Article pubs.acs.org/JAFC
Integrated Approach To Producing High-Purity Trehalose from Maltose by the Yeast Yarrowia lipolytica Displaying Trehalose Synthase (TreS) on the Cell Surface Ning Li,†,‡ Hengwei Wang,§ Lijuan Li,†,‡ Huiling Cheng,†,‡ Dawen Liu,†,‡ 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 (IAI), Zhejiang Ocean University, Zhoushan 316022, China S Supporting Information *
ABSTRACT: An alternative strategy that integrated enzyme production, trehalose biotransformation, and bioremoval in one bioreactor was developed in this study, thus simplifying the traditional procedures used for trehalose production. The trehalose synthase gene from a thermophilic archaea, Picrophilus torridus, was first fused to the YlPir1 anchor gene and then inserted into the genome of Yarrowia lipolytica, thus yielding an engineered yeast strain. The trehalose yield reached 73% under optimal conditions. The thermal and pH stabilities of the displayed enzyme were improved compared to those of its free form purified from recombinant Escherichia coli. After biotransformation, the glucose byproduct and residual maltose were directly fermented to ethanol by a Saccharomyces cerevisiae strain. Ethanol can be separated by distillation, and high-purity trehalose can easily be obtained from the fermentation broth. The results show that this one-pot procedure is an efficient approach to the economical production of trehalose from maltose. KEYWORDS: trehalose, trehalose synthase, Yarrowia lipolytica, cell surface display, ethanol, Picrophilus torridus
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INTRODUCTION Some unusual organisms, including resurrection plants, such as Selaginella pulvinata and Selaginella lepidophylla, nematodes, baker’s yeast cells, and fungal spores, have an amazing adaptation that allows them to survive for years under complete dehydration conditions.1−4 Although 99% of their body water may have been removed, they can resume their vital life functions promptly after rehydration. These unusual organisms can also withstand other abiotic stresses, such as extreme temperatures, strong vacuum, and high doses of ionizing radiation. It is reported that these desiccation-tolerant organisms accumulate some type of osmoprotector, such as a sugar or sugar alcohol, which plays an important role in the protection of cellular proteins, energy sources, and signaling pathways.5 Among these osmoprotectors, trehalose, a nonreducing disaccharide that has two glucose units linked by an α,α-1,1-glycosidic linkage, has attracted extensive interest. It seems to be one of the most efficient molecules protecting cells from stress and maintains membrane and protein stability in the state of desiccation by replacing water with osmolyte molecules or forming a glassy state.6 It is also widely observed in bacteria, archaebacteria, yeast, fungi, insects, and plants.7−12 Trehalose is widely applied in the agri-food, cosmetic, and pharmaceutical industries due to its excellent properties, which are shared by most chemically unreactive sugars, and strong stability.13,14 Although these properties have been recognized for many years, trehalose was not produced commercially for a long time. Recently, two enzymatic methods for the production of trehalose have been commercialized, and production costs have been dramatically reduced. One method produces trehalose from © XXXX American Chemical Society
starch or maltooligosaccharides using maltooligosyltrehalose synthase and maltooligosyltrehalose hydrolase (or MTS−MTH fusion enzyme) in combination with α-amylase and pullulanase.15−17 The other method produces trehalose directly from maltose using trehalose synthase (TreS or TSase) via intramolecular transglucosylation.18,19 Other enzymatic systems include (i) the TPS−TPP system, in which the TPS enzyme catalyzes the formation of trehalose-6-phosphate from UDPglucose and glucose-6-phosphate and TPP catalyzes the subsequent hydrolysis to trehalose and phosphate;20 (ii) the TreP enzyme, which synthesizes trehalose from glucose-1phosphate and glucose;10 and (iii) the TreT enzyme, which converts ADP-glucose and glucose into trehalose.21 These three enzymatic methods cannot be commercialized due to their high cost. Among the above-reported enzymatic methods, the enzyme TreS converts maltose to trehalose through an intramolecular rearrangement in a single-step process, which is a simple, fast, and low-cost method and shows great advantages and potential for commercial trehalose production. Maltose can be easily prepared from starch by β-amylase and pullulanase. A number of treS genes from different bacteria have been identified and characterized, and some have been applied in trehalose production.7,22−27 However, treS-containing bacteria produce trehalose from maltose with a very low yield. Thus, treS genes were cloned from those bacteria, overexpressed in Escherichia coli, Received: May 18, 2016 Revised: July 21, 2016 Accepted: July 22, 2016
