Genetic Modification of the Marine-Isolated Yeast ... - Springer Link

Mar Biotechnol (2015) 17:511–522 DOI 10.1007/s10126-015-9638-8

ORIGINAL ARTICLE

Genetic Modification of the Marine-Isolated Yeast Aureobasidium melanogenum P16 for Efficient Pullulan Production from Inulin Zai-Chao Ma 1 & Nan-Nan Liu 1 & Zhe Chi 1 & Guang-Lei Liu 1 & Zhen-Ming Chi 1

Received: 5 November 2014 / Accepted: 28 April 2015 / Published online: 19 May 2015 # Springer Science+Business Media New York 2015

Abstract In this study, in order to directly and efficiently convert inulin into pullulan, the INU1 gene from Kluyveromyces maximum KM was integrated into the genomic DNA and actively expressed in the high pullulan producer Aureobasidium melanogenum P16 isolated from the mangrove ecosystem. After the ability to produce pullulan from inulin by different transformants was examined, it was found that the recombinant strain EI36, one of the transformants, produced 40.92 U/ml of inulinase activity while its wildtype strain P16 only yielded 7.57 U/ml of inulinase activity. Most (99.27 %) of the inulinase produced by the recombinant strain EI36 was secreted into the culture. During the 10-l fermentation, 70.57±1.3 g/l of pullulan in the fermented medium was attained from inulin (138.0 g/l) within 108 h, high inulinase activity (42.03 U/ml) was produced within 60 h, the added inulin was actively hydrolyzed by the secreted inulinase, and most of the reducing sugars were used by the recombinant strain EI36. This confirmed that the genetically engineered yeast of A. melanogenum strain P16 was suitable for direct pullulan production from inulin.

Keywords Metabolic engineering . Pullulan production . Inulin . Inulinase gene . A. pullulans

* Zhen-Ming Chi [email protected] 1

Unesco Chinese Center of Marine Biotechnology, Ocean University of China, Yushan Road, No. 5, Qingdao, China

Introduction Pullulan which is essentially a linear glucan containing α-1,4 and α-1,6 linkages in the ratio of 2:1 is the water-soluble homopolysaccharide produced extracellularly by Aureobasidium spp. The regular alternation of α-1,4 and α1,6 bonds results in two distinctive properties of structural flexibility and enhanced solubility. The unique linkage pattern also endows pullulan with distinctive physical traits along with adhesive properties and its capacity to form fibers, compression moldings, and strong oxygen impermeable films. Consequently, this polysaccharide is of economic importance with increased application in food, pharmaceutical, agricultural, chemical, and cosmetic industries (Prajapati et al. 2013). The pullulans also have many physiological functions for the producers (Ma et al. 2014). It has been reported that pullulan is synthesized intracellularly at the cell wall or cell membrane and secreted out to the cell surface to form a loose and slimy layer. Therefore, a pathway for pullulan biosynthesis was proposed (Li et al. 2013a). Because the yeast-like cells cannot synthesize and secrete high level of amylase, inulinase, and cellulase, the pullulan is only produced from glucose, sucrose, coconut by-products, beet molasses, agro-industrial wastes, grape skin pulp, starch waste, olive oil wastes, carob pod, and hydrolysate of inulin (Prajapati et al. 2013; Ma et al. 2014; Shin et al. 1989). Therefore, it is very important to widen their substrate ranges for pullulan production by metabolic engineering of the producers. Inulin consists of linear chains of β-2,1-linked Dfructofuranose molecules terminated by a glucose residue through a sucrose-type linkage at the reducing end and is widely presented in the roots and tubers of plants such as Jerusalem artichoke, chicory, dahlia, and yacon. It has been reported that the dried materials of the roots and tubers of the

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plants contained over 70 % inulin. So far, inulin and inulincontaining materials have been used as raw materials for production of ethanol, single-cell protein, single cell oil, ultrahigh-fructose syrup, inulooligosaccharide, citric acid, 2,3butanediol, lactic acid, sugar alcohols, and other useful products (Chi et al. 2011). The exo-inulinase produced by microorganisms catalyzes removal of the terminal fructose residues from the non-reducing end of the inulin molecule to produce major fructose and minor glucose. In our previous studies (Duan et al. 2008), fructose and glucose were found to be the best sugars for production of pullulan by Aureobasidium spp. (Fig. 1). As stated above, the hydrolysates of inulin are also the mixture of glucose (minor) and fructose (major). Therefore, they may be the best sugars for production of pullulan. However, as shown in Fig. 1, inulin has not been used as a material for direct production of pullulan from inulin

