International Journal of Biological Macromolecules 117 (2018) 727–734

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

α-Amylase, glucoamylase and determine molecular weight of pullulan produced by Aureobasidium melanogenum P16

Nan-Nan Liu a, Zhe Chi a,c,⁎, Guang-Lei Liu a,c, Tie-Jun Chen a, Hong Jiang a,c, Zhong Hu b, Zhen-Ming Chi a,c a College of Marine Life Sciences, Ocean university of China, Yushan Road, No. 5, Qingdao, China b Department of Biology, Shantou University, Shantou 515063, China c Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, 266003 Qingdao, China article info abstract

Article history: A high molecular weight (Mw) pullulan has many potential applications in various fields. α-, Received 18 September 2017 glucoamylase and were thought to play an important role in high Mw pullulan biosynthesis. How- Received in revised form 29 May 2018 ever, there is no genetic evidence for this role. In this study, the genes encoding α-amylase, glucoamylase and Accepted 31 May 2018 pullulanase were cloned from Aureobasidium melanogenum P16, a high pullulan producing yeast and character- Available online 01 June 2018 ized. The proteins deduced from the cloned α-amylase gene, the glucoamylase gene and the isopullulanase gene, Keywords: not a pullululanse gene had their corresponding conserved amino acid sequences, respectively. After the single High Mw pullulan gene of them was deleted, the Mw of the pullulan produced by the single disruptants greatly increased and Aureobasidium spp. the pullulan concentration decreased. It was found that the triple mutant DT15 grown at the flask level could pro- 6 α-Amylase, glucoamylase duce 46.2 g/L of pullulan with a Mw of 3.02 × 10 Da and grown in the 10-L fermentor could yield 58.14 g/L of Isopullulanase pullulan with the same Mw while its wild type strain P16 produced 65.5 ± 3.5 g/L of pullulan with a Mw of 0.35 × 106 Da. After the genes were complemented, pullulan production, Mw of the produced pullulan and others were restored. All the results demonstrated that the α-amylase, glucoamylase and isopullulanase indeed could determine the Mw of the produced pullulan. © 2018 Published by Elsevier B.V.

1. Introduction the drug capsules made of pullulan have many advantages and useful properties and are warmly welcome by the vegetarians and Muslims, Pullulan is a linear and un-branched exo-polysaccharide which diabetics, and patients with restricted diet [3]. If pullulan is used as mainly consists of maltotriose units attached by α (1 → 6) glycosidic the material for making the drug capsule and as a component of linkage. The main producers are different strains of Aureobasdium the cosmetic, it is required that the molecular weight (Mw) of the spp. [1]. Pullulan is a water soluble, biodegradable, non-toxic, non- pullulan should be more than 2.0 × 106 Da due to its high viscosity, immunogenic, non-carcinogenic and non-mutagenic biopolymer, a very strong strength and high stability [4]. However, so far, the ap- can form thin films which are oil resistant, transparent and oxygen plication of pullulan in various fields was actually limited due to both impermeable and has adhesive property during drying so that it low production yield and low Mw of the pullulan. For example, the can be widely used in food, pharmaceutical and cosmetic industries commercial pullulans produced by Hayashibara Company, the prin- [2]. Each repeating unit of pullulan consists of nine hydroxyl groups ciple producer of commercial pullulan in the world include food for the chemical substitution reactions. After chemical modification, grade (designated as PF) and deionized (PI) products with mean pullulan can be used in tissue engineering and grafting, a carrier for Mw of 1.0 × 105 or 2.0 × 105 Da [5]. drug delivery and gene delivery, medical imaging and so on [2]. Therefore, it is very important how to yield the pullulan with high One of the most important applications of pullulan should be as a Mw. In our previous study [4], changes in a culture medium can increase major material of the drug capsules and cosmetics. To date, most of Mw of the produced pullulan by Aureobasidium melanogenum P16, a the drug capsules have been made of gelatin which has many draw- high pullulan producer. Yu et al. [6] and Wang et al. [7] also have the backs such as contamination of the animal pathogens since it is ob- similar findings. tained from several animal body parts. It has been reported that During cultivation and fermentation, it has been well docu- mented that the Mw of pullulan greatly decreases in the late station- ary growth phase due to the presence of isopullulanase, -amylase ⁎ Corresponding authors at: College of Marine Life Sciences, Ocean University of China, α Yushan Road, No. 5, Qingdao, China. and glucoamylase secreted into the medium (Fig. 1)[8, 9]. In our pre- E-mail addresses: [email protected] (Z. Chi), [email protected] (Z.-M. Chi). vious study [4], it was found that after the compositional change of a

https://doi.org/10.1016/j.ijbiomac.2018.05.235 0141-8130/© 2018 Published by Elsevier B.V.

