Alternative primers are required for pullulan biosynthesis in Aureobasidium melanogenum P16

International Journal of Biological Macromolecules 147 (2020) 10–17

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Alternative primers are required for pullulan biosynthesis in Aureobasidium melanogenum P16 Tie-Jun Chen a,1, Guang-Lei Liu a,b,1, Lu Chen a, Guang Yang a, Zhong Hu c, Zhen-Ming Chi a,b, Zhe Chi a,b,⁎ a b c

College of Marine Life Sciences, Ocean University of China, Yushan Road, No. 5, Qingdao, China Key Laboratory of Marine Genetics and Breeding, Ocean University of China, Yushan Road, No. 5, Qingdao 266003, China Department of Biology, Shantou University, Shantou 515063, China

a r t i c l e

i n f o

Article history: Received 16 November 2019 Received in revised form 28 December 2019 Accepted 6 January 2020 Available online 07 January 2020 Keywords: A. melanogenum α-Glucan synthetase Pullulan biosynthesis Pullulan primers

a b s t r a c t Although pullulan has many uses in industry, the detailed mechanisms of its biosynthesis still require clarification. In this study, it was found that a short α-1,4-glucosyl chain (pullulan primer) synthesized by the glycogenins Glg1 and Glg2 for initiation of glycogen biosynthesis was also needed for pullulan synthesis. The primers were also synthesized on sterol glycosides and glucosylceramides by catalysis of sterol glucosyltransferase (Sgt1) and ceramide β-glucosyltransferase (Gcs1). All the primers might be elongated to be long α-1,4-glucosyl chain (pullulan precursor) by catalysis of the glycogen synthetase domain of the AmAgs2 as previously reported. Then, the amylase domain of the same AmAgs2 was responsible for pullulan biosynthesis. Removal of all the genes encoding Glg1, Glg2, Gcs1 and Sgt1 made all the mutants produce much less pullulan than the strain P16. However, pullulan synthesis could not be stopped totally in these mutants, suggesting that any other unknown alternative pullulan primers may exist in the yeast cells. Complementation of all the genes in the mutants restored pullulan biosynthesis. This is the first time to report that like starch and glycogen biosynthesis, alternative primers are also required for pullulan biosynthesis in Aureobasidium melanogenum P16. © 2018 Elsevier B.V. All rights reserved.

1. Introduction It has been well established that regularly repeating structural unit of pullulan is maltotriose and pullulan is a maltotriose polymer with the chemical structure [α-(1 → 4)Glucose-α-(1 → 4)Glucose-α(1 → 6)Glucose], produced extracellularly by Aureobasidium spp. [1,2] while glycogen molecule produced by yeasts and filamentous fungi is characterized by glucose units linked by α-1,4 glycosidic bonds of linear chain and α-1,6 linked glucose at the branching points [3]. In both amylose and amylopectin, glucose residues are linked together by α(1 → 4) O-glycosidic linkages that are regularly branched in α-(1 → 6) positions [4]. Therefore, the only common features that pullulan, glycogen and starch have are their bond types and glucose unit. Each linear chain of glycogen molecule has an average length of 13 glucose units and contains two branching points by means of α-(1,6)glycosidic bonds, each branching point having approximately seven glucoses. It has been well documented that biosynthesis of glycogen and starch requires several enzymes for initiation, elongation, and branching. In all eukaryotic cells, the glycogen initiation step is performed by a ⁎ Corresponding author at: College of Marine Life Sciences, Ocean University of China, Yushan Road, No. 5, Qingdao, China. E-mail addresses: [email protected], [email protected] (Z. Chi). 1 Tie-Jun Chen and Guang-Lei Liu made equal contribution to this work.

https://doi.org/10.1016/j.ijbiomac.2020.01.049 0141-8130/© 2018 Elsevier B.V. All rights reserved.

glycogenin (Glg) to yield a short linear α-(1,4)-glucosyl chain (glycogen primer) by autoglucosylation activity using UDP-Glucose as the glucose donor [3]. In Saccharomyces cerevisiae, Glg is encoded by two genes, GLG1 and GLG2 [3]. However, removal of both the genes in the yeast could not make the mutants have a complete glycogen deficiency, suggesting that the initiation step can take place using any other unknown alternative primers [5]. Indeed, in our previous study [6], it was found that deletion of any known single gene in Aureobasidium melanogenum P16 did not result in a complete pullulan deficiency in the mutant. However, the complete removal of the AmAGS2 gene, the key gene responsible for pullulan biosynthesis could totally stop pullulan production [7]. Starch synthetase (SS) also requires a free, non-reducing end of the elongating α-(1 → 4)-D-glucan chain to which the glucosyl moiety is transferred from the activated ADP-glucose, not UDP-glucose. This feature means that the synthesis of starch also requires the previous formation of a primer that will be subsequently elongated and branched [4]. In plants, such starch primer or starch granule initiation may be synthesized by starch synthetase class IV (SSIV) [4]. The elongation step for glycogen biosynthesis is carried out by a glycogen synthetase that catalyzes the successive addition of α-1,4-linked glucose residues to the non-reducing end of the primers mentioned above using UDP-glucose as the donor substrate. The yeast S. cerevisiae contains two GSY1 and GSY2 genes encoding two glycogen synthetases [3]. Therefore, the initiation step can provide the non-

T.-J. Chen et al. / International Journal of Biological Macromolecules 147 (2020) 10–17

