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Xylitol production by NAD+-dependent xylitol dehydrogenase (xdhA)- and l -arabitol-4-dehydrogenase (ladA)-disrupted mutants of Aspergillus oryzae
Journal of Bioscience and Bioengineering VOL. 115 No. 4, 353e359, 2013 www.elsevier.com/locate/jbiosc
Xylitol production by NADþ-dependent xylitol dehydrogenase (xdhA)- and L-arabitol-4-dehydrogenase (ladA)-disrupted mutants of Aspergillus oryzae Asif Mahmud,1 Koji Hattori,2 Chen Hongwen,3 Noriyuki Kitamoto,4 Tohru Suzuki,1, * Kohei Nakamura,2 and Kazuhiro Takamizawa2 The United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan,1 Faculty of Applied Biological Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan,2 Department of Bioengineering and Biotechnology, Huaqiao University, Xiamen, Fujian 361021, China,3 and Food Research Center, Aichi Center for Industry and Science Technology, 2-1-1 Shinpukujicho, Nishi-ku, Nagoya, Aichi 451-0083, Japan4 Received 22 June 2012; accepted 19 October 2012 Available online 1 January 2013
Aspergillus oryzae can metabolize xylan to D-xylose and D-xylose to xylitol. However, accumulation of xylitol is controlled by dehydrogenases, such as xylitol dehydrogenase (XDH) and L-arabitol-4-dehydrogenase (LAD), and fluxed into the pentose phosphate pathway. In A. oryzae, XDH and LAD are encoded by xdhA and ladA, respectively. We disrupted the xdhA and ladA genes individually in an attempt to increase xylitol production. The xdhA- and ladA-disrupted mutants were constructed by homologous transformation into A. oryzae P5 (DpyrG), and pyrG was used as a selectable marker. The mutants were grown on different carbohydrate-containing media, colony diameters of mutants were measured, and gene disruption was confirmed by PCR. The xylitol productivity of the mutants was measured using D-xylose and oat spelt xylan as the sole sources of carbohydrates. The xdhA-disrupted mutant xdhA2-1 produced 16.6 g/L xylitol at a yield of 0.43 g/g D-xylose and productivity of 0.248 g/L. h from D-xylose, while 10.2 g/L xylitol was produced at a yield of 0.204 g/g xylan from oat spelt xylan. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Xylitol; Disrupted mutant; Aspergillus oryzae; Xylitol dehydrogenase; L-Arabitol-4-dehydrogenase]
Xylitol, a five-carbon sugar alcohol, is a natural carbohydrate abundantly present in fruits and vegetables. Its sweetening power is comparable to that of sucrose, while its endothermal hydration gives a cooling sensation for use as a sweetener. Xylitol inhibits dental cavities and acute otitis media (1,2) and is widely used in products, including chewing gums, sweets, toothpastes, and cosmetics. It is also used as a sweetener for diabetics because its metabolism is insulin independent. Xylitol is commercially produced by chemical hydrogenation of D-xylose derived from hemicelluloses, such as birch wood xylan and hydrolyzates of biomass material, which includes drawbacks, such as hydrolysis, expensive separation, purification steps, and the requirement of high temperature and pressure conditions (3). Therefore, use of microbial xylanolytic enzymes for the industrial hydrolysis of lignocelluloses in an environmentally friendly way could be a better option. In the last few decades, several reports were published on xylitol production using bacteria (4,5), and fungi (6e10). Among microorganisms, yeasts are the best xylitol producers. Candida tropicalis have been studied extensively for xylitol production as they have the advantage over the metabolically engineered Saccharomyces cerevisiae for being natural D-xylose consumers and maintaining the reduction oxidation balance during xylitol accumulation (11). * Corresponding author. Tel.: þ81 58 293 3171; fax: þ81 58 293 3172. E-mail address: [email protected] (T. Suzuki).
Candida is a very good candidate for xylitol production due to its high xylose uptake rate and xylitol production capacity (10,12). As xylitol has applications and potential uses in the food, dental, and pharmaceutical industries, usage of Candida yeasts, recognized as opportunistic pathogens, is a significant drawback. Moreover Candida yeasts lack the xylanolytic enzyme, which is recognized as one of the major enzymes for lignocellulose hydrolysis. For better commercial xylitol production, lignocelluloses should be used as raw materials as they are the most abundant renewable natural materials of the agricultural wastes and significant quantities of them are presently disposed of by burning. This clearly is a great loss of energy conserved through the process of biosynthesis by the green plants. Therefore, we selected Aspergillus oryzae since it possesses a better enzyme system to hydrolyze lignocelluloses than Candida and also has a long fermentation history as well as being generally recognized as safe. A. oryzae has long been used in Japan for food fermentation, such as the production of shoyu or soy sauce, miso, and sake as well as industrial enzyme production, such as a-amylase and glucoamylase (13,14). A. oryzae can produce D-xylose from xylan using xylanase (15) and convert D-xylose into xylitol using xylose reductase (XR) (EC 1.1.1.21). The accumulation of xylitol is also affected by the downstream activity of xylitol dehydrogenase (XDHA) (EC 1.1.1.9), which converts xylitol to D-xylulose. We have reported another enzyme, L-arabitol dehydrogenase (LADA) (EC 1.1.1.12) (16), which converts arabitol to L-xylulose (17), is highly
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.10.017
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similar (35% amino acid sequence similarity) to XDHA, and is able to convert xylitol to D-xylulose. In the case of Hypocrea jecorina, LAD-1, an ortholog of LADA, also contributes to xylitol conversion. These dehydrogenase enzymes can have a significant effect on production of xylitol by converting xylitol into xylulose. D-Xylulose can then be converted to xylulose 5-phosphate by xylulokinase (EC 2.7.1.17) and fluxed into the pentose phosphate pathway. In this research, we disrupted the xdhA and ladA genes of A. oryzae individually and determined the xylitol productivities of these mutants under laboratory conditions. MATERIALS AND METHODS Fungal strains A. oryzae P5 (18) is a pyrG-deficient strain developed from A. oryzae KBN616, which is a soy sauce koji isolated from a soy brewing plant and was obtained from Bio’c (Toyohashi, Aichi, Japan). A. oryzae P5 does not encode orotidine 50 -phosphate carboxylase, the terminal enzyme in uridine 50 -phosphate biosynthesis. Plasmid pYRG100 (19) was used for pyrG gene amplification, which was used as a selectable marker. Construction of the disruption cassettes for xdhA and ladA Genomic DNA of A. oryzae KBN616 was extracted (20) and used as the template for the amplification of the xdhA (AO090038000631) and ladA (AO090005001078) gene inserts. Using the xdhA-P1/xdhA-P2 and xdhA-P3/xdhA-P4 primer sets (Table 1), 1025 bp and 908 bp regions of xdhA, respectively, were amplified using standard PCR conditions. Primers were designed to include SacI and Eco47III restriction sites. The inserts were purified using a GENECLEANR Turbo kit (Qbiogene, Graffenstaden, France); then, the xdhA-P1 and xdhA-P4 primers were used to amplify a 1903 bp region of xdhA (Fig. 1) by overlap extension PCR. Similarly, using ladA-P1/ladA-P2 and ladA-P3/ladA-P4 primer sets (Table 1), 1079 bp and 1001 bp regions of ladA, respectively, were amplified, including kpnI and Eco47III restriction sites. After purification, these inserts were used as templates and a 2053 bp region of ladA was amplified using ladA-P1 and ladA-P4 primers by overlap extension PCR. The pUC19 vector was digested using SacI and kpnI restriction enzymes, and the xdhA and ladA fragments were ligated into the pUC19 vector using T4 Ligase (Takara, Shiga, Japan) and incubated at 16 C for 2 h to obtain pXDHA (4.6 kbp), as shown in Fig. 1 and pLADA (4.7 kbp). pYRG100 was double digested with HindIII and BamHI (Takara) to obtain the pyrG insert. After purification of the pyrG fragment, blunt ends were created using T4 polymerase (Takara) by incubating at 37 C for 30 min, and the fragment purified using a Qiagen reaction cleanup kit (Venlo, Netherlands). pXDHA and pLADA were digested using Eco47III and then purified. To prevent self-ligation, the digested vectors were dephosphorylated by incubation with calf intestine alkaline phosphatase (Takara) at 50 C for 30 min. The pyrG fragment was ligated into pXDHA and pLADA using T4 DNA ligase (Takara) by incubating at 16 C for 2 h to obtain the pYRGXDHA (Fig. 1) and pYRG-LADA vectors. Takara Ex Taq was used for amplification, using a Takara thermal cycler and the primers listed in Table 1.