Reproduced fom International Journal of Biological Macromolecules 117: 727-734 (2018). Zhen-Ming Chi: Participant of the 18th UM, 1990-1991.

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Fig. 1. Pullulan biosynthesis and degradation.

pullulan production medium, a Mw of the pullulan produced by Mw of the produced pullulan could be determined by the three A. melanogenum P16 increased, but pullulan titer decreased. The in- . creased Mw of the pullulan was due to the decreased activities of α-amylase, glucoamylase and pullulanase. It has been reported that 2. Materials and methods pullulan also contains as a minor structural feature, a low percentage of α-1,6-linked maltotetraose subunits which are substrates for α- 2.1. Microbial strains, media and plasmids amylase [9]. This was also proven by Prasongsuk et al. [10] who added the α-amylase inhibitor acarbose to the culture medium, One isolate of A. melanogenum P16, obtained from a mangrove showing that pullulan of slightly higher Mw was obtained from late ecosystem (at DongZaiGou, Haikou, Hainan Province, N19°53′ cultures. It appeared that the responsible for pullulan degra- E110°19) was used in this study. The strain was known to be a high dation during the cultivation was also likely glucoamylase B [11]. pullulan producing yeast [13]. It was cultivated in a YPD medium Furthermore, A. pullulans has been reported to produce other en- which contained 20.0 g/L of , 20.0 g/L of polypeptone, zymes that might attack pullulan, including glucoamylase [10] and 10.0 g/L of yeast extract for 48 h. The competent cells of Escherishia pullulanase [12]. However, so far, there have been no genetic evi- coli DH5α and the cloning vector of T-Vector pMD™19 (Simple) dences to show that the pullulanase, α-amylase and glucoamylase were purchased from TaKaRa Co., Ltd. (Japan). The plasmids pFL4a are implicated in high Mw pullulan biosynthesis. Therefore, further carrying the hypromycin B resistance gene (the HPT gene) and study of the relationship between pullulanase, α-amylase and pPWN302 carrying the nourseothricin resistance gene (the NAT glucoamylase activities and the Mw of pullulan is needed, including gene) constructed in this laboratory were used for disruption of the an analysis of α-amylase gene, glucoamylase gene and pullulanase target genes in A. melanogenum P16 [14]. A plasmid pNATX13 and a gene. Furthermore, a more practical approach for yielding a higher plasmid pNAT13-NS bearing the nourseothricin resistance gene Mw pullulan might be to use α-amylase, glucoamylase and pullulanase (the NAT gene) and a plasmid pAPX13-NS carrying the hypromycin negative mutants (Fig. 1). B resistance gene (the HPT gene) constructed in this laboratory The aims of the study were to clone, characterize, delete and were used for complementation of the target genes in different complement α-amylase, glucoamylase and isopullulanase genes in disruptants obtained in this study [14]. A fermentation medium for A. melanogenum P16 in order to confirm the hypothesis that the pullulan production was composed of 120.0 g/L glucose, 3.0 g/L