reducing ends that ensure the glycogen elongation by the glycogen synthetase (Gys) through the formation of the α-1,4-glycosidic linkages. The branching of the glycogen and starch chains was catalyzed by the glycogen or starch branching enzymes [3,4]. Bertolini et al. [8] and Simon et al. [9] found intracellular glycogen level and extracellular pullulan content were inversely correlated, and proposed a hypothesis of the cytosolic α (1 → 4) glucose units of glycogen involved in biosynthesis of the pullulan. So it was speculated that there would be some similarities in biosynthetic mechanisms of pullulan, starch and glycogen in fungi and plant and it was thought that like glycogen biosynthesis in fungi, the similar pullulan primer (a short α-(1,4)-glucosyl chain) might also be synthesized for pullulan biosynthesis in the wild type strain P16 used in this study [10]. In this study, we found that like glycogen and starch biosynthesis in fungi and plants, biosynthesis of pullulan primers with the short α-1,4glucosyl chain could take place on many compounds. Then, under catalysis of the glycogen synthetase_domain of the AmAgs2, the key enzyme for pullulan biosynthesis as previously reported [7], the pullulan primers could be elongated to form the long α-1,4-glucosyl chains (pullulan precursors) for pullulan biosynthesis by any other domains of the AmAgs2. 2. Materials and methods 2.1. Yeast strains, media and plasmids A. melanogenum P16, isolated from the mangrove systems in Hainan Province of China, was a high pullulan producing yeast [10]. All the yeast strains used in this study were listed in Table S1. A pullulan production medium was used for pullulan production and the YPD medium was used for yeast cultivation [10]. A plasmid pFL4A-NAT-loxp carrying a nourseothricin resistance gene (NAT gene) and a plasmid pNTX13-NSloxp carrying the NAT gene were used for construction of the disruption vectors and the expression vectors used in this study, respectively [11,12]. A plasmid pAMCRE-1 was used for deletion of the NAT gene mentioned above and was lost automatically during the yeast cell growth [12]. All the plasmids used in this study were listed in Table S2. 2.2. Pullulan production, isolation and purification A. melanognum P16, the disruptants, complementing strains and the transformants obtained in this study (Table S1) were grown in the pullulan production medium at 28 °C by shaking at 180 rpm for 5 days. The diluted cultures were centrifuged at 4 °C and 12,000 ×g for 15 min. Cell dry weights were prepared and determined as described by Chi et al. [13]. At the same time, the pullulan in the supernatants was prepared and purified based on the methods described by Chen et al. [1] and Ma et al. [9]. The purified pullulan in the eluate was concentrated by ethanol precipitation and centrifugation again and dried at 80 °C until its weights were constant. Finally, the amount (g/L) of pullulan per liter of the culture was calculated. 2.3. Cloning and characterization of the possible genes related to pullulan primer biosynthesis

11

sequence from all the genes, and database searches were conducted using the BLAST and ORF Finder programs at the National Center for Biotechnology Information (NCBI). Multiple sequence alignments of the amplified DNA and the amino acid sequences of the deduced proteins were carried out using the programs of a DNAMAN 6.0 (http://www. lynnon.com) and a Clustal X 1.8 [15]. 2.4. Construction of the disruption vectors for disruption of the target genes 5′-Arms and 3′-arms of all the cloned genes mentioned above, including UGT1 gene encoding UDP-glucose: glycoprotein glucosyltransferase (Accession number: KX421262.1) and PUL1 gene encoding pullulan synthetase (Accession number: KM258394.1) genes [6,16] were PCR amplified using the primers (Table S3). The 5′ arms and 3′ arms of all the genes were digested with the corresponding enzymes (Table S3) and the digests were ligated into the disruption plasmid pFL4A-NAT-loxp to form pFL4A-NAT-loxp-ΔGLG1, pFL4A-NATloxp-ΔGLG2, pFL4A-NAT-loxp-ΔGCS1, pFL4A-NAT-loxp-ΔSGT1, pFL4ANAT-loxp-ΔUGT1 and pFL4A-NAT-loxp-ΔPUL1. Then, the linear 3′ arm-loxp-PGK-NAT-polyA-loxp-5′ arm fragments from the recombinant vectors were prepared by digesting them with the corresponding DNA restriction enzymes (Table S3). Finally, the linear fragments obtained were transformed into the competent cells of A. melanogenum P16 and the disruptants as described below. 2.5. Construction of the expression vectors for complementation of the genes in the disruptants and overexpression of the genes in the wild type strain P16 All the genes mentioned above were PCR amplified with the primers (Table S3). The PCR products and the expression plasmid pNTX13-NSloxp were digested with the same corresponding enzymes and the digested PCR products were inserted into the expression plasmid pNTX13-NS-loxp digested with the same enzymes to form pNTX13NS-loxp-GLG1, pNTX13-NS-loxp-GLG2, pNTX13-NS-loxp-GCS1, pNTX13-NS-loxp-SGT1, pNTX13-NS-loxp-UGT1 and pNTX13-NS-loxpPUL1. The recombinant plasmids were digested with the corresponding DNA restriction enzymes (Table S3) and the linear fragments obtained were transformed into the competent cells of the different disruptants and the wild type strain P16. 2.6. Disruption of multi-genes in the yeast cells In order to simultaneously remove multi-genes in the yeast cells, the Cre/loxp system was used in this study. This system is applicable for marker recycling, which enables us to reuse a selectable marker repeatedly. The plasmid pAMCRE-1 carrying the HPT gene [11] was transformed into the different disruptants and transformants obtained above. After the NAT gene in the disruptants and transformants was removed and the pAMCRE-1 carrying the HPT gene was automatically cured through cell division, another DNA fragment carrying both a new target gene and the NAT gene was transformed into the yeast cells which were sensitive to both nourseothricin and hygromycin B as described above. 2.7. DNA transformation

As the whole genomic DNA sequence of A. melanogenum P16 has been finished [1], the GLG1 (MG825857.1), GLG2 (MG825858.1), GCS1 (MG208875.1) and SGT1 (MG208876.1) genes encoding the glycogenin 1 (Glg1), glycogenin 2 (Glg2), UDP-glucose:glucosylceramide synthetase (Gcs1) and UDP-glucose:sterol glucosyltransferase (Sgt1) were PCR amplified from the genomic DNA of A. melanogenum P16 using the primers GLG1-F/GLG1-R, GLG2-F/GLG2-R, GCS1-F/GCS1-R, SGT1-F/ SGT1-R and EPI1-F/EPI1-R (Table S3), respectively. The reaction system and the conditions for the PCR amplification were described by Chi et al. [14]. The nucleotide sequence analysis, deduction of the amino acid

The competent cells of A. melanogenum P16, the complementing strains and the different disruptants were transformed with the linear DNA fragments obtained above based on the methods described by Chi et al. [14]. The disruptants including the Δglg1–1 mutant, the Δglg2–16 mutant, the ΔDGLG-12 mutant (the double mutant Δglg1Δglg2), the Δgcs1–8 mutant, the Δsgt1–1 mutant, tΔGS-17 mutant (the double mutant Δgcs1Δsgt1), the ΔGSG-3 mutant (Δgcs1Δsgt1Δglg1Δglg2), the ΔMG-7 mutant (Δgcs1Δsgt1Δglg1Δglg2Δugt1) and the ΔMP-6 mutant (Δgcs1Δsgt1Δglg1Δglg2Δugt1Δpul1) and the transformants including

12

T.-J. Chen et al. / International Journal of Biological Macromolecules 147 (2020) 10–17

the GLG1B-18 strain expressing the GLG1 gene, the GLG2B-6 strain expressing the GLG2 gene, the GCS1B-25 strain expressing the GCS1 gene, the SGT1B-50 strain expressing the SGT1 gene) were obtained (Table S1).