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DOI: 10.1021/acs.jafc.6b02175 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 1. Scheme of construction of the pINA-Pir1-treS plasmid and the linearized functional elements designed for the introduction of TreS into Y. lipolytica cells. medium (YNG selective plates, 6.7 g/L yeast nitrogen base without amino acids, 20 g/L glucose, 5 g/L ammonium sulfate, 0.02 g/L leucine, 0.02 g/L histidine, and 20 g/L agar, pH 5.5) was used for transformant selection. Construction of the Y. lipolytica Expression Vector for Surface Display. The DNA for the anchor protein Pir1 was amplified using the genomic DNA of Y. lipolytica CLIB724 as the template with the following primers: Ppir1-F (5′-ATAAAGCTTATGGTGTTCAAGTCTGCTGCTGTTTC-3′ (the underlined is the HindIII restriction site) and Ppir1-R (5′-ATACTGCAGACAGCCTTCCAAGTTAACGATAG-3′ (the underlined is the PstI restriction site). The PCR reaction began with denaturation at 94 °C for 5 min, followed by 35 PCR cycles (30 s at 94 °C, 40 s at 58 °C, and 50 s at 72 °C) and a final extension step of 10 min at 72 °C. The 876-bp PCR product was cut with HindIII and PstI and ligated into the vector pINA1313 that had been cut with HindIII and PstI, yielding the plasmid pINA1313-Pir1. The treS gene from P. torridus DSM9790 (GenBank accession no. AE017261) was optimized according to the codon bias of Y. lipolytica and was chemically synthesized by the solid-phase phosphoramidite method (General Biosystems, Inc., Chuzhou, Anhui, China). Then, the DNA fragment was subcloned into the plasmid pINA1313-Pir1, thus yielding the yeast surface display vector pINA-Pir1-treS. The pINA-Pir1-treS plasmid was linearized with NotI, and the linearized fragment (5-′zeta-ura3d1-hp4d-Pir1-treS-LIP2t-zeta′-3) was transformed into Y. lipolytica CLIB724 using the method described by An et al.31 The construction of the expression vector for surface display and the linearized fragment are shown in Figure 1. Cells were spread on the above-mentioned YNG minimal medium to select the transformants and incubated at 30 °C for 3−5 days until colonies appeared. Identification of Y. lipolytica Transformants. The genomic DNA of the Y. lipolytica transformants was extracted using the method described by Cheng and Jiang.32 The primers PtreS‑F (5′-ATGCTTGATAACAACGGTCTG-3′) and PtreS‑R (5′-TTAATCTTCTCCAATGAGGTC-3′) were used to amplify the treS gene with the following thermal cycling conditions: an initial denaturation at 94 °C for 5 min, followed by 30 cycles at 94 °C for 35 s, 58 °C for 40 s, and 72 °C for 90 s. Positive transformants were randomly selected to convert maltose to trehalose under the same conditions. Briefly, 10 transformants were cultured in 10 tubes each containing 10 mL of YPM medium (10 g/L yeast extract, 5 g/L tryptone, 20 g/L glucose, and
purified, and used as free enzymes or biocatalysts to convert maltose to trehalose at yields of 40−70%.25−27 The E. coli-based trehalose production method entails cultivating E. coli in antibiotic-containing medium, followed by induction with IPTG, purification of the enzyme from the crude extract and, finally, conversion of maltose to trehalose by free or immobilized TreS. The final reaction products contain trehalose, as well as residual maltose and a glucose byproduct, and must be separated by an SMB process. Thus, these current methods are not only laborious and time-consuming but also very expensive, especially in the case of TreS purification. Recently, Zheng et al. have made progress in producing trehalose from maltose by using whole cells of permeabilized recombinant E. coli.18 In addition, E. coli is a conditionally pathogenic enterobacterium and may cause health problems if trehalose is contaminated with this bacterium during the production process.28−30 In this study, we aimed to develop a simple and low-cost method to produce trehalose from maltose. First, the enzyme TreS from the extreme thermoacidophilic euryarchaeon Picroplilus torridus DSM9790 was displayed on the cell surface of Yarrowia lipolytica and was used as a whole-cell biocatalyst to directly transform maltose to trehalose. Then, residual maltose and glucose byproducts left in the reaction mixtures were fermented into ethanol by yeast cells to obtain high-purity trehalose. Trehalose can be directly crystallized from concentrated fermentation broth, as opposed to the complex SMB separation process. According to the above design of experiments, we expect that this method has the potential for the industrial production of trehalose.