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yeast extract, 5.0 g/L K2HPO4, 0.2 g/L MgSO4·7H2O, 1.0 g/L NaCl, and MluI and the digested DNA fragments were ligated into the plasmid 0.6 g/L (NH4)2SO4 [13]. pNATX13, pNATX13-NS and pPAPX13-NS using a T4 DNA , forming pNATX13-AMY, pPAPX13-IPU and pNATX13-GLY (Supplemen- 2.2. Cloning and characterization of α-amylase gene, glucoamylase gene tary files 5–7). and iospullulanase gene 2.4. Transformation and isolation of various disruptants and transformants The genomic DNAs in A. melanogenum P16 were isolated using TIANamp Yeast Genomic DNA Kits (TIANGEN BIOTECH (Beijing) The linear DNA fragments (3′-arm-Tef-HPT-PolyA-5′-arm) and (3′- CO., LTD.) as described by Chi et al. [15]. An α-amylase gene (acces- arm-PGK-NAT-PolyA-5′-arm) were PCR amplified from the plasmid sion number: KU886204), an isopullulanase gene (accession num- pFL4a-Δ-α-amylase, the plasmid pFL4a-Δ-glucoamylase and ber: KX641590) and a glucoamylase gene (accession number: pPWN302-Δpullulanase (Supplementary files 2–4) using the primers KX578909) in the yeast were PCR amplified from its genomic 5F1/3R1, 5F2/3R2 and 5F3/3R3 (Supplementary file 1). The linear DNA DNA using the primers 3F/5R (Supplementary file 1) designed fragments amplified were transformed into the competent cells of based on an α-amylase gene (HM590632.1) in A. pullulans NRRL A. melanogenum P16 as described by Chi et al. [15], respectively. Mean- Y-12974 [9], the primers 3F1/5R1 (Supplementary file 1) designed while, the pNATX13-AMY, pNATX13-GLY and pPAPX13-IPU con- based on an isopullulanase gene (accession number: D85240) in structed above (Supplementary files 5–7) were digested with the A. niger ATCC 9642 [16] and the primers 3F2/5R2 (Supplementary enzymes SmaI and EcoRI. The linear DNA fragments 26S rDNA-PGK- file 1) designed based on a glucoamylase gene (accession number: NAT-PolyA-α-amylase gene-TEF-18S rDNA, 26S rDNA-Tef-HPT-PolyA- XM_001390493) in Aspergillus niger CBS 513.88. The reaction sys- isopullulanase gene -TEF-18S rDNA and 26S rDNA-PGK-NAT-PolyA- tem and the conditions for the PCR amplification were as described glucoamylase gene-TEF-18S rDNA were transformed into the compe- by Chi et al. [15]. In order to characterize the cloned genes, BLAST tent cells of the corresponding disruptants. The transformants were and ORF Finder programs (http://www.ncbi.nlm.nih.gov/Blastat) grown on onto a two- layer agar plate, with the bottom layer consisting from the National Center for Biotechnology Information (NCBI) of 15.0 g/L agar in 12.0 mL of the HCS (Hol-liday complete medium con- were used for the nucleotide sequence analysis, deduction of the taining 1.0 M sorbitol) containing 50.0 μg/mL of hygromycin B or amino acid sequence and database searches. Multiple sequence nourseothricin, and the top layer consisting of 15 g/L agar in 12.0 mL alignments of DNA and amino acid were carried out using the pro- of HCS. All the transformed cells were then kept at 28 °C for 3–4 days. grams of DNAMAN 6.0 (http://www.lynnon.com) and Clustal × 1.8 The putative disruptants including the strain DA16 in which the α- [17]. Prediction of ORFs and promoters and Mw, signal peptide, N- amylase gene was removed, the strain DPA2 in which the isopullulanase glycosylation sites and conserved domains of the deduced proteins gene was knocked-out, the strain DG21 in which the glucoamylase gene from the cloned genes was carried out using the softwares at ORF was deleted, the transformants, the strain EA20 in which the α-amylse Finder in the NCBI platform [18], Neural Network Promoter Predic- gene was complemented, the strain EG15 in which the glucoamylase tion at Berkeley Drosophila Genome Project [19], NetNglyc server gene was complemented and the strain EP4 in which the isopullulanase [20], SignalP 4.1 Server [21] and NCBI [22]. gene was complemented were verified by cultivation on a HCS agar (15.0 g/L agar in HCS) containing 100.0 μg/mL of hygromycin B