2.8. Pullulan hydrolysis using commercial pullulanase To measure whether the purified EPSs were pullulan which contained unique α-1,6-glucan linkages, the purified EPSs mentioned above were dissolved in 10.0 mL of deionized water in a 80 °C water bath. The EPS solution was hydrolyzed by incubating the mixture of 1.0 mL of the EPS solution, 0.9 mL of the acetate buffer (50 mM, pH 4.5) and 0.1 mL of the commercial pullulanase (Sigma) for 15 min at 60 °C [9]. The hydrolysates were analyzed using HPLC methods and maltotriose purchased from Sigma was used as the standard.

2.9. Assays of intracellular glycogen, trehalose, glycerol, polysaccharide and total sugar The washed yeast cells of A. melanogenum P16, all the disruptants and the transformants obtained above were treated following the procedures described by Parrou and Francois [17] and the glycogen in the treated cells were hydrolyzed at 57 °C for 10 h under constant agitation using a commercial amyloglucosidase (1.2 U/mL) from Aspergillus niger (lyophilized powder, 30–60 units/mg protein, ≤0.02% glucose, Cas: A7420 MSDS, Sigma), which could specifically transform glycogen into glucose. The released glucose was quantitatively determined using a glucose determination kit (Nanjing Jiancheng Bioeng Institute, Nanjing, China) and the amount (μg) of the glucose per 108 cells was calculated [17]. Meanwhile, all the intracellular trehalose in the washed cells was extracted and quantitatively assayed using the methods described by Chi et al. [18]. Glycerol extraction and its quantitative determination were performed based on the methods described by Kuhn et al. [19]. At the same time, the washed yeast cells mentioned above were suspended in a 20.0 mM potassium dihydrogen phosphate buffer (pH 6.2) and disrupted using a Constant Cell Disruption System (Constant System Ltd., UK) for twice at 40 kpsi. The disrupted cell suspension was centrifuged at 12,000 ×g and 4 °C for 10 min. The polysaccharide in the supernatants obtained was precipitated using cold 100% ethanol and quantitatively measured as described above. The total sugar in the same supernatants was assayed by using the Anthrone Method [20]. All the data obtained were subjected to a one-way analysis of variance (ANOVA). P values were calculated by a Student's t-test (n = 3). P values b .05 were considered statistically different and P values b .01 were considered statistically significantly different. A statistical analysis was conducted using a SPSS11.5 for Windows (SPSS Inc., Chicago, USA).

2.10. Fluorescent real-time PCR The total RNA isolation, purification and reverse transcription of the RNA to cDNA were performed according to the methods described by Liu et al. [21]. The fluorescent real-time RT-PCR assay was carried out in triplicate on a 96-well plate in 20 μL reaction volume per well containing 9.0 μL of SYBR Green PCR Master Mix (TIANGEN, China), 0.5 μL of 1:10 diluted cDNA, and 200.0 nM of each forward and reverse primer. All the primers for the quantitative real-time PCR measurements are shown in Table S3. 2.11. Observation and records of their colony and cell morphology The yeast cells of A. melanogenum P16, all the disruptants and transformants obtained above were cultivated on the YPD plates for 48 h at 28 °C. Color, margin and surface of the colonies of the different strains were observed and photographed using a normal camera. Meanwhile, the cell morphology of the different strains was observed and photographed using a phase microscope with a 100 × oil immersion objective. The images were recorded using a cellSens Standard software and photographed. 3. Results and discussion 3.1. Molecular characterization of the putative genes related to pullulan primer biosynthesis The biosynthesis pathway of glycogen has been almost completely resolved [3]. It also has been strongly demonstrated that a short linear α-(1,4)-glucosyl chain (8–12 glucosyl residues) (glycogen primer) covalently attached to a tyrosine residue of the glycogenin by autoglucosylation activity was the glycogen primer [3,8]. Based on these, molecular characteristics of all the putative genes that might be involved in pullulan primer biosynthesis were summarized in Table 1. At the same time, the molecular characteristics of the UGT1 gene coding for UDP-glucose:glycoprotein glucosyltransferase (Ugt1) and the PUL1 gene encoding pullulan synthetase (Pul1) [6,16] were also included in Table 1. It can be seen from Table 1, similar to the genomic DNA of S. cerevisiae, the genomic DNA of A. melanogenum P16 also contained two glycogenin isoforms (Glg1 and Glg2) for glycogen primer biosynthesis. However, the genomic DNA of the filamentous fungus Neurospora crassa carries only one isoform of glycogenin for glycogen primer biosynthesis (Glg1) [7]. Both the Glg1 and the Glg2 from A. melanogenum P16 had only one self-glucosylating Tyr residue (Tyr239 and Tyr247), respectively. In contrast, the Glg1 isoform from S. cerevisiae carries one self-glucosylating Tyr residue (Tyr239), but the Glg2 isoform from the same yeast cells has two adjacent residues (Tyr230 and Tyr232) [8]. Moreover, the only one Glg isoform from N. crassa also has two glucosylation sites–Tyr196 and Tyr198 [8].

Table 1 Characteristics of all the putative genes related to pullulan primer biosynthesis. Enzyme and gene name

Accession number

Conserved domain

Sterol glucosyltransferase (SGT1) Glycogenin glucosyltransferase (GLG1) Glycogenin glucosyltransferase (GLG2) UDP-glucose: glycoprotein glucosyltransferase (UGT1)

Glucosylceramide synthase MG208876.1 PH-GRAM_AGT26 MG825857.1 GT8_Glycogenin MG825858.1 GT8_Glycogenin DP-g GGTase; KX421262.1 GT8_HUGT1_C_like

Pullulan synthase (PUL1)

KM258394.1

Ceramide glucosyltransferase (GCS1)

MG208875.1

Unknown

Signal Subcellular localization peptide

Transmembrane region

N-Glycosylation site

O-Glycosylation site

NO

Endoplasmic reticulum

4

2

14

NO NO NO

Cytoplasmic Cytoplasmic Cytoplasmic

0 0 1

3 0 1

12 2 1

YES

Endoplasmic reticulum

0

3

7

YES

Extracellular, including cell wall

0

5

4 29

The software used in this study are: Conserved domain prediction website, https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml; Subcellular localization prediction website, https:// psort.hgc.jp/form2.html; Signal peptide prediction website, http://www.cbs.dtu.dk/services/SignalP/; N-glycosylation site prediction website, http://www.cbs.dtu.dk/services/ NetNGlyc/; O-glycosylation site prediction website, http://www.cbs.dtu.dk/services/YinOYang/.

T.-J. Chen et al. / International Journal of Biological Macromolecules 147 (2020) 10–17

13

Fig. 1. Pullulan production and cell growth by different mutants obtained during the single and double mutation and its wild type strain P16 (A), during the multi-gene mutation (B) and the released maltotriose from pullulan produced by the mutants and their wild type strain P16 after hydrolysis of pullulan with commercial pullulanase (C). All the yeast strains shown in this figure are described in Table S1 and the text. (n = 3, x ± S) Compared with those produced by the P16 strain, * P b .05, ** P b .01.