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MATERIALS AND METHODS
Strains and Culture Conditions. The yeast Y. lipolytica CLIB724 (PO 1f strain, MatA, leu2-270, ura3-302, xpr2-322, axp-2), an auxotrophic strain, was obtained from the International Center for Microbial Resources (CIRM-Levures, Thiverval-Grignon, France) and used as the host for cell surface display expression. An E. coli DH5α strain was used as the host for DNA manipulations. Minimal YNG B
DOI: 10.1021/acs.jafc.6b02175 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry 5 g/L maltose monohydrate, pH 6.5) at 30 °C for 50 h. The cells were collected, suspended in an appropriate amount of 100 g/L aqueous maltose solution to adjust the OD600 to 20, and incubated in 50-mL shake flasks for 50 h at 40 °C in an orbital shaker at a speed of 200 rpm. Trehalose was then analyzed by HPLC. Determination of Relative treS Gene Expression in Selected Transformants by RT-qPCR. The above 10 transformants were cultivated in 10 tubes, each containing 10 mL of YPD medium (10 g/L yeast extract, 5 g/L tryptone, and 20 g/L glucose, pH 6.5) at 30 °C for 24 h. The cells were collected, and the total RNA was extracted with the TRIzol total RNA extraction kit (catalog lot DP405-2, TianGen, China), and all total RNA samples were treated with TURBO DNase (Amicon). The cDNAs were synthesized from 1.0 μg of DNasetreated RNA using a TIANScript II RT kit (catalog no. KR107-01, TianGen, China), according to the manufacturer’s instructions. Quantitative real-time PCR (RT-qPCR) was performed in 96-well plates using SYBR Premix Ex-TaqII (Tli RNaseH Plus) (catalog no. RR82LR, TAKARA, Dalian, China). The primers used to amplify a 126-bp fragment of the treS gene, 5′-CGACGAGATAGGCATGGGTG-3′ and 5′-GGTGAGTAGAGCTGTTCCGAAT-3′, were designed using the treS gene sequence. The primers 5′-GCGAGAAATCGTCCGAGACAT-3′ and 5′-GCAGCCTCAAGACCCAGCAT-3′ were used to amplify a 201-bp fragment of the β-actin gene, which was used as a reference. The PCR program was run in an ABI 7500 Fast RealTime PCR System (Applied Biosystem), with a profile at 95 °C for 30 s, 45 cycles of 5 s at 95 °C, and 40 s at 60 °C. Three technical replicates were used for each biological sample. The normalized relative quantities were obtained using the 2−ΔΔCT method.33 Transformants with the highest relative expression of the treS gene and the highest trehalose yield were used in subsequent investigations. Characterization of Recombinant Yeast Cells. The engineered yeast Y. lipolytica transformant was cultivated in YPD medium at 30 °C for 2 days, collected by centrifugation, washed with PBS buffer (50 mM, pH 6.0) two times, and lyophilized for 20 h. The intramolecular transglucosylation and the formation of the glucose byproduct (hydrolysis activity) by TreS on the cell surface of Y. lipolytica were measured by assaying the amount of trehalose and glucose produced from maltose, respectively. One unit of activity was defined as the number of yeast cells (in DCW) that catalyzed the formation of 1 μmol of trehalose or glucose per minute. One milliliter of cells at an OD600 of 1 was equivalent to 0.25 mg of DCW. (i) To determine the optimal temperature for intramolecular transglucosylation, the reactions were performed in 5 mL of PBS buffer (50 mM, pH 6.0) containing 500 mg of maltose and 25 mg of resting cells (in DCW) at various temperatures (20−80 °C) at 200 rpm for 10 h. (ii) The effects of pH on the intramolecular transglucosylation of displayed TreS were determined at various pH values from 4.0 to 8.5 using 50 mM sodium acetate buffer (pH 4.0−5.5) and 50 mM sodium phosphate buffer (pH 6.0−8.5). Briefly, 25 mg of cells (in DCW) were suspended in 5 mL