TABLE 1. Primers used in this study. Gene/ Vector xdhA
ladA
pyrG
M13
Primer
Length (base)
Sequence
xdhA-P1 xdhA-P2 xdhA-P3 xdhA-P4 xdhA-S1 xdhA-S2 xdhA-PF xdhA-PR ladA-P1 ladA-P2 ladA-P3 ladA-P4 ladA-S1 ladA-S2 ladA-PF ladA-PR pyrG-Fed pyrG-Rev pyrG-S1 pyrG-S2 M13-P4 M13-P5
30 30 30 30 17 17 20 20 28 27 27 27 17 17 20 20 30 30 18 18 24 20
50 .GTCGAGCTCCCTGCTTTAATTTTCCGATTG.30 50 .TAATACTTGGCTAGCGCTCCGTCGTAAGGT.30 50 .ACCTTACGACGGAGCGCTAGCCAAGTATTA.30 50 .CGCGAGCTCCCTAGTCATCTACTAATTTTCTCC..30 50 .CTTCCCCCTTCAACTTT.30 50 .AAACTGCCCCTGACATT.30 50 .AACAAGCGTAGTTCCATGGT.30 50 .GCTGATTCCCTGATCTTGCC.30 50 .TTGGTACCCGCTCCTGGTTCCTATAGTTTCC.30 50 .CTCCATCCTCAGCGCTCATATCACCGA.30 50 .TCGGTGATATGAGCGCTGAGGATGGAG.30 50 .TTGGTACCGGCGGTCATACAAAAGCAG.30 50 .TGCTTGTCCCTCTTGCT.30 50 .TCTCTTGGGTGCTCAGA.30 50 .GCTTCACGTTTTGAGGACAT.30 50 .GTATCCATAGGAATAGGTGG.30 50 .GGAAGCGCTGCTGGAATTGACATTATTATG.30 50 .TTAAGCGCTCAATACCGTACGGGAGATTGT.30 50 .AATAAGCCAGTTTCAACC.30 50 .ACTGCCATAAAAGGAGTA.30 50 .CGCCAGGGTTTTCCCAGTCACGAC.30 50 .GGATAACAATTTCACACAGG.30
GAGCTC, SacI site; AGCGCT, Eco47III site; GGTACC, KpnI site.
FIG. 1. Construction of xdhA-disrupted vector. xdhA fragment (1.9 kbp) was amplified by PCR then ligated into the SacI site of pUC19. The obtained vector, pXDHA, was digested with Eco47III and ligated with the pyrG fragment of pYRG100 to generate the disrupted vector pYRG-XDHA (6.5 kb). The ladA-disrupted vector was also constructed in a similar manner.
Confirmation and sequence analysis of the disrupted constructs After transforming the pYRG-XDHA and pYRG-LADA vectors into Competent Quick DH5a (Toyobo, Osaka, Japan), the cells were cultured on LB agar media containing 2% X-gal and ampicillin at 37 C for 15 h. Transformed colonies appeared white, and a total of 16 colonies were confirmed by direct colony PCR using primers M13-P4 and M13-P5 (Table 1). Plasmids were extracted using a Qiagen Plasmid Mini extraction kit. To confirm the disruption of xdhA and ladA, pYRG-XDHA and pYRG-LADA were digested using SacI and KpnI (Takara) by incubating at 37 C for 2 h. The digests were run on agarose gel and the size of the bands was used to confirm the disruption of the cassettes. Confirmation of construct sequences was done by sequencing, especially of the terminal and linking regions. M13-P4, M13-P5, xdhA-S1, xdhA-S2, pyrG-Fed, pyrGRev, pyrG-S1, and pyrG-S2 primers were used for pYRG-XDHA and M13-P4, M13-P5, ladA-S1, ladA-S2, pyrG-Fed, pyrG-Rev, pyrG-S1, and pyrG-S2 primers (Table 1) were used for pYRG-LADA. For sequencing, BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA) and ABI PRISM 3100 sequence analyzer were used. Transformation of A. oryzae A. oryzae P5 was transformed according to a previously described method (21). P5 cells were cultured at 30 C overnight in Complete Media (20 g/L malt extract, 1 g/L bactopeptone, 20 g/L glucose, 1.5 g/L uridine, and 0.7 g/L uracil). The pH was adjusted to 6.5 with 0.1 M KOH. Five microliters of pYRG-XDHA and pYRG-LADA were used for transformation after being linearized by digestion with SacI and KpnI, respectively. The xdhA and ladA disruptants were selected as prototrophs using Czapek Dox (Difco, NJ, USA) agar media. Then, colonies were picked and spread on CP media (10 g/L polypeptone, 1 g/L NaNO3, 5 g/L KH2PO4, and 1 g/L MgSO4$7H2O, pH 6.0) containing 20 g/L of different carbohydrate sources, including D-glucose, D-xylose, L-arabinose, xylitol, and oat spelt xylan, as required. After 5 days incubation at 30 C, the diameter of the colonies was determined to evaluate substrate utilization ability. After extracting the genomic DNA (20) from the disrupted mutants, the genomic structures of the target genes were confirmed by PCR. The primer sets xdhA-PF/ xdhA-PR, and xdhA-P1/xdhA-PR (Table 1) were used for the xdhA-disrupted mutants (Fig. 2A and B), while ladA-PF/ladA-PR and ladA-P1/ladA-PR (Table 1)
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were used for the ladA-disrupted mutants (Fig. 3A and B). Considering the results from PCR and substrate utilization, candidate disruptants for xdhA and ladA were selected for further experiments. Culture conditions for fermentation experiments The stock strains were precultured in 100 mL preculture media (10 g/L D-glucose, 10 g/L yeast extract, 5 g/L KH2PO4, 1 g/L NaNO3, and 0.5 g/L MgSO4$7H2O) at 30 C and 150 rpm for 48 h using a 500 mL Erlenmeyer flask. Fungal mycelia were collected from the media and washed with 0.1 M potassium phosphate buffer (pH 6.0), and 5.0 g wet weight of mycelia were used to inoculate the fermentation media. The xylitol fermentation experiments from D-xylose and oat spelt xylan were performed in 500 mL Erlenmeyer flasks with 100 mL of xylitol fermentation media (50 g/L D-xylose, 10 g/L yeast extract, 5 g/L KH2PO4, 1 g/L NaNO3, and 0.5 g/L MgSO4$7H2O) at 30 C and 150 rpm. Oat spelt xylan (50 g/L) was used instead of D-xylose to determine xylitol production from oat spelt xylan. Assay of XR and XDH The XDH and XR activities were determined spectrophotometrically using Ultrospec 2100 pro (Amersham Bioscience, NJ, USA) by monitoring the change in A340 upon NAD reduction or NADPH oxidation at 25 C, respectively (22). Cells grown on xylitol fermentation medium were harvested by filtration. After the cells were washed with 100 mM potassium phosphate buffer (pH 7.0), 1.0 g of cells was collected, mixed with 1.0 g of sea sand (Nacalai Tesque, Kyoto, Japan) and crushed using a mortar and pastel with liquid nitrogen cooling. After adding 5.0 mL of 100 mM potassium phosphate buffer (pH 7.0), the cell debris was separated by centrifugation at 13,000 rpm for 15 min and the supernatant was then used to measure enzyme activity. The XDH assay mixture contained 1 M potassium phosphate buffer (pH 7.5), 0.1 M 2-merceptoethanol, 0.5 M xylitol, 3.4 mM NADþ, deionized water, and enzyme solution. The XR assay mixture contained 1 M potassium phosphate buffer (pH 7.0), 0.1 M 2merceptoethanol, 0.5 M D-xylose, 3.4 mM NADPH, deionized water and enzyme solution. The activity was expressed in units, where 1 U corresponded to the conversion of 1 mmol of NAD or NADPH per minute. Each measurement was
FIG. 3. PCR confirmation of the specific integration of pyrG into ladA-disrupted mutants. (A) Primer set ladA-PF and ladA-PR was used for PCR. Lane 1: l-EcoT14 I-digested marker; lanes 2e8: ladA disruptants; lane 9: KBN616. (B) Primer set ladA-P1 and ladA-PR was used for PCR. Lane 1: l-EcoT14 I-digested marker; lanes 2e8: ladA disruptants; lane 9: KBN616.