Fig. 1 Inulin hydrolysis and pullulan biosynthesis by the engineered Aureobasidium spp. carrying the INU1 gene. After the INU1 gene from K. maximum KM is actively expressed in Aureobasidium spp., the produced inulinase is secreted into medium (Gong et al. 2007). After the inulin is hydrolyzed into glucose and fructose by the secreted inulinase (Chi et al. 2011), the glucose and fructose are taken up by the cells of the engineered Aureobasidium spp. and transformed into pullulan which is secreted into the medium (Li et al. 2013a)

Mar Biotechnol (2015) 17:511–522

because of very low or no inulinase activity of Aureobasidium spp. In our previous studies (Ma et al. 2014), Aureobasidium pullulans var. melanogenum P16 strain isolated from mangrove system was found to be able to produce high level of pullulan. In recent studies (Gostinčar et al. 2014), the authors have thought that the four varieties A. pullulans var. pullulans, A. pullulans var. subglaciale, A. pullulans var. namibiae, and A. pullulans var. melanogenum of Aureobasidium spp. are large enough to justify their redefinition as four separate Aureobasidium species: A. pullulans, Aureobasidium m e l a n o g e n u m , A u re o b a s i d i u m s u b g l a c i a l e , a n d Aureobasidium namibiae. After analysis of the genomic DNA of A. melanogenum (http://genome.jgi-psf.org/Aurpu_ var_mel1/Aurpu_var_mel1.home.html), it was found that there was no the gene encoding inulinase in this yeast.


Therefore, in this study, in order to genetically engineer the pullulan producer, an inulinase gene (INU1) from Kluyveromyces maximum KM was actively expressed in A. melanogenum P16 strain and the recombinant strains which could produce high inulinase activity were used for efficient production of pullulan from inulin (Fig. 1).

Materials and Methods Strains and Cultivation Media The yeast A. melanogenum strain P16 isolated from the mangrove systems in Hainan Province of China was used in this study and stored at −80 °C in this laboratory (Ma et al. 2014). The medium for growth of the seed culture contained 60.0 g/l sucrose, 3.0 g/l yeast extract, 5.0 g/l K2HPO4, 0.2 g/l MgSO4· 7H2O, 1.0 g/l NaCl, and 0.6 g/l (NH4)2SO4 (Duan et al. 2008). The cultivation time and temperature were 48 h and 28 °C, respectively. The medium for pullulan production consisted of 138.0 g/l inulin, 3.0 g/l yeast extract, 5.0 g/l K2HPO4, 0.2 g/l MgSO4·7H2O, 1.0 g/l NaCl, and 0.6 g/l (NH4)2SO4 (Duan et al. 2008). K. maximum KM which is a high inulinase producer was grown in the inulinase production medium (Zhou et al. 2013). The Escherichia coli strain DH5α [F− endA1 hsdR17 (rK_/mK+) supE44 thi− λ−recA1gyr96DlacU169 (j80lacZDM15)] was used in this study and was grown in Luria-Bertani broth (LB). The E. coli transformants were grown in LB medium with 100 μg/ml of ampicillin. The yeast transformants were grown in the YPD medium containing 100 μg/ml of hygromycin B. The inulinase production medium was 10.0 g/l yeast extract, 20.0 g/l polypeptone, and 20.0 g/l inulin. The inulin used in this study was bought from Haida Biotech Company in Qingdao, China. Plasmids The expression vector pAPX13 for over-expression of heterologous gene in Aureobasidium spp. was constructed in this Table 1