or 2.3. Construction of the knock-out vectors and the plasmids for complemen- nourseothricin. The putative disruptants and transformants were aero- tation of the target genes in different disruptants bically cultivated in the pullulan production medium at 28 °C for 4 days and the titers, Mw of the produced pullulans and cell dry weight by dif- To delete the α-amylase, isopullulanase and glucoamylase genes, ferent putative disruptants, transformants and their wild type strain the 3′-arms and the 5′-arms were PCR amplified from the genomic P16 were determined as described below. DNA of A. melanogenum P16 as the template using the primers 3F1/ At the same time, after both the two linear DNA fragments 3′-arm- 3R2 and 5F1/5R1 (Supplementary file 1), the primers 3F2/3R2 and 5F2 Tef-HPT-PolyA-5′-arm PCR amplified from the plasmid pFL4a-Δ-α-am- and 5R2 and the primers 3F3/3R3 and 5F3/5R3 (Supplementary file ylase and the plasmid pFL4a-Δ-glucoamylase were simultaneously 1), respectively. The reaction system and the conditions for the PCR am- transformed into the disruptant DPA2, a strain DT15 in which all the plification were described by Chi et al. [15]. The amplified 3′-arms and α-amylase gene, glucoamylase gene and iospullulanase gene were to- 5′-arms were ligated into T-Vector pMD™19 (Simple), respectively. tally removed was obtained. The titers, Mw of the produced pullulans The recombinant plasmids were transformed into the competent cells and cell dry weight by the triple disruptant DT15 were also determined of E. coli DH5α and the extracted recombinant plasmids from the as described below. transformants were digested with BamHI/EcoRI and SphI/SalI. The digested 3′-arms and 5′-arms were ligated into the knock-out vector 2.5. Pullulan purification and determination of mw of the purified pullulan pFL4a carrying the hypromycin B resistance gene (the HPT gene), the knock-out vector pPWN302 carrying the nourseothricin resistance The wild type strain P16, the single disruptants DA16, DPA2, DG21 gene (the NAT gene) and the knock-out vector pFL4a carrying the and triple mutant DT15, the transformants EA20, EG15 and EP4 were hypromycin B resistance gene (the HPT gene), respectively, forming aerobically cultivated in the pullulan production medium at 28 °C and the plasmid pFL4a-Δ-α-amylase (5209 bp) (the Supplementary file 2), 180 rpm for 5 days and each culture was heated at 100 °C for 10 min the plasmid pPWN302-Δpullulanase (4557 bp) (the Supplementary to inactivate the enzymes and precipitate the proteins in the cultures. file 3) and the plasmid pFL4A-Δglucoamylase (5206 bp) (the Supple- The heated cultures were centrifuged at 6000 ×g for 20 min and the mentary file 4). pullulan in the supernatants was purified as described by Ma et al. In order to carry out the α-amylase, glucoamylase and [13] and Lee et al. [23]. The solution with 0.1 mg/L of the purified isopullulanase gene complementation, the genes encoding the α- pullulan was made with deionized water and the Mw of the purified amylase, iospullulanase and glucoamylase were PCR amplified from pullulan was also proved using a Gel Permeation Chromatography cDNAs of A. melanogenum P16 using the primers A1/A2, I1/I2 and G1/ (GPC) [4]. The solutions with 5.0 mg/L of the purified pullulan and stan- G2 (Supplementary file 1). The reaction system and the conditions for dard pullulans brought from Sigma (USA) were prepared with deion- the PCR amplification were described by Chi et al. [15]. The PCR prod- ized water and the solutions were filtrated using 0.22 μm membrane. ucts were ligated into the T-Vector pMD™19 (Simple). The recombinant The Mw of the pullulans in the filtrates were measured using the GPC plasmids were transformed into E. coli DH5α. The extracted and purified (Wyatt DAWN HELEOS type II and a Multi-Angle static Light Scattering recombinant plasmids were digested with SpeI/SacI, SpeI/SacI and SpeI/ detector, USA) with a Styagel R HMW 6E GPC column. The detection

