Cell wall integrity is required for pullulan biosynthesis and glycogen accumulation in Aureobasidium melanogenum P16

Accepted Manuscript Cell wall integrity is required for pullulan biosynthesis and glycogen accumulation in Aureobasidium melanogenum P16

Tie-Jun Chen, Zhe Chi, Hong Jiang, Guang-Lei Liu, Zhong Hu, Zhen-Ming Chi PII: DOI: Reference:

S0304-4165(18)30079-5 doi:10.1016/j.bbagen.2018.03.017 BBAGEN 29068

To appear in: Received date: Revised date: Accepted date:

14 December 2017 11 March 2018 13 March 2018

Please cite this article as: Tie-Jun Chen, Zhe Chi, Hong Jiang, Guang-Lei Liu, Zhong Hu, Zhen-Ming Chi , Cell wall integrity is required for pullulan biosynthesis and glycogen accumulation in Aureobasidium melanogenum P16. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Bbagen(2018), doi:10.1016/j.bbagen.2018.03.017

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ACCEPTED MANUSCRIPT Title page

Title: Cell wall integrity is required for pullulan biosynthesis and glycogen

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Running title: Cell wall integrity and pullulan biosynthesis

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accumulation in Aureobasidium melanogenum P16

Author list: Tie-Jun Chen1, Zhe Chi1,3, Hong Jiang1, Guang-Lei Liu1,3, Zhong Hu2,

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Zhen-Ming Chi*1-3

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Corresponding author: Dr and Professor Zhen-Ming Chi

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Mailing address: 1College of Marine Life Sciences, Ocean University of China, Yushan Road, No. 5, Qingdao, China

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E-mail: [email protected]

1

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Tel and Fax: 0086-532-82032266

College of Marine Life Sciences, Ocean University of China, Yushan Road, No. 5, Qingdao, China

2

Department of Biology, Shantou University, Shantou 515063, China

3

Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science

and Technology, 266003, Qingdao, China

1

ACCEPTED MANUSCRIPT ABSTRACT _______________________________________________________________ Background: Pullulan and glycogen have many applications and physiological functions. However, to date, it has been unknown where and how the pullulan is synthesized in the

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yeast cells and if cell wall structure of the producer can affect pullulan and glycogen

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biosynthesis.

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Methods: The genes related to cell wall integrity were cloned, characterized, deleted and

gene expression were examined. In

this

study,

the

GT6

and

GT7

genes

encoding

different

α1,2

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Results:

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complemented. The cell wall integrity, pullulan biosynthesis, glycogen accumulation and

mannosyltransferases in Aureobasidum melanogenum P16 were cloned and characterized. ___________________________________

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Tie-Jun Chen and Zhe Chi make equal contribution to this study, *Corresponding author. Fax: +86 532 82032266. E-mail address: [email protected] (Z.-M. Chi).

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The proteins deduced from both the GT6 and GT7 genes contained the conserved sequences YNMCHFWSNFEI and YSTCHFWSNFEI of a Ktr mannosyltransferase family.

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The removal of each gene and both the two genes caused the changes in colony and cell morphology and enhanced glycogen accumulation, leading to a reduced pullulan biosynthesis and the declined expression of many genes related to pullulan biosynthesis. The swollen cells of the disruptants were due to increased accumulation of glycogen, suggesting that uridine diphosphateglucose (UDP-glucose) was channeled to glycogen biosynthesis in the disruptants, rather than pullulan biosynthesis. Complementation of the GT6 and GT7 genes in the corresponding disruptants and growth of the disruptants in the 2

ACCEPTED MANUSCRIPT presence of 0.6 M KCl made pullulan biosynthesis, glycogen accumulation, colony and cell morphology be restored. General significance: This is the first report that the two α1,2 mannosyltransferases were required for colony and cell morphology, glycogen accumulation and pullulan biosynthesis

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in the pullulan producing yeast.