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laboratory. pMD 19-T simple vectors were purchased from TaKaRa (Japan). Isolation of DNA, Restriction Digestions, and Transformation The genomic DNAs were isolated from K. maximum KM and A. melanogenum strain P16 and its transformants, respectively, and DNA manipulations were carried out using the standard methods (Sambrook et al. 1989). Bacterial plasmid DNA was purified using TIANprep Mini Plasmid Kit (TIANGEN). Restriction endo-nuclease digestions and DNA ligations were performed according to the manufacturer’s recommendations. E. coli was transformed with plasmid DNA according to Sambrook et al. (1989). A. melanogenum strain P16 was transformed according to the methods described by Chi et al. (2012). Vector Construction for Expression of the INU1 Gene in A. melanogenum Strain P16 To express the INU1 gene from K. maximum KM in A. melanogenum strain P16, the primers for amplification of the INU1 gene encoding the inulinase gene were designed according to the sequence of the gene (accession no. AF135594.1) in K. maximum. The forward primer and the reverse primer were INU-se and INU-an (Table 1 and Fig. 2). The genomic DNA of K. maximum KM was used as the template for PCR. The reaction system was 50.0 μl containing 1.0 μl Takara EX Taq, 5.0 μl 10× La PCR buffer II (Mg2+ Plus), 8.0 μl 2.5 mM dNTPs, 1.0 μl 20.0 μM each primer, 1.0 μl of 10 ng/ml genomic DNA, and sterile deionized water up to 50.0 μl. The conditions for the PCR amplification were initial denaturation at 94 °C for 5 min, denaturation at 94 °C for 45 s, annealing temperature at 60 °C for 45 s, extension at 72 °C for 1 min 45 s, and final extension at 72 °C for 5 min. PCR was run for 30 cycles. The PCR products were separated by agarose gel electrophoresis and ligated into the plasmid pMD19-T simple vector. The recombinant vector was

Primers used in this study

Name

Sequences

INU-se INU-an YIF YIR INU RAse INU RAan AP26S AP26X

5′-GAGCTCGATGGTGACAGCAAGGCCATCACTA-3′ (bold and underlined bases encode SacI restriction site) 5′-GTCGACTCAAACGTTAAATTGGGTAACGTTA-3′ (bold bases encode SalI restriction site) 5′ -AAAACCCCAACTTCGGAAAGGGGTG-3′ 5′ –TGGTGTAAGCCTTGTCGTATGGCGA-3′ 5′-CACCGTCACCTCCGAAAAC-3′ 5′-GTTGTCGTCGTTGTTACCCTTG-3′ 5′- AGCCTTCGGGTTCGCATTG-3′ 5′-CCAGTCACATACGGGATTCTCAC-3′

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Fig. 2 Construction of the expression vector for overexpression of the INU1 gene in A. melanogenum strain P16. The DNA fragment encoding the signal peptide comes from the gene (accession number EF198023) encoding alkaline protease in A. pulllulans HN2-3. 26S rDNA, 18S rDNA, PolyA, and TEF come from the genomic DNA in A. pullulans. The plasmid was digested with the enzymes EcoRI and SphI. The linear fragments carrying the INU1 gene were used for yeast transformation

transformed into E. coli DH5α. The recombinant vectors carrying the PCR products from the E. coli transformants were extracted and purified. The purified recombinant vectors carrying the PCR products were digested with SacI and SalI, and the digests were ligated into the expression vector pAPX13 digested with the same enzymes (Fig. 2). The resulting plasmid carrying the INU1 gene was designated as pAPX13-INU (Fig. 2).

activity produced by the cells of different positive transformants was determined as described below, respectively. A. melanogenium strain P16 not to be transformed was used as a control. After determination of inulinase activity produced by the cells of over 200 positive transformants, it was found that inulinase activity produced by the recombinant strain EI36 among them was the highest. Therefore, the recombinant strain EI36 was used as the inulinase producer, subsequently.

Transformation and Selection The plasmid pAPX13-INU obtained above was digested with the enzymes EcoRI and SphI. The linear fragments carrying the INU1 gene were separated in agarose gel and recovered using TaKaRa Agarose Gel DNA Purification Kit Ver.2.0, respectively. The recovered linear fragments carrying the INU1 gene were transformed into the cells of A. melanogenum strain P16 by using the methods described by Chi et al. (2012). The putative transformants were verified by cultivation on YPD agar containing 100 μg/ml of hygromycin B. The positive transformants were grown in the inulinase production medium for 4 days and inulinase

Confirmation of the Integrated INU1 Gene in the Genomic DNA of the Recombinant Strain EI36 The genomic DNAs in the recombinant strain EI36 and A. melanogenum strain P16 were extracted as described above and used as the templates for PCR. The DNA fragments (2, 529 bp) containing the partial sequence of 18S rDNA and the whole INU1 gene were PCR amplified using the primers YIF and YIR (Table 1 and Fig. 2). The sizes of the PCR products were estimated using the Automated Documentation and Analysis System (Gene-Genius, USA). The PCR products


were sequenced by Shanghai Sangon Company (Zhao et al. 2010).