of the above buffers containing 100 g/L maltose and reacted at 50 °C and 200 rpm for 10 h. (iii) To determine the effect of temperature on the hydrolysis activity of displayed TreS, reactions were performed by incubating the resting cells (25 mg in DCW) in 5 mL of 50 mM sodium phosphate buffer (pH 6.0) containing 100 g/L maltose at various temperatures from 20 to 70 °C for 30 h. (iv) To determine the effects of the maltose concentration on the production and yield of trehalose catalyzed by displayed TreS, reactions were performed by incubating the resting cells (25 mg in DCW) in 50 mM sodium phosphate buffer (pH 6.0) containing various concentrations of maltose (20−500 g/L) at pH 6.0 and 50 °C for 30 h. In the above experiments, the final reaction mixtures were centrifuged for 10 min at 10000g, and aliquots of 20 μL were withdrawn for HPLC analysis. The yield of trehalose (in %) was calculated by using the formula 100 × trehalose produced/total maltose. Determination of the Stability of TreS on Lyophilized Yeast Cells. To analyze the stability of TreS-displaying yeast cells, lyophilized engineered Y. lipolytica cells were stored at 25 °C for 2, 5, 10, 15, 20, 25, 30, and 40 days. Then, 25 mg of cells from each treatment was resuspended in 5 mL of PBS buffer (50 mM, pH 6.0) containing 100 g/L maltose and reacted at 50 °C for 10 h with agitation at 150 rpm. Then, the trehalose synthase activity of TreS was determined.
Detection of TreS Enzyme Activity in Resting Cells and Culture Supernatants. Theoretically, the enzyme TreS was displayed on the cell wall of Y. lipolytica after being transported across the plasma membrane. In our study, the presence of the TreS enzyme (trehalose or maltose synthase) in the growth medium was also tested. The percentage of cell-bound synthase activity was expressed as the ratio of cell-bound activity to that of the entire culture (resting cells and culture supernatants). Five milliliters of engineered yeast cells was inoculated in 250-mL shake flasks containing 50 mL of YPD medium and cultured at 30 °C and 200 rpm until the OD600 reached 10. The cells were collected by centrifugation, and 50 mL of supernatants was then transferred to clean shake flasks and adjusted to pH 6.0. The cells were washed twice using PBS buffer (50 mM, pH 6.0), centrifuged at 6000g for 10 min, and resuspended in 50 mL of PBS buffer (50 mM, pH 6.0). To detect the trehalose synthesis activity of the cells and the supernatants, 100 g/L anhydrous maltose was added to the above cell suspensions and supernatants and catalyzed at 50 °C and 150 rpm for 10 h. To detect the maltose synthesis activity of the cells and supernatants, 100 g/L anhydrous trehalose was used. The forward activity of TreS was assayed by measuring the amount of trehalose produced from maltose, and the reverse activity was assayed by measuring the amount of maltose produced from trehalose. Screening of Yeast Strains for Bioremoval of Glucose and Maltose. To obtain a yeast strain that could utilize glucose and maltose but not trehalose, yeast strains from our laboratory stocks were screened by inoculating each culture on solid plates containing 10 g/L YNB (Difco, Detroit, MI, USA) and 20 g/L agar supplemented with 10 g/L glucose (YNG), 10 g/L maltose (YNM), or 10 g/L trehalose (YNT); the cells were cultured at 28 °C for 5 days. The yeast strains that could grow on the other plates but not on YNT plates were chosen. Target yeast strains were identified by the sequences of their 18S rDNA and internal transcribed spacer (ITS) region. Transformation, Purification, and Verification of Trehalose. Two hundred milliliters of recombinant Y. lipolytica culture was inoculated into a 3-L