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Keywords: A. melanogenum; α1,2 mannosyltransferases; Pullulan biosynthesis; Glycogen

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accumulation

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1. Introduction

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The yeast cell wall is composed of the major polymers, β-1, 6- and β-1, 3-glucans, glucomannoproteins, mannoproteins and chitin. This extracellular matrix forms an

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organelle that is dynamically involved with the plasma membrane and the underlying

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secretory organelles along with cytoskeletal and cytoplasm components in maintaining cell integrity during growth and morphogenesis. The inner layer of the yeast cell wall is

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comprised of glucan polymers (β-1, 6- and β-1, 3-glucans) and chitin while the outer cell

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wall layer is a lattice of highly glycosylated mannoproteins [1]. This suggests that different mannosyltransferases may play a role in biosynthesis of cell wall components of the outer layer and removal of the mannosyltransferases can cause defect in yeast cell surface integrity [2]. It has been proposed that different mannosyltransferase genes from Saccharomyces cerevisiae can be divided into KTR and MNN1 mannosyltransferase gene families. The KTR family contains many members, such as KRE2, YUR1, KTR1, KTR2, KTR3, KTR4, KTR5, KTR6 and KTR7 while the MNN1 family contains six members: 3

ACCEPTED MANUSCRIPT MNN1, TTP1, YGL257c, YNR059w, YIL014w and YJL86w. Among them, a typical Ktrp protein has a short amino-terminal cytoplasmic tail, a hydrophobic transmembrane domain, and a lumenal domain that includes a stem region which links a large catalytic domain to the membrane-spanning region. The most conserved region between all the Ktrps is

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423-YNLCHFWSNFEI-434 [2]. Although the position of the substrate or acceptor binding

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site or the mannosyltransferase catalytic residue is still unknown, the invariant glutamate at

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residue 433 of the 'NFEI' site mentioned above is a good candidate to be the active site nucleophile [2]. It has been reported that different members of each family has different

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functions. For example, Ktr1p and Ktr3p are Golgi α-1, 2-mannosyltransferases involved

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with Kre2p in adding the second and third mannose on O-linked glycans and also participating in N-linked outer chain synthesis [3].

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It has been confirmed that the yeast cells with perturbed cell walls are hypersensitive

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or resistant to calcofluor white, a cell surface polymer-intercalating drug, Congo red and H2O2 when compared with an isogenic wild-type control cell. A mutant with a disturbed or

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weakened cell wall is inhibited by the drugs that do not affect the growth of normal

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wild-type cells. For example, a ktr1 ktr3 double disruptant showed a level of sensitivity of the wild type, but a ktr1 ktr3 kre2 triple null mutant was more hypersensitive than a

kre2 mutant alone [2]. Pullulan, a best known yeast glucan, is a linear polysaccharide produced by different strains of Aureobasidium spp. The polymer consists of maltotriose units α-1, 4-linked to one another by α-1, 6-linkages [4]. Although its chemical structure was reported in 1960, its biosynthesis mechanism is still unclear [4]. So far, only α-phosphoglucose mutase 4

ACCEPTED MANUSCRIPT (Pgm), UDPG-pyrophosphorylase (Ugp), one glucosyltransferase (Ugt1) and pullulan synthase (Pul1) have been confirmed to be involved in pullulan biosynthesis [4-6]. However, to date, it has been unknown where and how the pullulan is synthesized in the yeast cells and if cell wall structure of the producer can affect its biosynthesis.

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Glycogen which is a carbon and energy store in yeast cells is a highly branched glucan

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of linear α-(1, 4)-glucosyl chains with α-(1, 6)-linkages. First, a glycogenin (Glg1 and

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Glg2) catalyzes synthesis of a short α-(1,4)-glucosyl chain by autoglucosylation using UDP-Glucose. Then, a glycogen synthase (Gsy1, Gsy2) catalyzes successive addition of

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α-1,4-linked glucose residues to the non-reducing end of glycogen, again using

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UDP-Glucose as the glucose donor. Finally, an amylo α-(1,4), α-(1,6)-transglucosidase (Glc3) transfers one linear α-(1, 4) glucosyl chain to another linear chain, making an α-(1,

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6) bond between glucosyl units [7]. Trehalose which is another reserve carbohydrate in

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yeast cells is a non-reducing disaccharide composed of two molecules of glucose linked at their 1-carbons. In yeast and filamentous fungi, trehalose is synthesized in a two-step First,

trehalose-6-phosphate

is

formed

from

UDP-glucose

and

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process.