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P16 was regarded as 100 %. The determinations were carried out in triplicate and all the data were the average of the three independent experiments.

Determination of the Recombinant Inulinase Activity Pullulan Production from Inulin at Flask Level The recombinant inulinase activity of different positive transformants including the recombinant strain EI36 obtained above was determined according to Gong et al. (2007). One inulinase unit (U) was defined as the amount of enzyme that produces 1 μmol of reducing sugar per minute under the assay conditions used in this study. At the same time, the yeast cells of the recombinant strain EI36 in 5.0 ml of the cultures were washed three times with sterile distilled water by centrifugation at 4 °C and 5,000×g for 10 min, respectively. The cellbound inulinase activity of the washed yeast cell suspensions (5.0 ml) was determined as described above. The determinations were carried out in triplicate and all the data were the average of the three independent experiments. Inulinase Production from Inulin at Flask Level The cells of the recombinant strain EI36 and A. melanogenum strain P16 were transferred to 50.0 ml of the sterile medium for the seed culture and cultivated at the shaking speed of 180 rpm and 28 °C for 24 h, respectively. Two milliliters of the culture (2.5×108 cells/ml) was transferred to 50.0 ml of the sterile inulinase production medium, and the yeast cells were cultivated at the shaking speed of 180 rpm and 28 °C for 96 h. The measurements of inulinase activity and cell mass were performed as described above and below. The cultivations were carried out in triplicate and all the data were the average of the three independent experiments. Fluorescent Real-Time PCR Fluorescent real-time PCR was performed according to the methods described by Liu et al. (2011). The seed cultures of the recombinant strain EI36 and A. melanogenum strain P16 were prepared as described above. The seed cultures were transferred into 50 ml of the sterile inulinase production medium and the cultures were aerobically grown for 72 h and, at this time, inulinase activity reached the highest. The cells were harvested and washed, and the washed cells were used for RNA extraction and cDNA preparation (Liu et al. 2011). All the primers used for fluorescent real-time PCR were designed according to the corresponding gene sequences of K. maximum and A. melanogenum strain P16. The primers INU RAse and INU RAan were designed according to the INU1 gene sequence (GenBank accession no. AF135594.1) in K. maximum, and the primers Ap26S and Ap26X were designed according to 26S rRNA gene sequence (GenBank accession no. EU622575) in A. pullulans (Table 1). The transcriptional level of the INU1 gene in A. melanogenum strain

The seed cultures of the recombinant strain EI36 and A. melanogenum strain P16 were prepared as described above. Two milliliters of the culture (2.5 × 10 8 cells/ml) were transferred to 50.0 ml of the sterile pullulan production medium, and the yeast cells were cultivated at the shaking speed of 180 rpm and 28 °C for 96 h. The determination of pullulan, inulinase activity, reducing sugar, total sugar, and cell mass was performed as described above and below. The cultivations were carried out in triplicate and all the data were the average of the three independent experiments. Pullulan Production from Inulin by Batch Fermentation Pullulan production by the recombinant strain EI36 was also carried out in the 10-l fermentor [BIOQ-6005-6010B, Huihetang Bio-Engineering Equipment (Shanghai) COLTD]. The seed cultures were prepared as described above. The fermentation was carried out in the sterile fermentor equipped with baffles, a stirrer, heating element, oxygen sensor, and temperature sensor. Three hundred milliliters of the seed culture were transferred into 6.5 l of the sterile pullulan production media containing 138.0 g/l inulin and 3.0 g/l yeast extract. The fermentation was performed under the conditions of agitation speed of 300 rpm, aeration rate of 6.5 l/min, temperature of 28 °C, and fermentation period of 132 h. Only 40.0 ml of the culture was collected in the interval of 12 h and was centrifuged at 5,000×g and 4 °C for 5 min, and pullulan, inulinase activity, reducing sugar, and total sugar in the supernatant obtained were determined as described above and below. The cell dry weight in 20.0 ml of the culture during the 10-l fermentation was also determined as described below. The fermentations were carried out in triplicate and all the data were the average of the three independent experiments. Purification and Quantitative Determination of EPS The cultures obtained during the 10-l fermentation were heated at 100 °C in water bath for 15 min, then were cooled to room temperature. The heated cultures were centrifuged at 14, 046×g and 4 °C for 5 min to remove cells and other precipitates. The supernatant (10.0 ml) was transferred into a test tube, then 20.0 ml of cold ethanol (absolute ethanol or 95 % ethanol) was added to the test tube and mixed thoroughly and held at 4 °C for 12 h to precipitate the extracellular polysaccharide. After removal of residual ethanol, the precipitate was dissolved in 10.0 ml of deionized water at 80 °C and the solution was dialyzed against deionized water for 48 h to