fermenter (Bioflo 110, New Brunswick Scientific) containing 1.8 L of fermentation medium (15 g/L yeast extract, 5 g/L tryptone, 10 g/L maltose, and 30 g/L glucose) and cultured at 30 °C and pH 6.5 with an aeration of 1.0 vvm and agitation at 200 rpm. When the cell density reached 20 at OD600, 600 g of maltose monohydrate was added to the fermenter to achieve a concentration of 300 g/L. The cellular viability of yeasts can be quantified by counting colony-forming units (CFU). From this time point, 100 μL of culture medium was withdrawn and spread directly onto a YPD plate and cultivated at 30 °C for 36 h. The parameters of pH, agitation, and aeration remained unchanged, but the temperature was increased to 50 °C, which was the optimal temperature for trehalose synthesis. Samples were withdrawn regularly and analyzed by HPLC. When the trehalose content remained constant for approximately 12 h, the temperature was decreased to 30 °C and the transformation was terminated. The agitation was then increased to 300 rpm, and 100 mL of the yeast culture used for the bioremoval of residual glucose and maltose was inoculated into the fermenter. Samples were withdrawn regularly and analyzed by HPLC to measure the concentrations of residual glucose and maltose and the production of ethanol. After the biopurification step, all of the yeast cells were removed by centrifugation, and the clear supernatant was decolorized with activated carbon at 80 °C. Then, the colorless solution was applied to ion-exchange resin columns to remove ions, which was verified by a conductivity of 70% of the TreS activities were cell-bound (Figure 5). Researchers have suggested that the YlPir1 gene should be deleted to improve the efficiency of enzyme display on the cell wall because the cell surface of Y. lipolytica can be saturated with the YlPir1 protein itself when overexpressed.41 Thus, more of target enzyme can be bound to the anchoring sites on yeast cells, and the amount of enzyme present in the medium can be decreased. Disruption of the YlPir1 gene did not cause changes in the cellular physiological characteristics, such as the cell morphology, growth rate, and morphological transition of Y. lipolytica.41 Another approach to improve display efficiency is to fuse target proteins to the C-terminus of YlPir instead of its N-terminus. It is reported that the target proteins fused to the N-terminus were not only anchored to the cell wall but also secreted into the medium in some cases.42 Screening and Identification of a Yeast Strain for Bioremoval of Glucose and Maltose. Among the 50 yeast
55 mL of culture medium containing approximately 125 mg of DCW cells and 50 mL of supernatant. The ratio of trehalose synthase activity anchored on resting cells to the total units in the entire culture was 74.6% (Figure 5). When trehalose was used as the substrate, maltose synthase activity (forward activity) reached 0.27 U/mg DCW and 0.2 U/mL supernatant, and the total activity reached 43.3 U in 55 mL of culture broth containing approximately 125 mg of DCW cells and 50 mL of supernatant. The ratio of trehalose synthase activity (reverse activity) anchored on the resting cells to the total units in entire culture was 76.9% (Figure 5). Our results demonstrate that the majority of enzyme TreS was anchored to the cells. Duquesne et al. reported that 75 and 96% activities of expressed xylanase were located in the culture supernatants when YlCWP1 and YlCBM were used as anchor proteins, and 63% of its activities were located in the culture supernatants when YlPir was used as the anchor protein, indicating that YlPir was a better system for anchoring xylanase on Y. lipolytica cells.40 F