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α-glucose-6-phosphate by a trehalose-6-phosphate synthase (Tps1). Then, this compound is dephosphorylated to trehalose and inorganic phosphate by a trehalose-6-phosphate phosphatase (Tps2) [8-9]. Therefore, in trehalose, pullulan and glycogen biosynthesis, the same UDP-Glucose is used as the glucose donor. However, it is still unknown if cell wall structure of the producer can affect glycogen and trehalose biosynthesis. In this study, two mannosyltransferase genes (the GT6 gene and the GT7 gene) in Aureobasdium melanogenum P16 were cloned and characterized. It was found that deletion 5

ACCEPTED MANUSCRIPT of the mannosyltransferase genes could negatively influence colony and cell morphology and pullulan biosynthesis, but enhance glycogen biosynthesis. This is the first report that the mannosyltransferases were related with glycogen accumulation and pullulan

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biosynthesis in A. melanogenum.

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2.1. Microbial strains, media, genes and plasmids

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2. Materials and methods

Aureobasdium melanogenum P16, a high pullulan producing yeast, was isolated from

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a mangrove ecosystem in Province of Hainan, China [10]. The yeast strain P16 was grown

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in a seed culture medium at 28 °C and 180 rpm for 24 h [10]. A pullulan production medium consisted of 120.0 g/L sucrose, 3.0 g/L yeast extract, 5.0 g/L K2HPO4, 0.2 g/L

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MgSO4·7H2O, 1.0 g/L NaCl, and 0.6 g/L (NH4)2SO4 [10]. An Escherichia coli DH5α

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bought from Beijing Tiangen Biotech Co. Ltd was grown in a Luria-Bertani broth (LB) and used for amplification of the recombinant plasmids. The E. coli transformants were grown

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in the LB medium with 100 μg/mL of ampicillin. The yeast transformants were grown in a

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YPD medium containing 100 μg/mL of hygromycin B or nourseothricin [11]. Two mannosyltransferase genes (the GT6 gene, accession number: KY31905 and the GT7 gene, accession number: MF289781) were PCR-amplified from the genomic DNA of A. melanogenum P16. A plasmid pMD 19-T simple vector for cloning of PCR products was purchased from TaKaRa (Japan). A plasmid pF14a-GT6 (4967 bp) carrying the hypromycin B resistance gene (the HPT gene) and a plasmid pWN302-GT7 (4510 bp) carrying the nourseothricin resistance gene (the NAT gene) for knock-out of the two 6

ACCEPTED MANUSCRIPT mannosyltransferase genes were constructed as described below. Two plasmids pNTX13-GT6 carrying the nourseothricin resistance gene (the NAT gene) and pAPX13-GT7 carrying the hypromycin B resistance gene (the HPT gene) were used for complementation of the two mannosyltransferase genes in the disruptants as described

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below.

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2.2. Bioinformatic analysis of the two mannosyltransferases genes The BLAST and ORF Finder programs at the National Center for Biotechnology

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Information (NCBI) were used for the nucleotide sequence analysis, deduction of the

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amino acid sequence from the two mannosyltransferases genes, and database searches. Multiple sequence alignments of the amplified DNA and the amino acid sequence of the

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deduced proteins were carried out using the programs of a DNAMAN 6.0

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(http://www.lynnon.com) and a Clustal X 1.8 [12]. A phylogenetic tree of the α-1, 2-mannosyltransferases from different yeasts, filamentous fungi and bacteria was

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constructed by using a MEGA7.0. Transmembrane helices prediction and three dimension

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analysis of the proteins were conducted on http://www.cbs.dtu.dk/services/TMHMM-2.0/ website

and

http://www.rcsb.org/pages/search_features#search_sequences

website,

respectively.