Therefore, the two Glg1 and Glg2 from A. melanogenum P16 used in this study were different from those from any other fungi. 3.2. Biosynthesis of the pullulan primers took place on various compounds As mentioned above, the glycogenin has been strongly demonstrated to be involved in glycogen primer biosynthesis in yeast and filamentous fungi [3,8]. This led to the question of whether the initiation of pullulan synthesis was also mediated through the self-glucosylation of the same glycogenin encoded by the GLG1 and GLG2 genes in A. melanogenum P16 (Table 1) because they have similar bond types and glucose units [3,22]. Therefore, these two genes were deleted in A. melanogenum P16. The results in Fig. 1A showed that the Δglg1–1 in which the GLG1 gene alone was removed only produced 33.90 ± 1.75 g/L of purified pullulan while the Δglg2–16 without the GLG2 gene only yielded 38.87 ± 0.79 g/L of purified pullulan (Fig. 1A). At the same time, complementation of the GLG1 gene in the Δglg1–1 and that of the GLG2 gene in the Δglg2–16 produced the recombinant yeast strains GLG1B-18 and GLG2B-6 by which pullulan production (54.08 ± 0.48 g/L and 53.10 ± 2.46 g/L pullulan, respectively) was restored compared with that (53.70 ± 2.37 g/L pullulan) produced by their wild type strain P16 (Table 2). Therefore, in both the disruptants, pullulan biosynthesis was negatively influenced. However, pullulan biosynthesis in them was not completely stopped (Fig. 1A). So, both the GLG1 gene and the GLG2 gene were simultaneously disabled and the obtained mutant ΔDGLG-12 still synthesized and secreted 35.34 ± 1.57 g/L of pullulan, confirming that simultaneous removal of both the GLG1 gene and the GLG2 gene did not further decrease pullulan biosynthesis (Fig. 1A). In addition, we found that deletion of the only single one

glycogen synthase (Gys) and the only single one glycogen branching enzyme in A. melanogenum P16 did not have any effects on pullulan production (data not shown), suggesting that the glycogen synthetase and the glycogen branching enzyme were not involved in pullulan biosynthesis. It can be seen from Table 3 that trehalose biosynthesis in the mutants Δglg1–1, Δglg2–16 and ΔDGLG-12 was enhanced, but glycogen biosynthesis in these mutants was reduced while complementation of the genes made the transformants GLG1B-18 and GLG2B-6 restore trehalose and glycogen biosynthesis (Table 3). Determination of the expression of various genes in the mutants Δglg1–1, Δglg2–16, ΔDGLG12 and their wild type strain P16 found that the transcriptional levels of the genes related to UDP-glucose and pullulan biosynthesis in the mutants Δglg1–1, Δglg2–16, ΔDGLG-12 were significantly decreased (Table 4). Especially, the GLG1 gene in the mutant Δglg1–1 and the GLG2 gene in the mutant Δglg2–16, both the GLG1 gene and the GLG2

Table 2 Pullulan production and cell dry weight (CDW) of the wild type strain P16 and the different complementary strains. Strains

Pullulan titer (g/L)

CDW (g/L)

P16 GLG1B-18 GLG2B-6 GCS1B-25 SGT1B-50

53.70 54.08 53.10 51.44 53.72

22.54 21.58 21.40 23.14 23.71

± ± ± ± ±

2.37 0.48 2.46 2.28 1.56

± ± ± ± ±

0.64⁎ 0.94⁎⁎ 0.28 0.62 0.56

Values are given as means from triplicate determination ± standard deviation (SD). Compared with those of the P16 strain. ⁎ P b .05. ⁎⁎ P b .01.

108.54 ± 3.91 69.16 ± 4.66⁎⁎ 72.34 ± 5.66⁎⁎ 107.26 ± 6.17

91.43 ± 3.56 93.33 ± 1.78 121.24 ± 3.67⁎⁎

Values are given as means from triplicate determination ± standard deviation (SD). Compared with those of the P16 strain, GluK: the gene encoding glucose kinase; PGM: the gene encoding phosphoglucose mutase; UGP: the gene encoding UDPGpyrophosphorylase; PUL1: the gene encoding pullulan synthetase; UGT1: the gene encoding UDP-glucosyltransferase; GCS1: the gene encoding ceramide glucosyltransferase; SGT1: the gene encoding sterol glucosyltransferase; GLG1, the gene encoding glycogenin1; GLG2, the gene encoding glycogenin2; AmAGS2: the gene encoding α-glucan synthase 2. ⁎ P b .05. ⁎⁎ P b .01.

99.03 ± 4.32 119.98 ± 3.33⁎⁎

4831.12 ± 203.67⁎⁎ 53.44 ± 2.77⁎⁎

94.16 ± 3.78 710.23 ± 30.56⁎⁎

99.40 ± 4.70

93.06 ± 9.21 183.30 ± 7.86⁎⁎ 0.49 ± 0.03⁎⁎ 28.08 ± 1.69⁎⁎ 118.26 ± 12.67 295.23 ± 2.65⁎⁎

89.23 ± 3.02 80.19 ± 5.25⁎⁎ 81.10 ± 3.95⁎⁎ 36.29 ± 2.88⁎⁎ 194.45 ± 25.83⁎⁎ 116.23 ± 7.08⁎⁎ 93.49 ± 7.12 117.33 ± 12.55⁎

AmAGS2 SGT1 GCS1 UGT1 PUL1 UGP PGM Genes Strains

Table 4 Transcriptional levels of various genes related to pullulan biosynthesis.