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maltose by ion-exchange approaches due to their similar physical and chemical characteristics. For these reasons, a onepot procedure integrating enzyme production, biotransformation, and bioremoval is desired. The byproduct glucose and residual maltose can both be converted into ethanol and carbon dioxide without any trehalose consumption. Chang and co-workers developed a combinatorial biocatalysis process to produce trehalose from starch using α-amylase, β-amylase, pullulanase, and TreS and converted the byproducts glucose, residual maltose, and maltotriose to ethanol using glucoamylase, Rhizopus crude enzyme, and S. cerevisiae.19,47 As an alternative method to eliminate residual maltose (up to 25% of reaction products), glucoamylase was added to hydrolyze maltose to glucose, which could be converted to ethanol by S. cerevisiae fermentation. In our experiments, the yeast strain S. cerevisiae Y1 was able to directly ferment maltose to ethanol, thus avoiding the use of glucoamylase and reducing the production cost. In conclusion, in our study an alternative approach to converting maltose to trehalose based on Y. lipolytica protein display technology was developed. Compared to the covalent linkage of proteins to support carriers, the immobilization used in this study offers proteins a physical support, which may improve their stability. The Y. lipolytica surface display technology does not require additional steps of protein purification and physical immobilization, thus greatly reducing production costs. In addition, our one-pot protocol consolidates enzyme production, trehalose biotransformation, and byproduct bioremoval in only one bioreactor, thus simplifying the trehalose production from maltose. Our study provides useful information in the combination of biotechnological processes for the biotransformation of similar sugars.
strains that were screened on YNB-based media supplemented with glucose, maltose, or trehalose, 15 strains were unable to grow on YNB media plus maltose, and 35 strains grew well on YNB media supplemented with maltose. Among the 35 strains that could utilize maltose, 5 strains could not utilize trehalose. The 5 yeast strains were identified as S. cerevisiae (2 strains), P. pastoris (1 strain), Wickerhamomyces anomalus (1 strain), and Schizosaccharomyces pombe (1 strain) on the basis of their 18S rDNA and ITS sequences. One of the S. cerevisiae strains was designated strain Y1 and was used in subsequent bioremoval experiments because it was a food-grade yeast. The growth of yeast strain Y1 on YNB plates containing glucose, maltose, or trehalose solid medium is shown in Figure S1. Profiles of Trehalose Production in a 3-L Fermenter. One-pot trehalose production was performed under the optimal pH (pH 6.0), temperature (50 °C), and maltose concentration (300 g/L) in a 3-L fermenter. The profiles of cell growth (enzyme production), trehalose production, and biopurification (or bioremoval) are depicted in Figure 6a. The engineered Y. lipolytica transformant 4 was inoculated into the fermenter and cultured at 30 °C for 45 h. The level of dissolved oxygen (DO) decreased to zero in 10 h. During this stage, cells grew well on the plates (Figure 6b), indicating that they were viable. When the OD600 reached 20 (from 45 h), the temperature was shifted to 50 °C. The DO level increased to 150% 3 h after the temperature shift, indicating that cellular respiration had ceased. The cell viability was almost completely lost when the temperature was maintained at 50 °C for 12 h (Figure 6c) and was completely lost within 24 h (Figure 6d). Although the cellular viability of transformant 4 was lost at 50 °C, the