repeated twice. The concentration of protein in the enzyme solution was measured using a Qubit protein assay system (Invitrogen). Sugar analysis The concentrations of D-glucose/D-mannose, D-xylose, xylitol, L-arabitol, and D-mannitol were analyzed by high-performance liquid chromatography (Shimadzu Corporation, Kyoto, Japan) using a Shimadzu RID10A detector and Shodex SUGAR SZ5532 column. Pure water was used as the mobile phase with a flow rate of 1.0 mL/min and column temperature of 60 C. L-arabinose,
RESULTS
FIG. 2. PCR confirmation of the specific integration of pyrG into the xdhA-disrupted mutants. (A) Primer set xdhA-PF and xdhA-PR was used for PCR. Lane 1: l-EcoT14 I-digested marker; lanes 2e6: xdhA disruptants; lane 7: KBN616. (B) Primer set xdhAP1 and xdhA-PR was used for PCR. Lane 1: l-EcoT14 I-digested marker; lanes 2e6: xdhA disruptants; lane 7: KBN616.
Construction of xdhA- and ladA- disrupted mutants A. oryzae P5 was transformed to uracil prototrophy with SacI- and KpnIdigested pYRG-XDHA and pYRG-LADA, respectively. Transformants were selected as prototrophs by growing on Czapek Dox agar media without uridine and uracil. A total of five xdhA and seven ladA transformants were selected initially by Colony Direct PCR. Genomic DNA was extracted and PCR was performed to confirm the disruption for each of these transformants. After electrophoresis on a 1.5% agarose gel, the predicted 4.5 kb (Fig. 2A) and 4.2 kb (Fig. 2B) bands, appeared for the two primer sets for the xdhA mutants xdhA2-1, 3-1, 4-4, 5-4, and 6-6, whereas A. oryzae KBN616 gave a band around 2.0 kb (Fig. 2A,B), indicating the successful disruption of the xdhA gene. For the ladA mutants, bands around 4.5 kb (Fig. 3A) and 4.3 kb (Fig. 3B) appeared for the two sets of primers for ladA2-1
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and 3-8, whereas the band for KBN616 was around 2.5 kb (Fig. 3A, B), indicating ladA disruption in the transformants. To assess the carbohydrate utilization ability, colony diameters of the mutants were measured on CP agar media containing different carbohydrate sources (Figs. 4 and 5). Observing the diameters of the colonies for different xdhA mutants, we found that all disruptants displayed considerably slower growth on D-xylose (Fig. 4A, B) and xylitol but were still able to grow on these carbon sources, which was understandable due to the xdhA disruption. In presence of other carbohydrates, all the disruptants displayed growth similar to KBN616 (Fig. 4B). Among the disruptants, xdhA21 was selected arbitrarily for a xylitol production experiment. All ladA transformants displayed considerable substrate utilization ability, much like the wild-type strain (Fig. 5A, B). Only ladA2-1 seemed unable to utilize L-arabinose (Fig. 5A), whereas ladA3-8 seemed to utilize L-arabinose like wild type. Though ladA3-8 gave a similar size PCR band as ladA2-1, we excluded ladA3-8 as a potential ladA-disrupted mutant due to its substrate utilization ability and selected ladA2-1 to carry on with xylitol production experiments. Xylitol production from D-xylose Mutants xdhA2-1 and ladA2-1 were tested for xylitol production from D-xylose-containing xylitol production media (Fig. 6). Consumption of D-xylose for xdhA2-1 and ladA2-1 was similar to that of KBN616. In terms of xylitol production, xdhA2-1 displayed the highest xylitol productivity of 0.25 g/L h at a concentration of 16.2 g/L xylitol (Fig. 6B), whereas the xylitol productivity of ladA2-1 decreased to 0.048 g/L h at a concentration of 4.2 g/L xylitol (Fig. 6C) in comparison to KBN616 (Fig. 6A).
FIG. 5. Colony diameters on different carbohydrate sources for ladA disruptants. (A) Growth of the parental strain and the ladA-disrupted mutants on CP medium with L-arabinose as the sole carbon and energy source. (B) The growth of colonies in the presence of different substrates was determined in cm. Gray bars, ladA2-1; open bars, ladA3-8; and closed bars, KBN616.
XR and XDH specific activity Specific enzyme activities for XR and XDH of KBN616 and xdhA2-1, grown on xylitol fermentation media were analyzed (Fig. 7A, B). The results indicate that KBN616 and xdhA2-1 do not have significant variation in specific XR activities (Fig. 7A). On the other hand, xdhA2-1 showed decreased XDH activity (Fig. 7B) compared to the wild-type strain. Xylitol production from oat spelt xylan xdhA-disrupted mutant xdhA2-1 exhibited the highest xylitol productivity from D-xylose and was selected for further production of xylitol from oat spelt xylan. Fig. 8A and B show the data for different carbohydrates when oat spelt xylan was used for xylitol fermentation using KBN616 and xdhA2-1. Mutant xdhA2-1 produced a maximum of 13.1 g/L D-xylose and 10.2 g/L xylitol at a yield of 0.204 g/g xylan (Fig. 8B), whereas KBN616 produced only 2.6 g/L D-xylose and 0.42 g/L xylitol. Trace amounts of D-glucose/D-mannose, L-arabitol, and D-mannitol were also produced by xdhA2-1. DISCUSSION
FIG. 4. Colony diameters on different carbohydrate sources for xdhA disruptants. (A) Growth of the parental strain and the xdhA-disrupted mutants on CP medium with D-xylose as the sole carbon and energy source. (B) The growth of colonies in the presence of different substrates was determined in cm. Open bars, xdhA2-1; filled square bars, xdhA3-1; gray bars, xdhA4-4; dotted bars, xdhA5-4; open square bars, xdhA6-6; and closed bars, KBN616.
Candida tropicalis has been reported to have the highest xylitol productivity (12 g/L h) using a cell recycle system (23). Xylose assimilating yeasts like C. tropicalis have an enzyme regime to metabolize xylitol from D-xylose, but most of them do not have the enzymes required for xylan degradation, such as xylanases and xylosidases, except for some special species (24e26). We selected A. oryzae because it has a suitable enzyme regime required for xylitol production from lignocellulosic biomass. It has a very long history of food fermentation in Japan and other Asian countries and is generally recognized as safe (GRAS). After disrupting the xdhA and ladA genes of A. oryzae, we found that xdhA-disrupted mutant xdhA2-1 had the highest xylitol productivity. Volumetric xylitol productivity increased 4 and 10 fold compared to the parental strain KBN616 when D-xylose and oat spelt xylan, respectively, were used as the substrate. The ladA-disrupted mutant, ladA2-1, showed lower xylitol productivity than KBN616. lad1-encoded L-arabitol-4-dehydrogenase in H. jecorina
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FIG. 7. Assay of XR and XDH activities. (A) Assay of XDH activities in KBN616 and xdhA2-1. Closed bars, KBN616; open bars, xdhA2-1. (B) Assay of XR activities in KBN616 and xdhA2-1. Closed bars, KBN616; open bars, xdhA2-1.