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wavelength was 658.0 nm, mobile phase was the pure water and flow 2.10. Determination of reducing sugar and total sugar in the fermented rate was 0.50 mL/min. medium

2.6. HILIC LC ESI-LTQ-Orbitrap-FT-MS analysis of the fructooligosaccharides Reducing sugar in the fermented media was measured by using the in the fermentation cultures Nelson–Somogyi method [28]. Residual total sugar was assayed as re- duction of sugar after of the fermented media (10 mL of the After all the pullulan and yeast cells in the fermentation cultures fermented media, 10 mL of 25% HCl and 30 mL of distilled water were were removed as described above, the supernatants obtained were mixed and heated in a boiling water bath for 3 h) [27]. taken to dryness on the rotary evaporator at 35–40 °C and reduced pres- sure. The residue was immediately dissolved in 2.0 mL of deionized 3. Results water. The fructooligosaccharides in the solutions were analyzed by using a liquid chromatography (HILIC LC ESI-LTQ-Orbitrap-FT-MS) [4, 3.1. Cloning and characterization of the α-amylase gene, the glucoamylase 24]. gene and the isopullulanase gene involved in pullulan degradation

It was found that the promoter of the cloned AMY gene had one con- 2.7. Assay of α-amylase, glucoamyalse and isopullulanase activities served sequence 5′-SYGGRG-3′ which was the of a Mig1, a global regulator in yeast cells, suggesting that expression of the gene The activities of α-amylase and glucoamyalse in the yeast cultures was repressed at a high concentration of glucose (Table 1)[14]. The were determined according to the methods described by Liu et al. cloned gene and the deduced protein had 93.10% and 98.08% similarities [25]. An isopullulanase activity was examined based on the methods de- to the α-amylase gene and their encoding protein in Aureobasidium scribed by Ma et al. [13]. One α-amylase activity unit, one glucoamylase pullulans NRRL Y-12974 (accession number: HM590632.1), respectively activity unit and one isopullulanase activity unit were defined as the (data not shown). Furthermore, the deduced protein had two aspartates amount of enzyme that hydrolyzes 1.0 mg of soluble starch per min; on the strands β-4 and β-7 (Table 1) and three conserced domains: an the amount of enzyme that produces 1.0 μM of reducing sugar from sol- α-amylase catalytic domain, a starch-binding domain and a domain uble starch per min and the amount of enzyme that produces 1.0 μM of which function was unknown (data not shown), indicating the cloned reducing sugar from pullulan per min under the assay conditions used gene indeed belonged to the α-amylase gene. The protein deduced in this study, respectively. The protein concentrations in the cultures from the cloned glucoamylase gene (GLY gene) had a conserved se- were measured using the methods described by Bradford, and the bo- quence KFEVDLTPFTGAWGRPQRD (Table 1), a catalytic domain and vine serum albumin served as the standard [26]. the starch binding domain connected by an O-glycosidic linker (data not shown). In contrast, after a bioinformatics analysis, the cloned IPU 2.8. Analysis of transcriptional levels of the genes related to pullulan biosyn- gene did encode an isopullulanase, rather than a pullululanase. The de- thesis, α-amylase, glucoamylase and pullulanase duced isopullulanase had the conserved sequences YKDVFYH, ESFM and TIE (Table 1) and glycosyl family 49 N-terminal Ig-like do- As mentioned above, the wild type strain P16, the single disruptants main and glycosyl hydrolase family 49 (data not shown). DA16, DPA2, DG21 and triple mutant DT15, the transformants EA20, EG15 and EP4 were aerobically cultivated in the pullulan production 3.2. Knock-out of each gene and all of the α-amylase gene, the glucoamylase medium at 28 °C and 180 rpm for 5 days. The cultures were centrifuged gene and the isopullulanase gene at 6000 ×g and 2 °C for 10 min and the pellets obtained were used as the samples for total RNA isolation. The total RNA was extracted by using a It was found that after removal of each α-amylase, or glucoamylase RNAprep pure Tissue Kit (TIANGEN, China). A reverse transcription was gene or isopullulanase gene from A. melanogenum P16, the pullulan titer conducted using a PrimeScript RT reagent Kit (TaKaRa, Japan) according and the Mw of the produced pullulan by the single disruptant DA16 to the manufacturer's protocol. The fluorescent real-time RT-PCR assay (removal of only the α-amylase gene), the single disruptant DG21 was conducted according to the methods described by Liu et al. [25]. (removal of only the glucoamylase gene) and the single disruptant All the primers used for the fluorescent real-time PCR were designed ac- DPA2 (removal of only the isopullulanase gene) were 48.5 ± 2.5 g/L, cording to the corresponding gene sequences of A. melanogenum P16 58.2 ± 3.2 g/L, 46.4 ± 1.8 g/L, 0.75 ± 0.06 × 106 Da, 0.57 ± 0.05 × [14] (Supplementary file 1). The relative transcriptional levels of differ- ent genes were calculated using the formula RATE = 2−ΔΔCt. The sam- ple data obtained from the real-time PCR analysis were subjected to a Table 1 Characteristics of the α-amylase gene, the glucoamylase gene and the isopullulanase gene one-way analysis of variance (ANOVA) [25]. P values were calculated involved in pullulan degradation. by a Student's t-test (n = 3). P values less than 0.05 were considered statistically significant. A statistical analysis was performed using a AMY GLY IPU SPSS11.5 for Windows (SPSS Inc., Chicago, USA). Accession KU886204 KX578909 KX641590 number Gene size 2245 bp 2125 bp 1941 bp 2.9. High Mw pullulan production using a 10-L fermenter TATA box 1 1 1 CAAT box 1 1 1 The triple mutant DT15 obtained above was aerobically grown in the The conserved 5′-SYGGRG-3′ No No DNA medium for growth of the seed culture at 28 °C for two days. Seven hun- sequence dred milliliters of the seed cultures were transferred into 6.3 L of the The conserved Two aspartates on the KFEVDLTPFTGAWGRPQRD YKDVFYH, pullulan production medium in the 10-L fermenter. The fermentation aa sequence strands β-4 and β-7 ESFM,TIE, was carried out for 120 h as described by Ma et al. [13]. During the fer- Intron 7 5 2 mentation, only 30.0 mL of the culture was harvested in the interval of Deduced aa 625aa 617aa 610aa Signal peptide 23 bp 16 bp 19 bp 12 h and was centrifuged at 5000 ×g and 4 °C for 10 min, and the Mw of deduced 67,758.50 Da 65,587.70 Da 66,863.71 pullulan titer, Mw of the pullulan, reducing sugar and total sugar in protein Da the supernatant obtained were determined as described above and PI 5.51 5.86 4.17 below. The cell dry weight in 10.0 mL of the culture during the 10-L fer- N-glycosylation 14 7 site mentation was also measured as described by Chi et al. [27].