gene in the double mutant ΔDGLG-12 were not expressed (Table 4), confirming that they were totally abolished. All these results demonstrated that the glycogenins (Glg1 and Glg2) in A. melanogenum P16 were indeed implicated with both pullulan and glycogen primer biosynthesis. This was reasonable because the short α-(1,4)-glucosyl chain (primer) synthesized on the glycogenins was necessary for biosynthesis of the primers of both pullulan and glycogen (Fig. 1 and Table 1) [3]. This suggested that during pullulan biosynthesis, the short α-(1,4)-glucosyl chain synthesized by the Glg1 and Glg2 was also utilized as one primer of pullulan biosynthesis. However, it should be noted that all the disruptants still produced a considerable amount of pullulan (Fig. 1A), meaning that biosynthesis of the pullulan primers could happen in any other positions in the absence of the functional glycogenins. Indeed, it has been well known that the initiation step for glycogen biosynthesis in the yeast S. cerevisiae can take place using alternative primers [3]. S. cerevisiae cells lacking either one of these two genes (GLG1 and GLG2 genes) can accumulate glycogen at levels comparable to its wild-type cells [8]. Glycogen biosynthesis in A. pullulans was also found to be closely related to pullulan biosynthesis [9]. It also has been reported that α-1,4-glucan of starch in plants and algae is also synthesized [23]. This meant that the short α-(1,4)glucosyl chain could be utilized as the primers of pullulan, glycogen and starch biosynthesis because they had the similar the bond types and glucose units. In order to know if any other primers can be used for biosynthesis of pullulan, the cloned GCS1 gene coding for UDP-Glc:ceramide βglucosyltransferase [24] and the cloned SGT1 gene encoding UDPglucose:sterol glucosyltransferase [25] (Table 1) were disrupted, respectively. It can be observed from the data in Fig. 1A that the obtained Δgcs1–8 mutant in which the GCS1 gene was removed only yielded 36.76 ± 1.49 g/L pullulan while the Δsgt1–1 mutant in which the SGT1 gene was cleared up only yielded 28.42 ± 1.62 g/L pullulan, much less than that (53.70 ± 2.37 g/L pullulan) produced by its wild type strain P16. At the same time, after both the GCS1 gene and the SGT1 gene were simultaneously knocked-out, pullulan production (22.32 ± 1.35 g/L) by the ΔGS-17 mutant was further declined (Fig. 1A). Complementation of the GCS1 gene in the Δgcs1–8 mutant and the SGT1 gene in the Δsgt1–1 mutant made the recombinant yeast strains GCS1B-25 and SGT1B-50 restore pullulan production (Table 2). Similarly, glycogen biosynthesis in the mutants Δgcs1–8, Δsgt1–1 and ΔGS-17 was enhanced, but trehalose biosynthesis was not changed (Table 3), suggesting that the reduced pullulan synthesis could promote glycogen biosynthesis. This was reasonable because when pullulan biosynthesis in the mutants Δgcs1–8, Δsgt1–1 and ΔGS-17 was inhibited, most of glucose in the cells was flowed to glycogen biosynthesis. Expression of most of the genes involved in UDPglucose biosynthesis in the Δgcs1–8 mutant and the Δsgt1–1 mutant

GLG1

Values are given as means from triplicate determination ± standard deviation (SD). Compared with those of the P16 strain. ⁎ P b .05. ⁎⁎ P b .01.

100.24 ± 8.07 72.50 ± 1.17⁎⁎ 0.14 ± 0.02⁎⁎

GLG2

0.02 0.08⁎⁎ 0.01⁎⁎ 0.06⁎⁎

100.08 ± 4.61 1.11 ± 0.13⁎⁎ 224.52 ± 24.95⁎⁎ 126.37 ± 2.56⁎⁎ 41.13 ± 5.14⁎⁎ 0.81 ± 0.06⁎⁎ 32.13 ± 2.78⁎⁎ 900.06 ± 61.72⁎⁎ 135.57 ± 7.16⁎⁎ 145.11 ± 7.56⁎⁎

0.05 0.06⁎⁎ 0.17 0.04⁎⁎ 0.04⁎⁎ 0.05⁎⁎ 0.15⁎⁎

GluK

± ± ± ± ± ± ± ± ± ± ±

100.04 ± 3.31 79.72 ± 6.14⁎⁎ 33.18 ± 2.58⁎⁎ 120.45 ± 4.89⁎⁎ 0.12 ± 0.00⁎⁎ 37.47 ± 3.18⁎⁎ 0.10 ± 0.00⁎⁎ 73.87 ± 2.95⁎⁎ 70.24 ± 8.42⁎⁎

1.09 0.67 1.09 1.98 2.53 0.52 2.55 1.07 1.35 2.03 1.48

100.21 ± 7.51 74.45 ± 1.08⁎⁎ 235.64 ± 12.80⁎⁎ 2.01 ± 0.03⁎⁎ 54.20 ± 1.89⁎⁎ 180.30 ± 5.03⁎⁎ 4.02 ± 0.14⁎⁎ 255.96 ± 20.18⁎⁎ 219.91 ± 13.23⁎⁎

8.27 ± 0.91 12.48 ± 0.71⁎⁎ 15.87 ± 1.00⁎⁎ 10.28 ± 0.09⁎⁎ 9.85 ± 0.03⁎⁎ 4.61 ± 0.32⁎⁎ 12.53 ± 0.31⁎⁎ 12.78 ± 0.53⁎⁎ 6.86 ± 0.48⁎ 12.65 ± 0.27⁎⁎ 9.24 ± 0.15⁎

100.01 ± 1.95 81.70 ± 0.33⁎⁎ 264.08 ± 12.06⁎⁎ 78.24 ± 4.12⁎⁎ 36.12 ± 3.23⁎⁎ 149.27 ± 7.25⁎⁎ 57.44 ± 1.12⁎⁎ 177.29 ± 7.96⁎⁎

P16 Δglg1–1 Δglg2–16 Δgcs1–8 Δsgt1–1 ΔDGLG-12 ΔGS-17 GLG1B-18 GLG2B-6 GCS1B-25 SGT1B-50

100.02 ± 2.64 123.89 ± 6.98⁎⁎ 89.11 ± 4.11⁎ 198.23 ± 8.65⁎⁎ 240.56 ± 6.67⁎⁎ 96.41 ± 3.09 30.19 ± 2.64⁎⁎ 92.00 ± 5.99 70.47 ± 1.22⁎⁎ 154.37 ± 2.99⁎⁎ 191.45 ± 11.77⁎⁎

Intracellular glycogen content (μg glucose/108 cells)

100.04 ± 3.46 45.45 ± 3.09⁎⁎ 50.05 ± 6.42⁎⁎ 114.10 ± 7.21⁎⁎ 62.12 ± 3.89⁎⁎ 55.36 ± 1.23⁎⁎ 27.16 ± 1.77⁎⁎ 89.47 ± 2.33⁎ 65.54 ± 4.41⁎⁎ 86.40 ± 5.36⁎⁎

Intracellular trehalose content (μg/108 cells)

100.15 ± 3.67 82.51 ± 1.43⁎⁎ 172.55 ± 4.56⁎⁎ 83.01 ± 1.02⁎⁎ 47.41 ± 4.05⁎⁎ 118.15 ± 4.23⁎⁎ 26.34 ± 1.23⁎⁎ 116.23 ± 10.40⁎⁎

Strains

100.35 ± 5.74 58.33 ± 3.25⁎⁎ 78.93 ± 3.51⁎⁎

Table 3 Intracellular trehalose and glycogen contents of various mutants obtained during single and double gene deletion, transformant strains and their wild type strain P16.