FIG. 6. Production of xylitol from D-xylose. (A) Consumption of D-xylose and production of xylitol by KBN616. Closed circles, D-xylose; closed triangles, xylitol. (B) Consumption of D-xylose and production of xylitol by xdhA2-1. Closed circles, D-xylose; closed triangles, xylitol. (C) Consumption of D-xylose and production of xylitol by ladA2-1. Circles, D-xylose; triangles, xylitol.
can compensate partially for the metabolism of D-xylose in mutants with a loss of xdh1 function (17). Since the xdhA and ladA genes of A. oryzae were disrupted separately in this study, ladA-disrupted mutant did not show any significant impact on D-xylose metabolism when xdhA was intact (Fig. 5B), and hence the xylitol productivity also did not increase (Fig. 6C). To evaluate the effect of ladA disruption on xylitol accumulation, a double disrupted mutant lacking both XDHA and LADA might be necessary, which we will report in our next paper. Therefore, we excluded ladA-disrupted mutant ladA2-1 as a potential xylitol producer and did not measure its specific XDH and XR activities. Xylose reductase (XR) and xylitol dehydrogenase (XDH) constitute the initial metabolic pathway for xylose in fungi. The overall efficiency of xylose metabolism is connected through a complex regulatory network with the ability of XR and XDH to provide a high flux of carbon through the initial pathway (16). Therefore, XDHA is an important target for the metabolic engineering of A. oryzae
toward the utilization of xylose in a manner that meets the requirements of the biotech industry for increased xylitol production. XDHA is encoded by xdhA in A. oryzae, and the function of XDHA is thought to be compensated for by LADA (17), which has 35% similarity with XDHA (16). LADA is encoded by ladA in A. oryzae and is up regulated in the presence of D-xylose even though it does not contribute to its catabolism (27,28). The main yield-limiting factor for xylitol production is its consumption for cell growth and maintenance. Therefore, if the metabolic step from xylitol to D-xylulose could be blocked by disruption of the corresponding xdhA and ladA genes, and if cosubstrates, such as glucose, galactose, or glycerol, were supplied for cell growth, the yield of xylitol should reach a theoretical level of 100% (29). Antisense inhibition of XDH in Trichoderma reesei reduced XDH activity to 48% and enhanced xylitol productivity (30). Similar studies were also done on C. tropicalis, which has higher xylitol productivity and a xylitol yield of 98% was achieved using glycerol as a cosubstrate (31). This report is an attempt to apply gene disruption techniques to analyze pentose metabolism in A. oryzae. We constructed A. oryzae mutant by disrupting either the xdhA or ladA gene in order to diminish XDH activity. Disruption of xdhA in the mutant xdhA2-1 decreased XDH activity compared to the wild-type strain KBN616 (Fig. 7B) however, we did not measure the XDH activity of the ladAdisrupted mutant. xdhA2-1 still showed some XDH activity, which could indicate the presence of other homologous genes present in A. oryzae (32). For the same reason, xylitol produced by xdhA2-1 was gradually consumed after 168 h (Fig. 6B) though the xdhA2-1 mutant accumulated 4-times more xylitol without the use of a cosubstrate or controlled aeration under batch fermentation (Fig. 6B). Xylitol production by xdhA2-1 had a yield of 33% from D-xylose. Though we did not measure the fungal mycelia
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J. BIOSCI. BIOENG., resulting in the presence of homologous genes (32). Disrupting only xdhA or ladA might not be enough to create a xylitol producer. Considering the results of this research, the organism might have bypassed the utilization of xylitol, expressing other homologous genes and hampering xylitol accumulation. Further research will target the homologous genes by disruption to construct a potential xylitol producer. ACKNOWLEDGMENT We thank Dr. Yasuda, Dr. Tran Ha Lien, Mr. Masanori Yogo, and Ms. Keiko Ushida for their technical assistance. References
FIG. 8. Production of xylitol from oat spelt xylan. (A) Oat spelt xylan was used as a substrate to measure the metabolites by KBN616. Squares, D-xylose; circles, xylitol; triangles, D-glucose/D-mannose; diamonds, L-arabitol; asterisks, D-mannitol; crosses, L-arabinose. (B) Oat spelt xylan was used as a substrate to measure the metabolites by xdhA2-1. Squares, D-xylose; circles, xylitol; triangles, D-glucose/D-mannose; diamonds, L-arabitol; asterisks, D-mannitol; crosses, L-arabinose.
concentrations for different time intervals, the amount of mycelium inoculated from preculture media into xylitol fermentation media for each flask culture experiment was constant around 5.0 g wet weight of the respective mycelia. For preculture conditions, we used C-40 (Type C) silicone stoppers where KLa (h1) was 145 14 and for xylitol fermentation experiments, we used T-32 (Type T) silicone stoppers, where KLa (h1) was 50 12 at 150 rpm. The sulfite oxidation method was used to measure the oxygen transfer rate for the flask experiments (33) and 150 rpm was kept constant for aeration of the flask experiments. We previously reported an improvement in the method of xylitol production using C. tropicalis (34). Fine tuning of aeration, glucose addition as a cosubstrate, and immobilization of the cells proved efficient for increasing xylitol production (35). Using similar techniques, it would not be difficult to increase the xylitol productivity of the xdhA-disrupted mutant. This is the first attempt to apply metabolic engineering to the xylose metabolic pathway in A. oryzae to enhance xylitol yield and productivity. This strategy for strain development will contribute greatly to other attempts for improving the metabolite production of microorganisms. We understand that our xylitol productivity was not as high as in C. tropicalis (30). The reason could be that the A. oryzae genome is one third larger than that of two relevant Aspergillus species (36)
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22. Yokoyama, S. I., Suzuki, T., Kawai, K., Horitsu, H., and Takamizawa, K.: Purification, characterization and structure analysis of NADP-dependant D-xylose from Candida tropicalis, J. Ferment. Bioeng., 79, 217e223 (1995). 23. Kwon, S. G., Park, S. W., and Oh, D. K.: Increase of xylitol productivity by cellrecycle fermentation of Candida tropicalis using submerged membrane bioreactor, J. Biosci. Bioeng., 101, 13e18 (2006). 24. Bhadra, B., Rao, S. R., Singh, P. K., Sarkar, P. K., and Shivaji, S.: Yeasts and yeast-like fungi associated with the bark of trees: diversity of yeasts and identification of yeasts producing extracellular endo-xylanases, Curr. Microbiol., 56, 489e494 (2008). 25. Beg, Q. K., Kapoor, M., Mahajan, L., and Hoondal, G. S.: Microbial xylanases and their industrial applications: a review, Appl. Microbiol. Biotechnol., 56, 326e338 (2001). 26. Boonmak, C., Limtong, S., Jindamorakot, S., In, S. A., Yongmanitchai, W., Suzuki, K., Nakase, T., and Kawasaki, H.: Candida xylanilytica sp. nov., a xylandegrading yeast species isolated from Thailand, Int. J. Syst. Evol. Microbiol., 61, 1230e1234 (2011). 27. Tran, L. H., Kitamoto, N., Kawai, K., Takamizawa, K., and Suzuki, T.: Cloning and expression of a NADþ-dependent xylitol dehydrogenase gene (xylA) of Aspergillus oryzae, J. Biosci. Bioeng., 97, 419e422 (2004). 28. Andersen, M. R., Vongsangnak, W., Panagiotou, G., Salazar, M. P., Lehmann, L., and Nielsen, J.: A trispecies Aspergillus microarray: comparative transcriptomics of three Aspergillus species, Proc. Natl. Acad. Sci. USA, 105, 4387e4392 (2008).