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106 Da and 1.36 ± 0.21 × 106 Da, respectively whereas the pullulan titer to be used to make the drug capsule and as a component of the cosmetic and the Mw of the produced pullulan by their wild type strain P16 were [4]. Therefore, high Mw pullulan production by the triple mutant DT15 65.5 ± 3.5 g/L and 0.35 ± 0.05 × 106 Da (Table 2). This meant that after was carried out in the 10-L fermentation as described in Materials and removal of the target genes, the pullulan titer produced by all the single methods. The results in Fig. 2 showed that within 96 h, 58.14 g/L of disruptants greatly decreased, but the Mw of the produced pullulan ob- pullulan, 19.8 g/L of cell dry weight and 38.38 g/L of oligosaccharide viously increased. This also meant that in order to further get higher Mw were achieved by the triple mutant DT15, leaving 0.87 g/L of total of the produced pullulan, it was necessary that all of the α-amylase sugar and 0.37 g/L of glucose in the fermented medium. This meant gene, the glucoamylase gene and the isopullulanase gene were simulta- that 99% of the total sugar was used for high Mw pullulan and oligosac- neously knocked out from A. melanogenum P16. Indeed, it can be very charide biosynthesis and cell growth. Therefore, the yield and produc- clearly seen from data in Table 2 that the pullulan titer and the Mw of tivity of pullulan were 0.49 g/g sugar and 0.61 g/h/L while the yield the produced pullulan by the triple disruptant DT15 were 46.2 ± and productivity of oligosaccharide were 0.32 g/g sugar and 2.7 g/L and 3.02 ± 0.28 × 106 Da. These data demonstrated that the 0.40 g/h/L. After the pullulan was purified and the Mw of the purified Mw of the produced pullulan by the triple disruptant DT15 was much pullulan was determined by using GPC as described in Materials and higher than that of the pullulan produced by the single disruptant methods, it was found that the Mw of the purified pullulan was also DA16, the single disruptant DG21, the single disruptant DPA2 and 3.02 × 106 Da (data not shown). their wild type strain P16. It was also evidenced that removal of all the α-amylase, the glucoamylase and the isopullulanase indeed could 3.4. Complementation of the α-amylase gene, the glucoamylase gene and have significant and positive influence on production of high Mw the isopullulanase gene in the corresponding disruptants pullulan. However, the data in Table 2 also showed that after removal of each gene and all the genes mentioned above, the pullulan titer de- After the α-amylase gene was complemented in the disruptant DA16, creased as the Mw of the produced pullulan increased compared to the glucoamylase gene was complemented in disruptant DG21 and the those of the produced pullulan by their wild type strain P16. But the isopullulanase gene was complemented in disruptant DPA2 as described data in Table 2 showed that cell growth of all the disruptants was not af- in Materials and methods, the α-amylase gene complemented EA20, the fected after each gene and all the genes were knocked out. glucoamylase gene complemented EG15 and the isopullulanase gene The results in Table 3 indicated that the transcriptional levels of the complemented EP4 were obtained. It can be observed from the data in α-amylase gene in the strain DA16, the glucoamylase gene in the strain Table 2 that pullulan production and the Mws of the produced pullulan DG21, the isopullulanase gene in the strain DPA2 and all the α-amylase by the strains EA20, EG15 and EP4 were restored compared to those of gene, glucoamylase gene, isopullulanase gene in the triple mutant DT15 the produced pullulan by their wild type strain P16. At the same time, ac- were significantly decreased compared to those of the α-amylase gene, tivities (3.02 ± 0.1 U/mg; 1.81 ± 0.08 U/mg and 0.51 ± 0.05 U/mg) of α- the glucoamylase gene, the isopullulanase gene in the strain P16. This amylase, the glucoamylase and the isopullulanase produced by the demonstrated that the α-amylase gene in the strain DA16, the strains EA20, EG15 and EP4 (data not shown) and the transcriptional glucoamylase gene in the strain DG21, the isopullulanase gene in the levels (330%, 210% and 206%) of the α-amylase gene, the glucoamylase strain DPA2 and all the α-amylase gene, glucoamylase gene, gene and the isopullulanase gene in the strains EA20, EG15 and EP4 isopullulanase gene in the triple mutant DT15 had been removed. In (data not shown) were greatly enhanced compared to those of α- contrast, the transcriptional level of the glucoamylase gene in the strain amylase, the glucoamylase and the isopullulanase produced by the strain DA16, the transcriptional levels of the α-amylase gene and the P16 and those of the α-amylase gene, the glucoamylase gene and the isopullulanase gene in the strain DG21 and the transcriptional levels isopullulanase gene in the strain P16 (Table 3). of the α-amylase gene and the glucoamylase gene in the strain DPA2 were increased (Table 3). The results in Table 3 also revealed that the α-amylase activity in the 4. Discussion strain DA16, the glucoamylase activity in the strain DG21, the isopullulanase activity in the strain DPA2 and all the α-amylase activity, Many researchers thought that α-amylase, glucoamylase and glucoamylase activity, isopullulanase activity in the triple mutant DT15 pullulanase were involved in pullulan degradation, leading to produc- were obviously decreased compared to the α-amylase activity, the tion of low Mw pullulan (Fig. 1)[4, 8–12]. Therefore, the α-amylase glucoamylase activity, the isopullulanase activity in the strain P16 be- gene, the glucoamylase gene and the isopullulanase gene were cloned cause of the removal of the corresponding gene(s) in different from A. melanogenum P16, a high pullulan producing yeast, character- disruptants. In contrast, it can be observed from the data in Table 3 ized (Table 1) and deleted [13]. that the glucoamylase activity in the strain DA16, the α-amylase activity It has been well known that the glucoamylases are exoamylases ca- and the isopullulanase activity in the strain DG21 were increased. pable of hydrolyzing glycosidic linkages of α-1,4 type which also occurs All the results in Table 3 were consistent with those in Table 2. in pullulan molecules by the successive removal of glucose units from the non-reducing end of the chain, releasing D-glucose. All the 3.3. High molecular weight pullulan production by the triple mutant DT15 glucoamylases from Aspergillus japonicas, A. niger, A. awomori, A. ficus through 10-L fermentation had the conserved sequences of KFNVDETAYTGSWGRPQRD. All the glucoamylases from microorganisms comprise two binding domains, a Table 2 showed that the triple mutant DT15 could produce 46.2 g/L catalytic domain and the starch binding domain connected by an O- of pullulan with the Mw of 3.02 × 106 Da. This Mw of pullulan is enough glycosidic linker [29]. The cloned GLY gene also had such conserved