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remove small molecules in the solution. After the EPS was precipitated again by using 20.0 ml of the cold ethanol, the precipitate was dried at 80 °C to a constant weight (Lee et al. 2001). Hydrolysis of the Purified Extracellular Polysaccharide and Assay of Reducing Sugar Hydrolysis of the purified extracellular polysaccharide and assay of reducing sugar were done as described by Ma et al. (2014). Fourier-Transform Infrared Analysis of the Purified Pullulan The purified pullulan obtained was characterized using Fourier-transform infrared (FT-IR) spectroscopy (a Nicolet Nexus FTIR 470 spectrophotometer). Two milligrams of the purified pullulan sample were mixed with 60 mg of 95 % potassium bromide powder. The mixture was desiccated overnight at 50 °C under vacuum. The FT-IR spectra were taken using potassium bromide pellets of the purified pullulan and standard pullulan obtained from sigma over arrange of 4,000– 400 cm−1 at a rate of 16 scans with a resolution of 2 cm−1.


Results Expression of the INU1 Gene in the High Pullulan Producing Yeast A. melanogenum Strain P16 In order to genetically engineer the high pullulan producer of A. melanogenum strain P16, the INU1 gene from K. maximum KM was integrated into the genomic DNA and actively expressed in A. melanogenum strain P16 as described in BMaterials and Methods^ (Fig. 2). After determination of inulinase activities of the different transformants and A. melanogenum strain P16, it was found that the recombinnat strain EI36 among them produced the highest inulinase activity (40.92 U/ml) as supported by ANOVA analysis (P=0.01 that differences were significant), and the inulinase activities produced by the 200 transformants were in the range from 11.72±0.25 U/ml to 40.92±0.43 U/ml (Fig. 3). It was strange that its wild-type strain P16 also produced inulinase activity of 7.57 U/ml (Fig. 3). At the same time, the transcriptional level of the INU1 gene in the recombinant strain EI36 was also much higher than that (almost 0) of the inulinase gene in A. melanogenum strain P16 (Fig. 4) as indicated from ANOVA analysis that found differences to be significant (P=0.01).

Determination of Reducing Sugar and Total Sugar

Confirmation of the Integrated INU1 Gene in the Genomic DNA of the Recombinant Strain EI36

Reducing sugar in the fermented media was determined by the Nelson–Somogyi method (Spiro 1966). Residual total sugar was measured as reduction of sugar after hydrolysis of the fermented media (10.0 ml of the fermented media, 10.0 ml of 25 % HCl, and 30 ml of distilled water were mixed and heated in a boiling water bath for 3 h) (Chi et al. 2001). The determinations were carried out in triplicate and all the data were the average of the three independent experiments.

Our data showed that the INU1 gene was amplified from the genomic DNA in the recombinant strain EI36 (data not shown). However, no such PCR products were amplified from the genomic DNA of A. melanogenum strain P16 (data not shown). This demonstrated that the INU1 gene was indeed integrated into the genomic DNA in the recombinant strain EI36, leading to stable occurrence of the gene in the genomic DNA of the recombinant strain EI36 according to the Fig. 2.

Measurement of Cell Dry Weight Cell dry weight was measured according to the methods described by Chi et al. (2001). The determinations were carried out in triplicate and all the data were the average of the three independent experiments. Statistic Analysis Statistical tests (two-tailed paired t test and two-tailed twosample t test) were performed using Statistical Package for the Social Sciences (SPSS) (ver.19; IBM corporation; USA) with significance set at α=0.05. The results of two independent experiments, each carried out in duplicate (means±SD), are presented in the figures.

Fig. 3 Inulinase activities of the different transformants including the recombinant strain EI36 and A. melanogenum strain P16. Data are given as mean±SD, n=3 *(P