activity of enzyme TreS was maintained at its peak. After biotransformation, the temperature was decreased to 30 °C, and the cells of S. cerevisiae Y1 were inoculated and started to grow rapidly (Figure 6e). Maltose was converted to trehalose with a yield of 73% at 50 °C during the 50 h of biotransformation, and the concentration of produced trehalose remained constant during the subsequent bioremoval procedure (Figure 6f). The maximum production of trehalose reached 219 g/L after 50 h of transformation, with a yield of 73% and a productivity of 4.5 g/(L·h). It has been reported that the crystallization of sugar alcohol, such as xylitol, is negatively affected by the content of byproduct sugars.43,44 The total concentration of residual glucose and maltose in the broth was approximately 90 g/L (Figure 6f). Therefore, to achieve large-scale industrial production of trehalose from the broth, the removal of the byproduct sugars is recommended. Residual glucose and maltose were simultaneously converted to ethanol by S. cerevisiae strain Y1, with a yield of 40−45% after 24 h of fermentation (Figure 6f,k). The ethanol can simply be separated from trehalose by distillation. The HPLC profiles for the one-pot trehalose production are shown in Figure 6g−k. The trehalose crystals obtained using our integrated strategy were of 99.5% purity (Figure 6l). The processes of enzyme production, trehalose biotransformation, and byproduct bioremoval were integrated in this study, thus simplifying the traditional procedures used for trehalose production from maltose. In the traditional methods used to produce trehalose from maltose, the recombinant enzyme was purified from E. coli cells and used to convert maltose to trehalose with a yield of 60−70%.7,18,26,27,45,46 To obtain highpurity trehalose, the byproduct glucose and residual maltose must be separated from the reaction mixtures by simulated moving bed chromatography (SMBC), which is very costly and complex. It is even more difficult to separate trehalose and
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b02175. Growth of the selected yeast strain Y1 on plates containing glucose, maltose, or trehalose as the sole carbon sources (PDF)
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AUTHOR INFORMATION
Corresponding Author
*(Hairong Cheng) Phone: +86-21-34206722. Fax: +86 2134206722. E-mail:
[email protected]. Author Contributions
Ning Li, Lijuan Li, Huiling Cheng, and Dawen Liu performed this study; Hengwei Wang and Hairong Cheng analyzed the data; Hairong Cheng designed the study and wrote the paper; and Zixin Deng discussed the results. Funding
We acknowledge financial support through grants from the National Basic Research Program of China (No. 2013CB733903), the Scientific Research Foundation (SRF, No. 22215010114), and Zhejiang Ocean University. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED TPS, trehalose-6-phosphate synthase; TPP, trehalose-6-phosphate phosphatase; TreP, trehalose phosphorylase; TreT, G
DOI: 10.1021/acs.jafc.6b02175 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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bioethanol, and high-protein product from rice by an enzymatic process. J. Agric. Food Chem. 2010, 58, 2908−2914. (20) Avonce, N.; Mendoza-Vargas, A.; Morett, E.; Iturriaga, G. Insights on the evolution of trehalose biosynthesis. BMC Evol. Biol. 2006, 6, 109. (21) Ryu, S. I.; Park, C. S.; Cha, J.; Woo, E. J.; Lee, S. B. A novel trehalose-synthesizing glycosyltransferase from Pyrococcus horikoshii: molecular cloning and characterization. Biochem. Biophys. Res. Commun. 