29. Jorgensen, T. R., Goosen, T., Hondel, C. A., Ram, A. F., and Iversen, J. J.: Transcriptomic comparison of Aspergillus niger growing on two different sugars reveals coordinated regulation of the secretory pathway, BMC Genomics, 10, 44 (2009). 30. Wang, T., Penttila, M., Gao, P., Wang, C., and Zhong, L.: Isolation and identification of xylitol dehydrogenase gene from Trichoderma ressei. Chin. J. Biotechnol., 14, 179e185 (1998). 31. Byoung, S. K., Jinmi, K., and Jung, H. K.: Production of xylitol from D-xylose by a xylitol dehydrogenase gene disrupted mutant of Candida tropicalis. Appl. Environ. Microbiol., 72, 4207e4213 (2006). 32. Machida, M.: Progress of Aspergillus oryzae genomics, Adv. Appl. Microbiol., 51, 81e106 (2002). 33. Cooper, C., Fernstorm, G., and Miller, A.: Gas liquid contactors, Ind. Eng. Chem., 36, 504e509 (1944). 34. Yahashi, Y., Horitsu, H., Kawai, K., Suzuki, T., and Takamizawa, K.: Production of xylitol from D-xylose by Candida tropicalis: the effect of D-glucose feeding, J. Ferment. Bioeng., 81, 148e152 (1996). 35. Yahashi, Y., Hatsu, M., Horitsu, H., Kawai, K., Suzuki, T., and Takamizawa, K.: D-glucose feeding for improvement of xylitol productivity from D-xylose using Candida tropicalis immobilized on a non-woven fabric, Biotechnol. Lett., 18, 1395e1400 (1996). 36. Galagan, J. E., Calvo, S. E., Cuomo, C., Ma, L. J., Wortman, J. R., Batzoglou, S., Lee, S. I., Bas¸türkmen, M., Spevak, C. C., Clutterbuck, J., and other 40 authors: Sequencing of Aspergillus nidulans and comparative analysis with Aspergillus fumigatus and Aspergillus oryzae, Nature, 438, 1105e1115 (2005).
Xylitol production by NADþ-dependent xylitol dehydrogenase (xdhA)- and L-arabitol-4-dehydrogenase (ladA)-disrupted mutants of Aspergillus oryzae Asif Mahmud,1 Koji Hattori,2 Chen Hongwen,3 Noriyuki Kitamoto,4 Tohru Suzuki,1, * Kohei Nakamura,2 and Kazuhiro Takamizawa2 The United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan,1 Faculty of Applied Biological Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan,2 Department of Bioengineering and Biotechnology, Huaqiao University, Xiamen, Fujian 361021, China,3 and Food Research Center, Aichi Center for Industry and Science Technology, 2-1-1 Shinpukujicho, Nishi-ku, Nagoya, Aichi 451-0083, Japan4 Received 22 June 2012; accepted 19 October 2012 Available online 1 January 2013
Aspergillus oryzae can metabolize xylan to D-xylose and D-xylose to xylitol. However, accumulation of xylitol is controlled by dehydrogenases, such as xylitol dehydrogenase (XDH) and L-arabitol-4-dehydrogenase (LAD), and fluxed into the pentose phosphate pathway. In A. oryzae, XDH and LAD are encoded by xdhA and ladA, respectively. We disrupted the xdhA and ladA genes individually in an attempt to increase xylitol production. The xdhA- and ladA-disrupted mutants were constructed by homologous transformation into A. oryzae P5 (DpyrG), and pyrG was used as a selectable marker. The mutants were grown on different carbohydrate-containing media, colony diameters of mutants were measured, and gene disruption was confirmed by PCR. The xylitol productivity of the mutants was measured using D-xylose and oat spelt xylan as the sole sources of carbohydrates. The xdhA-disrupted mutant xdhA2-1 produced 16.6 g/L xylitol at a yield of 0.43 g/g D-xylose and productivity of 0.248 g/L. h from D-xylose, while 10.2 g/L xylitol was produced at a yield of 0.204 g/g xylan from oat spelt xylan. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Xylitol; Disrupted mutant; Aspergillus oryzae; Xylitol dehydrogenase; L-Arabitol-4-dehydrogenase]
Xylitol, a five-carbon sugar alcohol, is a natural carbohydrate abundantly present in fruits and vegetables. Its sweetening power is comparable to that of sucrose, while its endothermal hydration gives a cooling sensation for use as a sweetener. Xylitol inhibits dental cavities and acute otitis media (1,2) and is widely used in products, including chewing gums, sweets, toothpastes, and cosmetics. It is also used as a sweetener for diabetics because its metabolism is insulin independent. Xylitol is commercially produced by chemical hydrogenation of D-xylose derived from hemicelluloses, such as birch wood xylan and hydrolyzates of biomass material, which includes drawbacks, such as hydrolysis, expensive separation, purification steps, and the requirement of high temperature and pressure conditions (3). Therefore, use of microbial xylanolytic enzymes for the industrial hydrolysis of lignocelluloses in an environmentally friendly way could be a better option. In the last few decades, several reports were published on xylitol production using bacteria (4,5), and fungi (6e10). Among microorganisms, yeasts are the best xylitol producers. Candida tropicalis have been studied extensively for xylitol production as they have the advantage over the metabolically engineered Saccharomyces cerevisiae for being natural D-xylose consumers and maintaining the reduction oxidation balance during xylitol accumulation (11). * Corresponding author. Tel.: þ81 58 293 3171; fax: þ81 58 293 3172. E-mail address: [email protected] (T. Suzuki).