2.3. Construction

of

the

knock-out

vectors

for

disruption

of

the

two

mannosyltransferases genes and the expression vectors for complementation of the two genes 7

ACCEPTED MANUSCRIPT The DNA fragments 5’-arm-Poly(A)-hygromycin B phosphotransferase (HPT) gene–TEF promoter-arm-3’ and 5’-arm-Poly(A)-nourseothricin sulfate (NAT) gene–PGK promoter-arm-3’ for disruption of the GT6 gene and GT7 gene were constructed based on the procedures described by Chi et al. [13] (Supplementary file 1). For example, the 5’-arm

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and 3’-arm of the GT6 gene (accession number: KY31905) cloned above were PCR

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amplified from the genomic DNA of A. melanogenum P16 using the primers

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GT6-5F/GT6-5R and GT6-3F/GT6-3R (Supplementary file 1). The 5’-arm and 3’-arm of the GT7gene (accession number: MF289781) cloned above were PCR amplified from the

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genomic DNA of A. melanogenum P16 using the primers GT7-5F/GT7-5R and

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GT7-3F/GT7-3R (Supplementary file 2). The 5’-arm and 3’-arm of the GT6 gene obtained were digested with SphI/SalI and BamHI/EcoRI, respectively. Meanwhile, the 5’-arm and

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3’-arm of the GT7 gene obtained were also digested with PstI/SalI and BamHI/EcoRI,

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respectively. The digested 5’-arm and 3’-arm of the GT6 gene were ligated into the plasmid pFL4a (digested with the same enzymes) to form pFL4a-GT6 (Supplementary

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file 1). Similarly, the digested 5’-arm and 3’-arm of the GT7 gene were ligated into the

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plasmid pWN302 (digested with the same enzymes) to form pWN302-GT7 (Supplementary file 2). Finally, the linear DNA fragments the 5’-arm-Poly(A)-hygromycin B

phosphotransferase

(HPT)

gene–TEF

promoter-arm-3’

and

5’-arm-Poly(A)-nourseothricin sulfate (NAT) gene–PGK promoter-arm-3’ were obtained by digestion of the plasmid pFL4a-GT6 with the enzymes SphI and EcoRI (Supplementary file 1), and of the plasmid pWN302-GT7 with the enzymes PstI and EcoRI (Supplementary file 2). 8

ACCEPTED MANUSCRIPT To further know the functions of the mannosyltransferases genes in pullulan biosynthesis in A. melanogenum P16, an expression vector pNTX13-GT6 for complementation of the disrupted GT6 gene was constructed (Supplementary file 3). The CDSs of the GT6 gene were PCR-amplified from the cDNAs of A. melanogenum P16

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using the primers GT6-F/GT6-R with the enzyme sites of MluI and KpnI (Supplementary

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file 5). The PCR products were digested with MluI and KpnI and the digested CDSs were

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ligated into the plasmid pNTX13 digested with the same restriction endonucleases, resulting in formation of the expression vector pNTX13-GT6 (Supplementary file 3).

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Meanwhile, another expression vector pAPX13-GT7 for complementation of the disrupted

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GT7 gene was yielded (Supplementary file 4). The CDSs of the GT7 gene were PCR-amplified from the cDNAs of A. melanogenum P16 using the primers GT7-F/GT7-R

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with the enzyme sites of PstI/SpeI (Supplementary file 5). The PCR products were digested

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with MluI and KpnI and the digested CDSs were ligated into the plasmid pAPX13 digested with the same restriction endonucleases, resulting in formation of the expression vector

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pAPX13-GT7 (Supplementary file 4). The plasmids were amplified in the E. coli DH5α

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and purified from the E. coli transformants.