100.00 ± 1.43 47.75 ± 1.16⁎⁎ 106.2 ± 1.28⁎ 215.99 ± 3.32⁎⁎ 13.45 ± 0.39⁎⁎ 158.2 ± 7.64⁎⁎ 6.97 ± 0.47⁎⁎ 170.2 ± 8.03⁎⁎ 124.12 ± 3.54⁎⁎ 116.98 ± 6.07⁎⁎ 156.08 ± 5.39⁎⁎

T.-J. Chen et al. / International Journal of Biological Macromolecules 147 (2020) 10–17

P16 Δglg1–1 Δglg2–16 Δgcs1–8 Δsgt1–1 ΔDGLG-12 ΔGS-17 GLG1B-18 GLG2B-6 GCS1B-25 SGT1–50

14

T.-J. Chen et al. / International Journal of Biological Macromolecules 147 (2020) 10–17

15

Fig. 2. The colony (A) and cell morphology (B) of the wild type strain P16 and different mutants obtained during the multi-genes disruption.

was down-regulated (Table 4). Especially, expression of the GCS1 gene in the Δgcs1–8 mutant, the SGT1 gene in the Δsgt1–1 mutant and both the GCS1 gene and the SGT1 gene in the ΔGS-17 strain were almost zero, In contrast, expression of the GCS1 gene in the recombinant yeast strain GCS1B-25 and the SGT1 gene in the recombinant yeast strain SGT1B-50 was highly up-regulated (Table 4). All the data mentioned above suggested that the Gcs1 and Sgt1 were also participated in biosynthesis of the pullulan primers and β-glucosylceramide and steryl βglycosides formed might also be used as the primers for biosynthesis of pullulan as mentioned below. Indeed, a CesA glucosyltransferase in plant initiates glucan polymerization by using sitosterol-β-glucoside (SG) as primer [26]. In addition, the colonies of the mutant ΔGS-17 became rough, matt and dried and the surface of the colonies was changed to be ruffle and irregular (Fig. 2) because of only low level (22.32 ± 1.35 g/L) of the produced pullulan by the this mutant (Fig. 1A). Furthermore, its cells became swollen (Fig. 2). 3.3. Abolishment of multi-genes in the pullulan producing yeast P16 As shown above, since pullulan biosynthesis in all the mutants obtained above could not be totally stopped (Fig. 1A), biosynthesis of the pullulan primers might occur on various compounds [3]. If these would happen, after all the multi-genes in the pullulan producing yeast P16 were simultaneously abolished, pullulan biosynthesis in the mutants might be completely inhibited. Therefore, the Cre/loxp system (Table S2) was used to continuously remove the GLG1 gene, the GLG2 gene, the UGT1 gene and the PUL1 gene shown in Table 1 based on the ΔGS-17 mutant in which both the GCS1 gene and SGT1 gene had been disabled (Fig. 1A). Finally, the ΔGSG-3 mutant without the GCS1 gene, SGT1 gene, GLG1 gene and GLG2 gene, the ΔMG-7 mutant in which all

the GCS1, SGT1, GLG1, GLG2 and UGT1 genes were disrupted and the ΔMP-6 mutant in which all the GCS1, SGT1, GLG1, GLG2, UGT1 and PUL1 genes were knocked-out were obtained (Table S1). It can be clearly noted from Fig. 1B that they only produced 11.98 ± 0.59 g/L, 8.36 ± 0.62 g/L and 5.12 ± 0.29 g/L pullulan, respectively. In contrast, glycogen, trehalose, intracellular polysaccharide biosynthesis and intracellular total sugar and reducing sugar in all the mutants were significantly enhanced, but the amount of intracellular glycerol was greatly reduced (Table 5). Table 6 indicated that expression of all the genes responsible for pullulan biosynthesis in the ΔGSG-3 mutant, the ΔMG-7 mutant and the ΔMP-6 mutant was greatly reduced compared to that of all the corresponding genes in their wild type strain P16. Especially, no expression of the GCS1 gene, the SGT1 gene, the GLG1 gene and the GLG2 gene in the ΔGSG-3 mutant, no expression of the GCS1, SGT1, GLG1, GLG2 and UGT1 genes in the ΔMG-7 mutant and no expression of the GCS1, SGT1, GLG1, GLG2, UGT1 and PUL1 genes in the ΔMP-6 mutant were detected (Table 6). However, after the remaining pullulan produced by all the mutants including the ΔGS-17, ΔGSG-3, ΔMG-7 and ΔMP-6 mutants and pullulan produced by their wild type strain P16 were hydrolyzed using the commercial pullulanase specific for hydrolysis of α-1,6-glycosidic linkages in pullulan molecules, maltotriose was still released from all the produced pullulan (Fig. 1C), demonstrating that the EPSs synthesized by all the mutants and their wild type strain P16 as shown in Fig. 1A and B were still pullulan. Therefore, other unknown glucosyltransferases which catalyze formation of the pullulan primers may exist in the yeast cells. Indeed, the initiation step of glycogen biosynthesis can take place using alternative primers [3]. Furthermore, in plants, more than one nonidentical CesA may be required for cellulose synthesis and the GhCesA-2 is a likely candidate among them [26]. As the amount of pullulan produced by different disruptants such as the mutants ΔGS-17, ΔGCS-3, ΔMG-7 and ΔMP-6 was continuously

Table 5 Intracellular trehalose, glycogen, polysaccharide, total sugar, glycerol contents and extracellular reducing sugar contents of the different disruptants obtained during the multi-genes disruption. Strains Intracellular trehalose content (μg/108 cells)

Intracellular glycogen content (μg glucose/108 cells)

Intracellular polysaccharide content (g/g CDW)

Intracellular total sugar content (g/g CDW)

Extracellular reducing sugar content (μg/mL)

Intracellular glycerol content (μg/μg protein)

P16 GSG-3 MG-7 MP-6

9.83 ± 0.82 37.36 ± 2.16⁎⁎ 50.05 ± 5.57⁎⁎ 49.86 ± 3.50⁎⁎

0.12 0.37 0.44 0.26

0.13 0.38 0.46 0.28

9.28 ± 0.22 24.72 ± 0.33⁎⁎ 26.70 ± 1.03⁎⁎ 31.47 ± 1.57⁎⁎

6.22 ± 0.02⁎ 2.08 ± 0.01⁎⁎ 1.9 3 ± 0.01⁎⁎ 2.80 ± 0.05⁎⁎

21.89 50.71 68.25 61.68

± ± ± ±

1.47 1.90⁎⁎ 3.65⁎⁎ 7.08⁎⁎

± ± ± ±

0.01 0.01⁎⁎ 0.02⁎⁎ 0.01⁎⁎

± ± ± ±

0.01 0.03⁎⁎ 0.03⁎⁎ 0.02⁎⁎

Values are given as means from triplicate determination ± standard deviation (SD). Compared with those of the P16 strain. ⁎ P b .05. ⁎⁎ P b .01.