Table 2 Pullulan production, its molecular weight and cell growth by different disruptants, their wild type strain P16 and different transformants.

Strain P16 DA16 DG21 DPA2 DT15 EA20 EG15 EP4

Knocked out/complemented gene No AMY GLY IPU AMY&GLY&IPU AMY GLY IPU Molecular weight (×106) 0.35 ± 0.05 0.75 ± 0.06 0.57 ± 0.05 1.36 ± 0.21 3.02 ± 0.28 0.21 ± 0.03 0.31 ± 0.02 0.19 ± 0.02 Pullulan (g/l) 65.5 ± 3.5 48.5 ± 2.5 58.2 ± 3.2 46.4 ± 1.8 46.2 ± 2.7 58.4 ± 3.1 62.4 ± 2.7 64.3 ± 3.1 DCW(g/l) 23.7 ± 0.38 21.72 ± 0.41 22.29 ± 0.35 22.81 ± 0.26 22.68 ± 1.02 23.12 ± 0.58 22.74 ± 0.71 23.45 ± 0.84

Data are given as mean ± SD, n = 3.

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Table 3 Enzyme activity and relative transcription level of the genes involved in pullulan degradation.

Relative transcription levels (%) Enzyme activity (U/mg)

P16 DA16 DG21 DPA2 DT15 P16 DA16 DG21 DPA2 DT15

Alpha-amylase 100 ± 7.1 2 ± 0.03 349 ± 31 103 ± 7.9 5 ± 0.7 0.78 ± 0.09 0.15 ± 0.02 0.86 ± 0.09 0.62 ± 0.08 0.16 ± 0.02 Glucoamylase 100 ± 7.3 117 ± 11.7 2 ± 0.05 126 ± 12.1 0.77 ± 0.08 0.89 ± 0.11 1.14 ± 0.2 0 0.97 ± 0.1 0.11 ± 0.02 Isopullulanase 100 ± 8.0 96 ± 8.2 170 ± 20 0.12 ± 0.01 0.16 ± 0.03 0.27 ± 0.04 0.24 ± 0.04 0.32 ± 0.05 0.11 ± 0.03 0.09 ± 0.02

Data are given as mean ± SD, n = 3.