Candida is a very good candidate for xylitol production due to its high xylose uptake rate and xylitol production capacity (10,12). As xylitol has applications and potential uses in the food, dental, and pharmaceutical industries, usage of Candida yeasts, recognized as opportunistic pathogens, is a significant drawback. Moreover Candida yeasts lack the xylanolytic enzyme, which is recognized as one of the major enzymes for lignocellulose hydrolysis. For better commercial xylitol production, lignocelluloses should be used as raw materials as they are the most abundant renewable natural materials of the agricultural wastes and significant quantities of them are presently disposed of by burning. This clearly is a great loss of energy conserved through the process of biosynthesis by the green plants. Therefore, we selected Aspergillus oryzae since it possesses a better enzyme system to hydrolyze lignocelluloses than Candida and also has a long fermentation history as well as being generally recognized as safe. A. oryzae has long been used in Japan for food fermentation, such as the production of shoyu or soy sauce, miso, and sake as well as industrial enzyme production, such as a-amylase and glucoamylase (13,14). A. oryzae can produce D-xylose from xylan using xylanase (15) and convert D-xylose into xylitol using xylose reductase (XR) (EC 1.1.1.21). The accumulation of xylitol is also affected by the downstream activity of xylitol dehydrogenase (XDHA) (EC 1.1.1.9), which converts xylitol to D-xylulose. We have reported another enzyme, L-arabitol dehydrogenase (LADA) (EC 1.1.1.12) (16), which converts arabitol to L-xylulose (17), is highly
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.10.017
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similar (35% amino acid sequence similarity) to XDHA, and is able to convert xylitol to D-xylulose. In the case of Hypocrea jecorina, LAD-1, an ortholog of LADA, also contributes to xylitol conversion. These dehydrogenase enzymes can have a significant effect on production of xylitol by converting xylitol into xylulose. D-Xylulose can then be converted to xylulose 5-phosphate by xylulokinase (EC 2.7.1.17) and fluxed into the pentose phosphate pathway. In this research, we disrupted the xdhA and ladA genes of A. oryzae individually and determined the xylitol productivities of these mutants under laboratory conditions. MATERIALS AND METHODS Fungal strains A. oryzae P5 (18) is a pyrG-deficient strain developed from A. oryzae KBN616, which is a soy sauce koji isolated from a soy brewing plant and was obtained from Bio’c (Toyohashi, Aichi, Japan). A. oryzae P5 does not encode orotidine 50 -phosphate carboxylase, the terminal enzyme in uridine 50 -phosphate biosynthesis. Plasmid pYRG100 (19) was used for pyrG gene amplification, which was used as a selectable marker. Construction of the disruption cassettes for xdhA and ladA Genomic DNA of A. oryzae KBN616 was extracted (20) and used as the template for the amplification of the xdhA (AO090038000631) and ladA (AO090005001078) gene inserts. Using the xdhA-P1/xdhA-P2 and xdhA-P3/xdhA-P4 primer sets (Table 1), 1025 bp and 908 bp regions of xdhA, respectively, were amplified using standard PCR conditions. Primers were designed to include SacI and Eco47III restriction sites. The inserts were purified using a GENECLEANR Turbo kit (Qbiogene, Graffenstaden, France); then, the xdhA-P1 and xdhA-P4 primers were used to amplify a 1903 bp region of xdhA (Fig. 1) by overlap extension PCR. Similarly, using ladA-P1/ladA-P2 and ladA-P3/ladA-P4 primer sets (Table 1), 1079 bp and 1001 bp regions of ladA, respectively, were amplified, including kpnI and Eco47III restriction sites. After purification, these inserts were used as templates and a 2053 bp region of ladA was amplified using ladA-P1 and ladA-P4 primers by overlap extension PCR. The pUC19 vector was digested using SacI and kpnI restriction enzymes, and the xdhA and ladA fragments were ligated into the pUC19 vector using T4 Ligase (Takara, Shiga, Japan) and incubated at 16 C for 2 h to obtain pXDHA (4.6 kbp), as shown in Fig. 1 and pLADA (4.7 kbp). pYRG100 was double digested with HindIII and BamHI (Takara) to obtain the pyrG insert. After purification of the pyrG fragment, blunt ends were created using T4 polymerase (Takara) by incubating at 37 C for 30 min, and the fragment purified using a Qiagen reaction cleanup kit (Venlo, Netherlands). pXDHA and pLADA were digested using Eco47III and then purified. To prevent self-ligation, the digested vectors were dephosphorylated by incubation with calf intestine alkaline phosphatase (Takara) at 50 C for 30 min. The pyrG fragment was ligated into pXDHA and pLADA using T4 DNA ligase (Takara) by incubating at 16 C for 2 h to obtain the pYRGXDHA (Fig. 1) and pYRG-LADA vectors. Takara Ex Taq was used for amplification, using a Takara thermal cycler and the primers listed in Table 1.
TABLE 1. Primers used in this study. Gene/ Vector xdhA
ladA
pyrG
M13
Primer
Length (base)
Sequence
xdhA-P1 xdhA-P2 xdhA-P3 xdhA-P4 xdhA-S1 xdhA-S2 xdhA-PF xdhA-PR ladA-P1 ladA-P2 ladA-P3 ladA-P4 ladA-S1 ladA-S2 ladA-PF ladA-PR pyrG-Fed pyrG-Rev pyrG-S1 pyrG-S2 M13-P4 M13-P5
30 30 30 30 17 17 20 20 28 27 27 27 17 17 20 20 30 30 18 18 24 20
50 .GTCGAGCTCCCTGCTTTAATTTTCCGATTG.30 50 .TAATACTTGGCTAGCGCTCCGTCGTAAGGT.30 50 .ACCTTACGACGGAGCGCTAGCCAAGTATTA.30 50 .CGCGAGCTCCCTAGTCATCTACTAATTTTCTCC..30 50 .CTTCCCCCTTCAACTTT.30 50 .AAACTGCCCCTGACATT.30 50 .AACAAGCGTAGTTCCATGGT.30 50 .GCTGATTCCCTGATCTTGCC.30 50 .TTGGTACCCGCTCCTGGTTCCTATAGTTTCC.30 50 .CTCCATCCTCAGCGCTCATATCACCGA.30 50 .TCGGTGATATGAGCGCTGAGGATGGAG.30 50 .TTGGTACCGGCGGTCATACAAAAGCAG.30 50 .TGCTTGTCCCTCTTGCT.30 50 .TCTCTTGGGTGCTCAGA.30 50 .GCTTCACGTTTTGAGGACAT.30 50 .GTATCCATAGGAATAGGTGG.30 50 .GGAAGCGCTGCTGGAATTGACATTATTATG.30 50 .TTAAGCGCTCAATACCGTACGGGAGATTGT.30 50 .AATAAGCCAGTTTCAACC.30 50 .ACTGCCATAAAAGGAGTA.30 50 .CGCCAGGGTTTTCCCAGTCACGAC.30 50 .GGATAACAATTTCACACAGG.30
GAGCTC, SacI site; AGCGCT, Eco47III site; GGTACC, KpnI site.
FIG. 1. Construction of xdhA-disrupted vector. xdhA fragment (1.9 kbp) was amplified by PCR then ligated into the SacI site of pUC19. The obtained vector, pXDHA, was digested with Eco47III and ligated with the pyrG fragment of pYRG100 to generate the disrupted vector pYRG-XDHA (6.5 kb). The ladA-disrupted vector was also constructed in a similar manner.