2005, 329, 429−436. (22) Nishimotoa, T.; Nakanoa, M.; Ikegamia, S.; Chaena, H.; Fukudaa, S.; Sugimotoa, T.; Kurimotoa, M.; Tsujisakaab, Y. Existence of a novel enzyme converting maltose into trehalose. Biosci., Biotechnol., Biochem. 1995, 59, 2189−2190. (23) Jiang, L.; Lin, M.; Zhang, Y.; Li, Y. P.; Xu, X.; Li, S. Identification and characterization of a novel trehalose synthase gene derived from saline-alkali soil metagenomes. PLoS One 2013, 8, e77437. (24) Koh, S.; Shin, H.-J.; Kim, J. S.; Lee, D.-S.; Lee, S. Y. Trehalose synthesis from maltose by a thermostable trehalose synthase from Thermus caldophilus. Biotechnol. Lett. 1998, 20, 757−761. (25) Koh, S.; Kim, J.; Shin, H.; Lee, D.; Bae, J.; Kim, D.; Lee, D. Mechanistic study of the intramolecular conversion of maltose to trehalose by Thermus caldophilus GK24 trehalose synthase. Carbohydr. Res. 2003, 338, 1339−1343. (26) Kim, T.-K.; Jang, J.-H.; Cho, H.-Y.; Lee, H.-S.; Kim, Y.-W. Gene cloning and characterization of a trehalose synthase from Corynebacterium glutamicum ATCC13032. Food Sci. Biotechnol. 2010, 19, 565− 569. (27) Chen, Y.-S.; Lee, G.-C.; Shaw, J.-F. Gene cloning, expression, and biochemical characterization of a recombinant trehalose synthase from Picrophilus torridus in Escherichia coli. J. Agric. Food Chem. 2006, 54, 7098−7104. (28) Efimochkina, N. R.; Sheveleva, S. A.; Kuvaeva, I. B.; Fluer, F. S.; Batishcheva, Slu.; Salova, N.; Andrusenko, E. E.; Bykova, I. V.; Markova, T. V. Detection and serological screening of conditionallypathogenic enterobacteria isolated from food products and the environment. Vopr. Pitan. 2002, 71, 29−34. (29) Herzog, K.; Dusel, J. E.; Hugentobler, M.; Beutin, L.; Sägesser, G.; Stephan, R.; Hächler, H. Diarrheagenic enteroaggregative Escherichia coli causing urinary tract infection and bacteremia leading to sepsis. Infection 2014, 42, 441−444. (30) Pearson, H. The dark side of E. coli. Nature 2007, 445, 8−9. (31) An, J.; Zhang, L.; Li, L.; Liu, D.; Cheng, H.; Wang, H.; Nawaz, M. Z.; Cheng, H.; Deng, Z. An alternative approach to synthesizing galactooligosaccharides by cell-surface display of β-galactosidase on Yarrowia lipolytica. J. Agric. Food Chem. 2016, 64, 3819−3827. (32) Cheng, H.-R.; Jiang, N. Extremely rapid extraction of DNA from bacteria and yeasts. Biotechnol. Lett. 2006, 28, 55−59. (33) Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 2001, 25, 402−408. (34) Roth, R.; Moodley, V.; van Zyl, P. Heterologous expression and optimized production of an Aspergillus aculeatus endo-1,4-βmannanase in Yarrowia lipolytica. Mol. Biotechnol. 2009, 43, 112−120. (35) Nordén, K.; Agemark, M.; Danielson, J. Å.; Alexandersson, E.; Kjellbom, P.; Johanson, U. Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris. BMC Biotechnol. 2011, 11, 47. (36) Madzak, C. Yarrowia lipolytica: recent achievements in heterologous protein expression and pathway engineering. Appl. Microbiol. Biotechnol. 2015, 99, 4559−4577. (37) Koh, S.; Kim, J.; Shin, H. J.; Lee, D.; Bae, J.; Kim, D.; Lee, D. S. Mechanistic study of the intramolecular conversion of maltose to trehalose by Thermus caldophilus GK24 trehalose synthase. Carbohydr. Res. 2003, 338, 1339−1343. (38) Sierks, M. R.; Sico, C.; Zaw, M. Solvent and viscosity effects on the rate-limiting product release step of glucoamylase during maltose hydrolysis. Biotechnol. Prog. 1997, 13, 601−608.
trehalose glycosyltransferring synthase; ADP-glucose, adenosine diphosphate glucose; UDP-glucose, uridine 5-diphosphoglucose; OD600, optical density measured at a wavelength of 600 nm; HPLC, high-performance liquid chromatography; rpm, revolutions per minute; SMB, simulated moving bed; NMR, nuclear magnetic resonance; CLIB, Collection de Levures d’Intérêt Biotechnologique
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