and α-amylase. Enzyme Microb. Technol. 2012, 50, 343−347. (39) Menéndez, C.; Martínez, D.; Trujillo, L. E.; Mazola, Y.; González, E.; Pérez, E. R.; Hernández, L. Constitutive high-level expression of a codon-optimized β-fructosidase gene from the hyperthermophile Thermotoga maritima in Pichia pastoris. Appl. Microbiol. Biotechnol. 2013, 97, 1201−1212. (40) Xu, L.; Wang, D.; Lu, L.; Jin, L.; Liu, J.; Song, D.; Guo, Z.; Xiao, M. Purification, Cloning, Characterization, and N-Glycosylation Analysis of a Novel β-Fructosidase from Aspergillus oryzae FS4 Synthesizing Levan-and Neolevan-Type Fructooligosaccharides. PLoS One 2014, 9, e114793. (41) Wang, X. L.; Shang, W. J.; Wang, D. X.; Wu, L.; Tu, C. H. Characterization and applications of nanofiltration membranes: State of the art. Desalination 2009, 236, 316−326. (42) Wang, K. Y.; Chung, T. S. The characterization of flat composite nanofiltration membranes and their applications in the separation of Cephalexin. J. Membr. Sci. 2005, 247, 37−50. (43) Mussatto, S. I.; Ballesteros, L. F.; Martins, S.; Maltos, D. A. F.; Aguilar, C. N.; Teixeira, J. A. Maximization of fructooligosaccharides and β-fructofuranosidase production by Aspergillus japonicus under solid-state fermentation conditions. Food Bioprocess Technol. 2013, 6, 2128−2134. (44) Mussatto, S. I.; Teixeira, J. A. Increase in the fructooligosaccharides yield and productivity by solid-state fermentation with Aspergillus japonicus using agro-industrial residues as support and nutrient source. Biochem. Eng. J. 2010, 53, 154−157. (45) Sangeetha, P. T.; Ramesh, M. N.; Prapulla, S. G. Production of fructosyl transferase by Aspergillus oryzae CFR 202 in solid-state 3837


Highly Efficient Fructooligosaccharides Production by an Erythritol

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