16

T.-J. Chen et al. / International Journal of Biological Macromolecules 147 (2020) 10–17

Table 6 Transcriptional levels of various genes related to pullulan biosynthesis in wild type strain P16 and different disruptants obtained during the multi-gene disruption. Strains

P16 ΔGSG-3 ΔMG-7 ΔMP-6

Genes GluK

PGM

UGP

PUL1

UGT1

GCS1

SGT1

GLG1

GLG2

AmAGS2

100.07 ± 4.39 27.14 ± 2.24⁎⁎ 13.29 ± 0.51⁎⁎ 10.28 ± 0.37⁎⁎

100.02 ± 2.06 57.94 ± 3.98⁎⁎ 55.29 ± 2.88⁎⁎ 20.29 ± 0.98⁎⁎

100.10 ± 5.33 38.98 ± 3.05⁎⁎ 35.05 ± 1.27⁎⁎ 23.63 ± 1.06⁎⁎

100.03 ± 3.05 64.33 ± 7.07⁎⁎ 40.49 ± 6.73⁎⁎ 0⁎⁎

100.11 ± 5.73 58.23 ± 1.45⁎⁎ 0⁎⁎ 0⁎⁎

100.02 ± 2.64 0⁎⁎ 0⁎⁎ 0⁎⁎

100.07 ± 4.56 0⁎⁎ 0⁎⁎ 0⁎⁎

100.01 ± 1.70 0⁎⁎ 0⁎⁎ 0⁎⁎

100.12 ± 5.90 0⁎⁎ 0⁎⁎ 0⁎⁎

100.05 ± 3.69⁎ 0.99 ± 0.19⁎⁎ 0.97 ± 0.05⁎⁎ 0.67 ± 0.03⁎⁎

Values are given as means from triplicate determination ± standard deviation (SD). Compared with those of the P16 strain. All the genes are described in Table 4. ⁎ P b .05. ⁎⁎ P b .01.

reduced, their colonies became rough, matt and dried and the surface of the colonies was changed to be ruffle (Fig. 2). Similarly, the yeast cells of all the mutants became swollen compared to those of their wild type strain P16, suggesting that their cell wall integrity was deficient in addition to the enhanced accumulation of intracellular sugars (Fig. 2 and Table 5). Although the Pul1 had some influences on pullulan biosynthesis in A. melanogenum P16 (Table 1 and Fig. 1B), its function is still unclear. It might be an auxiliary protein for one component required for the αglucan synthetase 2 activity as stated previously in our studies [7] because both Pul1 and the amylase domain of the α-glucan synthetase 2 (AmAgs2) were extracellular. Indeed, an auxiliary protein called GNIP (for glycogenin interacting protein) occurs in human cells, which enhances the self-glucosylation reaction upon the binding of GNIP to the glycogenin [8]. In our previous studies [6], total removal of the UGT1 gene in the same yeast was found to only cause the reduced pullulan production and the enzyme encoded by the UGT1 gene was predicted to be a UDP-glucose:glycoprotein glucosyltransferase-like protein in endoplasmic reticulum (Table 1). We thought that this Ugt1 might serve as a “folding sensor” involved in efficiently glucosylating incompletely folded glycoproteins related to pullulan biosynthesis [27]. Indeed, the results in Table 1 showed that all the Gcs1, Sgt1, Glg2, Ugt1 and Pul1 were glycoproteins with more than one glycosylation sites.

3.4. The whole pathway for pullulan biosynthesis was proposed According to all the results mentioned above and those obtained in our previous studies [7], a new pullulan biosynthesis pathway was given in Fig. 3. This was the first time to give the evidence that the primers [the short α-(1,4)-glucosyl chains] formed on the Glg1, Glg2, Gcs1 and Sgt1 could be elongated to be the long α-(1,4)-glucosyl chains (pullulan precursors). Then, the long α-(1,4)-glucosyl chains were transported to outside the plasma membrane and the long α-(1,4)glucosyl chains in the periplasmic space were transformed into pullulan by the AmAgs2. Based on this new pullulan biosynthesis pathway, it could be easily explained why loss of function of some genes shown in Table 1 and the UGT1 gene alone did not result in a complete pullulan deficiency (Fig. 1A) [6]. Moreover, the detailed functions of some enzymes such as Ugt1 and Pul1 are still unclear. The complete elucidation of the pullulan biosynthesis pathway was of significant for enhancing pullulan biosynthesis by metabolic engineering and synthetic biology.

4. Conclusions This is the first time to have elucidated that the primers [the short α(1,4)-glucosyl chains] could be synthesized by the Glg1, Glg2, Gcs1, Sgt1

Fig. 3. The proposed pullulan biosynthesis pathway using various primers. AmAgs2: α-glycan synthetase 2; Amy_D: extracellular α-amylase catalytic domain; Gsy_D: intracellular GT1_Glycogen_synthetase domain and EPST_D: EPS_sugtrans domain (EPST_D) embedded in multiple transmembrane regions. Gys: glycogen synthetase. Glg1 and Glg2: glycogenin isoforms; Gcs1: ceramide β-glucosyltransferase; Sgt1: sterol glucosyltransferase; short α-(1,4)-glucosyl chain: pullulan primer; long α-(1,4)-glucosyl chain: pullulan precursor; Lph-G: phospholipid intermediate-glucose; HXT: Hexose transporter.

T.-J. Chen et al. / International Journal of Biological Macromolecules 147 (2020) 10–17