Confirmation and sequence analysis of the disrupted constructs After transforming the pYRG-XDHA and pYRG-LADA vectors into Competent Quick DH5a (Toyobo, Osaka, Japan), the cells were cultured on LB agar media containing 2% X-gal and ampicillin at 37 C for 15 h. Transformed colonies appeared white, and a total of 16 colonies were confirmed by direct colony PCR using primers M13-P4 and M13-P5 (Table 1). Plasmids were extracted using a Qiagen Plasmid Mini extraction kit. To confirm the disruption of xdhA and ladA, pYRG-XDHA and pYRG-LADA were digested using SacI and KpnI (Takara) by incubating at 37 C for 2 h. The digests were run on agarose gel and the size of the bands was used to confirm the disruption of the cassettes. Confirmation of construct sequences was done by sequencing, especially of the terminal and linking regions. M13-P4, M13-P5, xdhA-S1, xdhA-S2, pyrG-Fed, pyrGRev, pyrG-S1, and pyrG-S2 primers were used for pYRG-XDHA and M13-P4, M13-P5, ladA-S1, ladA-S2, pyrG-Fed, pyrG-Rev, pyrG-S1, and pyrG-S2 primers (Table 1) were used for pYRG-LADA. For sequencing, BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA) and ABI PRISM 3100 sequence analyzer were used. Transformation of A. oryzae A. oryzae P5 was transformed according to a previously described method (21). P5 cells were cultured at 30 C overnight in Complete Media (20 g/L malt extract, 1 g/L bactopeptone, 20 g/L glucose, 1.5 g/L uridine, and 0.7 g/L uracil). The pH was adjusted to 6.5 with 0.1 M KOH. Five microliters of pYRG-XDHA and pYRG-LADA were used for transformation after being linearized by digestion with SacI and KpnI, respectively. The xdhA and ladA disruptants were selected as prototrophs using Czapek Dox (Difco, NJ, USA) agar media. Then, colonies were picked and spread on CP media (10 g/L polypeptone, 1 g/L NaNO3, 5 g/L KH2PO4, and 1 g/L MgSO4$7H2O, pH 6.0) containing 20 g/L of different carbohydrate sources, including D-glucose, D-xylose, L-arabinose, xylitol, and oat spelt xylan, as required. After 5 days incubation at 30 C, the diameter of the colonies was determined to evaluate substrate utilization ability. After extracting the genomic DNA (20) from the disrupted mutants, the genomic structures of the target genes were confirmed by PCR. The primer sets xdhA-PF/ xdhA-PR, and xdhA-P1/xdhA-PR (Table 1) were used for the xdhA-disrupted mutants (Fig. 2A and B), while ladA-PF/ladA-PR and ladA-P1/ladA-PR (Table 1)
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were used for the ladA-disrupted mutants (Fig. 3A and B). Considering the results from PCR and substrate utilization, candidate disruptants for xdhA and ladA were selected for further experiments. Culture conditions for fermentation experiments The stock strains were precultured in 100 mL preculture media (10 g/L D-glucose, 10 g/L yeast extract, 5 g/L KH2PO4, 1 g/L NaNO3, and 0.5 g/L MgSO4$7H2O) at 30 C and 150 rpm for 48 h using a 500 mL Erlenmeyer flask. Fungal mycelia were collected from the media and washed with 0.1 M potassium phosphate buffer (pH 6.0), and 5.0 g wet weight of mycelia were used to inoculate the fermentation media. The xylitol fermentation experiments from D-xylose and oat spelt xylan were performed in 500 mL Erlenmeyer flasks with 100 mL of xylitol fermentation media (50 g/L D-xylose, 10 g/L yeast extract, 5 g/L KH2PO4, 1 g/L NaNO3, and 0.5 g/L MgSO4$7H2O) at 30 C and 150 rpm. Oat spelt xylan (50 g/L) was used instead of D-xylose to determine xylitol production from oat spelt xylan. Assay of XR and XDH The XDH and XR activities were determined spectrophotometrically using Ultrospec 2100 pro (Amersham Bioscience, NJ, USA) by monitoring the change in A340 upon NAD reduction or NADPH oxidation at 25 C, respectively (22). Cells grown on xylitol fermentation medium were harvested by filtration. After the cells were washed with 100 mM potassium phosphate buffer (pH 7.0), 1.0 g of cells was collected, mixed with 1.0 g of sea sand (Nacalai Tesque, Kyoto, Japan) and crushed using a mortar and pastel with liquid nitrogen cooling. After adding 5.0 mL of 100 mM potassium phosphate buffer (pH 7.0), the cell debris was separated by centrifugation at 13,000 rpm for 15 min and the supernatant was then used to measure enzyme activity. The XDH assay mixture contained 1 M potassium phosphate buffer (pH 7.5), 0.1 M 2-merceptoethanol, 0.5 M xylitol, 3.4 mM NADþ, deionized water, and enzyme solution. The XR assay mixture contained 1 M potassium phosphate buffer (pH 7.0), 0.1 M 2merceptoethanol, 0.5 M D-xylose, 3.4 mM NADPH, deionized water and enzyme solution. The activity was expressed in units, where 1 U corresponded to the conversion of 1 mmol of NAD or NADPH per minute. Each measurement was
FIG. 3. PCR confirmation of the specific integration of pyrG into ladA-disrupted mutants. (A) Primer set ladA-PF and ladA-PR was used for PCR. Lane 1: l-EcoT14 I-digested marker; lanes 2e8: ladA disruptants; lane 9: KBN616. (B) Primer set ladA-P1 and ladA-PR was used for PCR. Lane 1: l-EcoT14 I-digested marker; lanes 2e8: ladA disruptants; lane 9: KBN616.
repeated twice. The concentration of protein in the enzyme solution was measured using a Qubit protein assay system (Invitrogen). Sugar analysis The concentrations of D-glucose/D-mannose, D-xylose, xylitol, L-arabitol, and D-mannitol were analyzed by high-performance liquid chromatography (Shimadzu Corporation, Kyoto, Japan) using a Shimadzu RID10A detector and Shodex SUGAR SZ5532 column. Pure water was used as the mobile phase with a flow rate of 1.0 mL/min and column temperature of 60 C. L-arabinose,
RESULTS
FIG. 2. PCR confirmation of the specific integration of pyrG into the xdhA-disrupted mutants. (A) Primer set xdhA-PF and xdhA-PR was used for PCR. Lane 1: l-EcoT14 I-digested marker; lanes 2e6: xdhA disruptants; lane 7: KBN616. (B) Primer set xdhAP1 and xdhA-PR was used for PCR. Lane 1: l-EcoT14 I-digested marker; lanes 2e6: xdhA disruptants; lane 7: KBN616.
Construction of xdhA- and ladA- disrupted mutants A. oryzae P5 was transformed to uracil prototrophy with SacI- and KpnIdigested pYRG-XDHA and pYRG-LADA, respectively. Transformants were selected as prototrophs by growing on Czapek Dox agar media without uridine and uracil. A total of five xdhA and seven ladA transformants were selected initially by Colony Direct PCR. Genomic DNA was extracted and PCR was performed to confirm the disruption for each of these transformants. After electrophoresis on a 1.5% agarose gel, the predicted 4.5 kb (Fig. 2A) and 4.2 kb (Fig. 2B) bands, appeared for the two primer sets for the xdhA mutants xdhA2-1, 3-1, 4-4, 5-4, and 6-6, whereas A. oryzae KBN616 gave a band around 2.0 kb (Fig. 2A,B), indicating the successful disruption of the xdhA gene. For the ladA mutants, bands around 4.5 kb (Fig. 3A) and 4.3 kb (Fig. 3B) appeared for the two sets of primers for ladA2-1
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and 3-8, whereas the band for KBN616 was around 2.5 kb (Fig. 3A, B), indicating ladA disruption in the transformants. To assess the carbohydrate utilization ability, colony diameters of the mutants were measured on CP agar media containing different carbohydrate sources (Figs. 4 and 5). Observing the diameters of the colonies for different xdhA mutants, we found that all disruptants displayed considerably slower growth on D-xylose (Fig. 4A, B) and xylitol but were still able to grow on these carbon sources, which was understandable due to the xdhA disruption. In presence of other carbohydrates, all the disruptants displayed growth similar to KBN616 (Fig. 4B). Among the disruptants, xdhA21 was selected arbitrarily for a xylitol production experiment. All ladA transformants displayed considerable substrate utilization ability, much like the wild-type strain (Fig. 5A, B). Only ladA2-1 seemed unable to utilize L-arabinose (Fig. 5A), whereas ladA3-8 seemed to utilize L-arabinose like wild type. Though ladA3-8 gave a similar size PCR band as ladA2-1, we excluded ladA3-8 as a potential ladA-disrupted mutant due to its substrate utilization ability and selected ladA2-1 to carry on with xylitol production experiments. Xylitol production from D-xylose Mutants xdhA2-1 and ladA2-1 were tested for xylitol production from D-xylose-containing xylitol production media (Fig. 6). Consumption of D-xylose for xdhA2-1 and ladA2-1 was similar to that of KBN616. In terms of xylitol production, xdhA2-1 displayed the highest xylitol productivity of 0.25 g/L h at a concentration of 16.2 g/L xylitol (Fig. 6B), whereas the xylitol productivity of ladA2-1 decreased to 0.048 g/L h at a concentration of 4.2 g/L xylitol (Fig. 6C) in comparison to KBN616 (Fig. 6A).
FIG. 5. Colony diameters on different carbohydrate sources for ladA disruptants. (A) Growth of the parental strain and the ladA-disrupted mutants on CP medium with L-arabinose as the sole carbon and energy source. (B) The growth of colonies in the presence of different substrates was determined in cm. Gray bars, ladA2-1; open bars, ladA3-8; and closed bars, KBN616.