amino sequences (Table 1). These meant that the cloned gene in this respectively [31]. In fact, the are divided into two study was indeed a glucoamylase gene (Table 1). subgroups: pullulanase type I, commonly called pullulanase and II called In general, the α-amylase that hydrolyzes endo bonds of large, amylopullulanase whereas the pullulan are subdivided into alpha-linked polysaccharides, such as starch, glycogen and pullulan is three types: types I (), II (isopullulanase) and III. composed of 6–7 highly conserved sequence regions (CSR I to VII). Pullulanases and pullulan hydrolase type I (neopullulanase) are pro- The CSRs I–VII are located on strands β-3, β-4, β-5, β-7, β-2, β-8 and duced by and . Among bacteria, many mesophilic, ther- on loop 3 and the catalytically active amino acid residues, two aspartate mophilic and hyperthermophilic bacteria produce pullulanases and and one glutamate, are located within the CSRs on the strands β-4, β-5 neopullulanases. While pullulan hydrolase type II (isopullulanase) and and β-7 [30]. For example, the cloned α-amylase gene from A. pullulans type III are produced by fungi and archaea [30]. However, we found strain NRRL Y-12974 had a putative mRNA of 1,878 bp encoding an α- that except A. niger ATCC 9642, the sequenced genomic DNAs of any amylase of 625 amino acid residues and the complete genomic DNA se- other filamentous fungi did not contain the gene encoding the quence contains 2,247 bp, also including 7 introns and 8 exons. The de- isopullulanase (data not shown). In contrast, all the strains of duced protein likely contained a 22 amino acid signal peptide, an α- Aureobasidium spp., including A. melanogenum P16 in which the genomic amylase superfamily domain and C-terminal carbohydrate-binding DNAs have been sequenced, were found to have such a gene [33]. There- module and family 20 domain [9]. The results in Table 1 revealed that fore, only the isopullulanase, not pullulanase played a role in degradation the cloned AMY gene in this study also had such characteristics. of the produced pullulan by different strains of Aureobasidium spp. It has been reported that an isopullulanase (IPU) (GenBank, (Tables 1, 2 and 3) and the results obtained in this study were not in BAA19473.1) from A. niger ATCC9642 hydrolyzes α-1,4-glucosidic link- agreement with those reported by any other researchers [4, 12]. ages of pullulan to produce isopanose (Glcα1-4Glcα1-6Glc) and is highly Table 2 showed that the disruptants deficiency in any gene of them glycosylated. The IPU consists of two domains, domain N (residues and all the genes could produce higher Mw pullulan and less pullulan 20–182) and domain C (residues 197–564), which are joined by a yields. Many results from any other researchers indeed confirmed that short linker (residues 183–196) and the domain C of the isopullulanases the higher Mws, the lower the pullulan titer [4, 6, 7]. However, the rea- forms the catalytic domain [31]. In contrast, a pullulanase catalyzes the son for this is still unclear. This may be due to the facts that more energy hydrolysis of the α-1,6-glucosidic linkage of pullulan, releasing is needed for biosynthesis of high Mw pullulan than biosynthesis of low maltotriose (Glcα1-4Glcα1-4Glc) [32]. The catalytic residues Glu706 Mw pullulan, leading to the decreased titer of the produced pullulan. and Asp677 are conserved between pullulanase and α-amylase. Unlike Table 3 revealed that the expression of the α-amylase gene, the the pullulanase and α-amylase, the isopullulanase did not have the con- glucoamylase gene and the isopullulanase gene and activities of α- served sequences GFRFDLMGI and IEFLHSGQ on the strands β-4 and β-8, amylase, glucoamylase and isopullulanase in all the disruptants were much lower than that of the α-amylase gene and the glucoamylase gene and the isopullulanase gene and those of α-amylase, glucoamylase and isopullulanase in the wild type strain P16. These demonstrated that it was important to clean up all the α-amylase gene, glucoamylase gene and isopullulanase gene in order to avoid the degradation of the pro- duced pullulan by any enzymes mentioned and further increase the Mw of the produced pullulan in the pullulan producing yeasts (Table 2). It can be seen from the results in Table 3 that the glucoamylase activ- ity in the strain DA16, the α-amylase activity and the isopullulanase ac- tivity in the strain DG21 were increased. This may be associated with a coordinate regulation that occurs in these yeasts. However, the exact mechanism of the coordinate regulation is still completely unclear. In fact, the coordinate regulation also happens in the lipid and melanin metabolism in yeasts [34, 35]. The data in Table 3 also indicated that only a single copy of the α- amylase gene, the glucoamylase gene and the isopullulanase gene existed in A. melanogenum P16. Similarly it has been reported that A. pullulans strain NRRL Y-12974 also carries only a single copy of this α-amylase gene [9]. The pullulan titer and Mw of the produced pullulan reached 58.14 g/l and 3.02 × 106 Da in the culture of the triple mutant DT15 (Fig. 2). In our previous study [4], it was found that when the yeast strain P16 was grown in the compositionally changed medium, the pullulan titer was 46.4 g/L and the Mw of the produced pullulan was 2.6 × 106 Da. So far, the Mw of the most of pullulans produced by A. pullulans is about 362–480 kDa. For example, Hayashibara Company Fig. 2. The time course of pullulan and oligosaccharide production, cell growth, the changes in total sugar and reducing sugar during the 10-L fermentation. Data are given Limited, Japan is producing the pullulans with a mean Mw of 100,000 as mean ± SD, n = 3. or 200,000 Da [36]. A. pullulans SZU 1001 mutant produced a Mw

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