and still unknown proteins. Then, the primers could be elongated to be the long α-(1,4)-glucosyl chains (pullulan precursors) for pullulan biosynthesis. The complete elucidation of the pullulan biosynthesis pathway was of significant for understanding detailed mechanisms of yeast pullulan synthesis and regulation,. This also will be helpful for promoting pullulan biosynthesis by metabolic engineering and molecular editing using synthetic biology. Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2020.01.049. Acknowledgements This study was financially supported by National Natural Science Foundation of China (Grant No. 31770061). Declaration of competing interest The authors declare that there is no conflict of interest. References [1] L. Chen, Z. Chi, G.L. Liu, S.J. Xue, Z.P. Wang, Z. Hu, Z.M. Chi, Improved pullulan production by a mutant of Aureobasidium melanogenum TN3-1 from a natural honey and capsule shell preparation, Internat. J. Biol. Macromole. 141 (2019) 268–277. [2] R.S. Singh, G.K. Saini, Biosynthesis of pullulan and its applications in food and pharmaceutical industry, in: T. Satyanarayana (Ed.), Microorganisms in Sustainable Agriculture and Biotechnology, Springer Science+Business Media 2012, pp. 509–552. [3] C. D’Hulst, A. Merida, The priming of storage glucan synthesis from bacteria to plants: current knowledge and new developments, New Phytol. 188 (2010) 13–21. [4] J.M. Francois, T. Walther, J.L. Parrou, Genetics and regulation of glycogen and trehalose metabolism in Saccharomyces cerevisiae, in: Z.L. Liu (Ed.), Microbial Stress Tolerance for Biofuels, Microbiology Monographs 22, Springer-Verlag, Berlin Heidelberg 2012, pp. 29–54. [5] M.J. Torija, M. Novo, A. Lemassu, W. Wilson, P.J. Roach, J.M. Francois, J.L. Parrou, Glycogen synthesis in the absence of glycogenin in the yeast Saccharomyces cerevisiae, FEBS Lett. 579 (2005) 3999–4004. [6] X. Chen, Q.Q. Wang, N.N. Liu, G.L. Liu, Z. Chi, Z.M. Chi, A glycosyltransferase gene responsible for pullulan biosynthesis in Aureobasidium melanogenum P16, Internat. J. Biol. Macromole. 95 (2017) 539–549. [7] T.J. Chen, G.L. Liu, X. Wei, K. Wang, Z. Hu, Z. Chi, Z.M. Chi, A multidomain α-glucan synthetase 2 (AmAgs2) is the key enzyme for pullulan biosynthesis in Aureobasidium melanogenum P16, Intern. J. Biol. Macromole. (2019) https://doi. org/10.1016/j.ijbiomac.2019.10.108. [8] M.C. Bertolini, F.Z. Freitas, R.M. de Paula, F.B. Cupertino, R.D. Goncalves, Glycogen metabolism and regulation in Neurospora crassa, in: G. Witzany (Ed.), Biocommunication of Fungi, #Springer Science+Business Media, Dordrecht 2012, pp. 39–55.

17

[9] L. Simon, B. Bouchet, K. Bremond, D.L. Gallant, M. Bouchonneau, Studies on pullulan extracellular production and glycogen intracellular content in Aureobasidium pullulans, Canad. J. Microbiol 44 (1998) 1193–1199. [10] Z.C. Ma, W.J. Fu, G.L. Liu, Z.P. Wang, Z.M. Chi, High-level pullulan production by Aureobasidium pullulans var. melanogenum P16 isolated from mangrove system, Appl. Microbiol. Biotechnol. 98 (2014) 4865–4873. [11] Q.Q. Wang, Y. Lu, Z.Y. Ren, Z. Chi, G.L. Liu, Z.M. Chi, CreA is directly involved in pullulan biosynthesis and regulation of Aureobasidium melanogenum P16, Curr. Genet. 63 (2017) 471–485. [12] Z. Zhang, Y. Lu, Z. Chi, G.L. Liu, H. Jiang, Z. Hu, Z.M. Chi, Genome editing of different strains of Aureobasidium melanogenum using an efficient Cre/loxp site-specific recombination system, Fung. Biol. 123 (2019) 723–731. [13] Z.M. Chi, J. Liu, W. Zhang, Trehalose accumulation from soluble starch by Saccharomycopsis fibuligera sdu, Enzy. Microb. Technol. 38 (2001) 240–246. [14] Z. Chi, X.X. Wang, Z.C. Ma, M.A. Buzdar, Z.M. Chi, The unique role of siderophore in marine-derived Aureobasidium pullulans HN6.2, Biomet 25 (2012) 219–230. [15] J.D. Thompson, T.J. Gibson, F. Plewniak, F. Jeanmougin, D.G. Higgins, The clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools, Nucleic Acids Res. 24 (1997) 4876–4882. [16] Z.C. Ma, Z. Chi, Q. Geng, F. Zhang, Z.M. Chi, Disruption of the pullulan synthetase gene in siderophore-producing Aureobasidium pullulans enhances siderophore production and simplifies siderophore extraction, Process Biochem. 47 (2012) 1807–1812. [17] J.L. Parrou, J.M. Francois, A simplified procedure for a rapid and reliable assay of both glycogen and trehalose in whole yeast cells, Anal. Biochem. 248 (1997) 186–188. [18] Z.M. Chi, J. Liu, J.R. Ji, Z. Meng, Enhanced conversion of soluble starch to trehalose by a mutant of Saccharomycopsis fibuligera sdu, J. Biotechnol. 102 (2003) 135–141. [19] J. Kuhn, H. Müller, D. Salzig, P. Czermak, A rapid method for an offline glycerol determination during microbial fermentation, Electron. J. Biotechnol. 18 (2015) 252–255. [20] P.R. Stewart, in: D.M. Prescott (Ed.), Methods in Cell Biology, 12, Academic Press, London and New York 1982, pp. 111–147. [21] G.L. Liu, D.S. Wang, L.F. Wang, S.F. Zhao, Z.M. Chi, Mig1 is involved in mycelial formation and expression of the genes encoding extracellular enzymes in Saccharomycopsis fibuligera A11, Fung. Genet. Biol. 48 (2011) 904–913. [22] Y. Li, Z. Chi, G.Y. Wang, Z.P. Wang, G.L. Liu, C.F. Lee, Z.C. Ma, Z.M. Chi, Taxonomy of Aureobasidium spp. and biosynthesis and regulation of their extracellular polymers, Crit. Review. Microbiol. 41 (2015) 228–237. [23] S.G. Ball, Regulation of starch biosynthesis, in: J.D. Rochaix, M. GoldschmidtClermont, S. Merchant (Eds.), The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, Kluwer Academic Publishers, The Netherlands 1998, pp. 549–567 , Printed in. [24] Y. Hirabayashi, Y. Ishibashi, UDP-glucose: Ceramide glucosyltransferase (UGCG), in: N. Taniguchi, et al., (Eds.), Handbook of Glycosyltransferases and Related Genes, # Springer, Japan 2014, pp. 3–13. [25] S. Grille, A. Zaslawski, S. Thiele, J. Plat, D. Warnecke, The functions of steryl glycosides come to those who wait: recent advances in plants, fungi, bacteria and animals, Prog. Lipid Res. 49 (2010) 262–288. [26] L. Peng, Y. Kawagoe, P. Hogan, D. Delmer, Sitosterol-β-glucoside as primer for cellulose synthesis in plants, Sci 295 (2002) 147–150. [27] K. Totani, Y. Ihara, T. Tsujimoto, I. Matsuo, Y. Ito, The recognition motif of the glycoprotein-folding sensor enzyme UDP-Glc:glycoprotein glucosyltransferase, Biochem 48 (2009) 2933–2940.