XR and XDH specific activity Specific enzyme activities for XR and XDH of KBN616 and xdhA2-1, grown on xylitol fermentation media were analyzed (Fig. 7A, B). The results indicate that KBN616 and xdhA2-1 do not have significant variation in specific XR activities (Fig. 7A). On the other hand, xdhA2-1 showed decreased XDH activity (Fig. 7B) compared to the wild-type strain. Xylitol production from oat spelt xylan xdhA-disrupted mutant xdhA2-1 exhibited the highest xylitol productivity from D-xylose and was selected for further production of xylitol from oat spelt xylan. Fig. 8A and B show the data for different carbohydrates when oat spelt xylan was used for xylitol fermentation using KBN616 and xdhA2-1. Mutant xdhA2-1 produced a maximum of 13.1 g/L D-xylose and 10.2 g/L xylitol at a yield of 0.204 g/g xylan (Fig. 8B), whereas KBN616 produced only 2.6 g/L D-xylose and 0.42 g/L xylitol. Trace amounts of D-glucose/D-mannose, L-arabitol, and D-mannitol were also produced by xdhA2-1. DISCUSSION
FIG. 4. Colony diameters on different carbohydrate sources for xdhA disruptants. (A) Growth of the parental strain and the xdhA-disrupted mutants on CP medium with D-xylose as the sole carbon and energy source. (B) The growth of colonies in the presence of different substrates was determined in cm. Open bars, xdhA2-1; filled square bars, xdhA3-1; gray bars, xdhA4-4; dotted bars, xdhA5-4; open square bars, xdhA6-6; and closed bars, KBN616.
Candida tropicalis has been reported to have the highest xylitol productivity (12 g/L h) using a cell recycle system (23). Xylose assimilating yeasts like C. tropicalis have an enzyme regime to metabolize xylitol from D-xylose, but most of them do not have the enzymes required for xylan degradation, such as xylanases and xylosidases, except for some special species (24e26). We selected A. oryzae because it has a suitable enzyme regime required for xylitol production from lignocellulosic biomass. It has a very long history of food fermentation in Japan and other Asian countries and is generally recognized as safe (GRAS). After disrupting the xdhA and ladA genes of A. oryzae, we found that xdhA-disrupted mutant xdhA2-1 had the highest xylitol productivity. Volumetric xylitol productivity increased 4 and 10 fold compared to the parental strain KBN616 when D-xylose and oat spelt xylan, respectively, were used as the substrate. The ladA-disrupted mutant, ladA2-1, showed lower xylitol productivity than KBN616. lad1-encoded L-arabitol-4-dehydrogenase in H. jecorina
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FIG. 7. Assay of XR and XDH activities. (A) Assay of XDH activities in KBN616 and xdhA2-1. Closed bars, KBN616; open bars, xdhA2-1. (B) Assay of XR activities in KBN616 and xdhA2-1. Closed bars, KBN616; open bars, xdhA2-1.
FIG. 6. Production of xylitol from D-xylose. (A) Consumption of D-xylose and production of xylitol by KBN616. Closed circles, D-xylose; closed triangles, xylitol. (B) Consumption of D-xylose and production of xylitol by xdhA2-1. Closed circles, D-xylose; closed triangles, xylitol. (C) Consumption of D-xylose and production of xylitol by ladA2-1. Circles, D-xylose; triangles, xylitol.
can compensate partially for the metabolism of D-xylose in mutants with a loss of xdh1 function (17). Since the xdhA and ladA genes of A. oryzae were disrupted separately in this study, ladA-disrupted mutant did not show any significant impact on D-xylose metabolism when xdhA was intact (Fig. 5B), and hence the xylitol productivity also did not increase (Fig. 6C). To evaluate the effect of ladA disruption on xylitol accumulation, a double disrupted mutant lacking both XDHA and LADA might be necessary, which we will report in our next paper. Therefore, we excluded ladA-disrupted mutant ladA2-1 as a potential xylitol producer and did not measure its specific XDH and XR activities. Xylose reductase (XR) and xylitol dehydrogenase (XDH) constitute the initial metabolic pathway for xylose in fungi. The overall efficiency of xylose metabolism is connected through a complex regulatory network with the ability of XR and XDH to provide a high flux of carbon through the initial pathway (16). Therefore, XDHA is an important target for the metabolic engineering of A. oryzae
toward the utilization of xylose in a manner that meets the requirements of the biotech industry for increased xylitol production. XDHA is encoded by xdhA in A. oryzae, and the function of XDHA is thought to be compensated for by LADA (17), which has 35% similarity with XDHA (16). LADA is encoded by ladA in A. oryzae and is up regulated in the presence of D-xylose even though it does not contribute to its catabolism (27,28). The main yield-limiting factor for xylitol production is its consumption for cell growth and maintenance. Therefore, if the metabolic step from xylitol to D-xylulose could be blocked by disruption of the corresponding xdhA and ladA genes, and if cosubstrates, such as glucose, galactose, or glycerol, were supplied for cell growth, the yield of xylitol should reach a theoretical level of 100% (29). Antisense inhibition of XDH in Trichoderma reesei reduced XDH activity to 48% and enhanced xylitol productivity (30). Similar studies were also done on C. tropicalis, which has higher xylitol productivity and a xylitol yield of 98% was achieved using glycerol as a cosubstrate (31). This report is an attempt to apply gene disruption techniques to analyze pentose metabolism in A. oryzae. We constructed A. oryzae mutant by disrupting either the xdhA or ladA gene in order to diminish XDH activity. Disruption of xdhA in the mutant xdhA2-1 decreased XDH activity compared to the wild-type strain KBN616 (Fig. 7B) however, we did not measure the XDH activity of the ladAdisrupted mutant. xdhA2-1 still showed some XDH activity, which could indicate the presence of other homologous genes present in A. oryzae (32). For the same reason, xylitol produced by xdhA2-1 was gradually consumed after 168 h (Fig. 6B) though the xdhA2-1 mutant accumulated 4-times more xylitol without the use of a cosubstrate or controlled aeration under batch fermentation (Fig. 6B). Xylitol production by xdhA2-1 had a yield of 33% from D-xylose. Though we did not measure the fungal mycelia
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FIG. 8. Production of xylitol from oat spelt xylan. (A) Oat spelt xylan was used as a substrate to measure the metabolites by KBN616. Squares, D-xylose; circles, xylitol; triangles, D-glucose/D-mannose; diamonds, L-arabitol; asterisks, D-mannitol; crosses, L-arabinose. (B) Oat spelt xylan was used as a substrate to measure the metabolites by xdhA2-1. Squares, D-xylose; circles, xylitol; triangles, D-glucose/D-mannose; diamonds, L-arabitol; asterisks, D-mannitol; crosses, L-arabinose.
concentrations for different time intervals, the amount of mycelium inoculated from preculture media into xylitol fermentation media for each flask culture experiment was constant around 5.0 g wet weight of the respective mycelia. For preculture conditions, we used C-40 (Type C) silicone stoppers where KLa (h1) was 145 14 and for xylitol fermentation experiments, we used T-32 (Type T) silicone stoppers, where KLa (h1) was 50 12 at 150 rpm. The sulfite oxidation method was used to measure the oxygen transfer rate for the flask experiments (33) and 150 rpm was kept constant for aeration of the flask experiments. We previously reported an improvement in the method of xylitol production using C. tropicalis (34). Fine tuning of aeration, glucose addition as a cosubstrate, and immobilization of the cells proved efficient for increasing xylitol production (35). Using similar techniques, it would not be difficult to increase the xylitol productivity of the xdhA-disrupted mutant. This is the first attempt to apply metabolic engineering to the xylose metabolic pathway in A. oryzae to enhance xylitol yield and productivity. This strategy for strain development will contribute greatly to other attempts for improving the metabolite production of microorganisms. We understand that our xylitol productivity was not as high as in C. tropicalis (30). The reason could be that the A. oryzae genome is one third larger than that of two relevant Aspergillus species (36)
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