2.4. Disruption and complementation of the two mannosyltransferases genes Preparation and transformation of the competent yeast cells of A. melanogenum P16 were performed using the procedures described by Chi et al. [13]. The transformation of the competent yeast cells of A. melanogenum P16 was accomplished by incubating 0.2 mL of

the

spheroplast

suspension

at

22 9

°C

with

at

least

1.0

μg

of

the

ACCEPTED MANUSCRIPT 5’-arm-polyA-HPT-TEF-3’-arm fragment and 5’-arm-polyA-NAT-PGK-3’-arm fragments obtained above according to the methods described by Chi et al. [13]. The cell suspension was then spread onto a two-layer HCS (Holliday complete medium containing 1.0 M sorbitol) agar plate, with the bottom layer containing 50.0 μg/mL of hygromycin B or

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nourseothricin sulfate, respectively, and the top layer consisting of the HCS without the

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antibiotics. The cells were then kept at 28 °C for 3–4 days and the transformed colonies

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generally appeared after 3 days of the incubation. The putative disruptants including a strain 6-1 were verified by cultivation on a HCS agar plate containing 100.0 μg/mL of

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hygromycin B. Meanwhile, the putative disruptants including a strain 6727 were verified

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by cultivation on a HCS agar plate containing 100.0 μg/mL of nourseothricin sulfate. In order to remove both the two genes, 5’-arm-polyA-NAT-PGK-3’-arm fragments

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obtained above were transformed into the competent cells of the strain 6-1 obtained above.

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Many putative disruptants including a strain P2 were verified by cultivation on the HCS agar plate containing both 100.0 μg/mL of hygromycin B and nourseothricin sulfate. the

same

time,

the

linear

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At

rDNA

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rDNA-PGK-NAT-PolyA-GT6-TEF1-18S

DNA

rDNA-TEF1-HPT-PolyA-GT7-TEF1-18S

rDNA

from

the

fragments

26S

and

26S

expression

vectors

pAPX13-GT6 and pAPX13-GT7 (Supplementary files 3 and 4) were transformed into the competent cells of the disruptant 6-1 and the disruptant 6727, respectively, as described above. Finally, two transformants B61 and B76 were obtained. All the disruptants, the transformants and their wild type strain P16 were aerobically cultivated in the pullulan production medium at 28 °C and 180 rpm for 5 days. At the same 10

ACCEPTED MANUSCRIPT time, all the disruptants were also cultivated in the pullulan production medium with 0.6 M KCl at 28 °C and 180 rpm for 5 days. The purification and quantitative determination of the produced pullulan were carried out as described below.

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2.5. Purification and quantitative determination of the produced pullulan

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The purification and quantitative determination of the produced pullulan by A.

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melanogenum P16, the positive disruptants 6-1, 6727 and P2, the transformants B61 and

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B76 were carried out according to the methods described by Ma et al. [10].

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2.6. Observation of their colony morphology

The yeast cells of A. melanogenum strain P16, the positive disruptants 6-1, 6727 and

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the disruptant P2, the transformants B61 and B76 obtained above were cultivated on the

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YPD plates for 48 h at 28 °C. Color, margin and surface of the colonies of the different

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strains were observed and photographed.

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2.7. Susceptibility of the disrupted mannosyltransferases mutant strains to cell wall disturbing compounds To test susceptibility to the cell wall disturbing compounds (Congo Red and H 2O2), the yeast cells of A. melanogenum P16, all the disruptants 6-1, 6727, P2 and the transformants B61 and B76 were cultivated on the YPD plates with Congo Red (its concentrations were 200 μg/mL, 300 μg/mL and 400 μg/mL) and H2O2 (its concentrations were 0.48 mM and 0.975 mM) at 28 °C for 4 days. 11

ACCEPTED MANUSCRIPT Meanwhile, the yeast cells of A. melanogenum P16, all the disruptants 6-1, 6727, P2 and the transformants B61 and B76 were cultivated on the YPD plates with 0.6 M KCl and colony morphology and cell growth of the different strains were observed and photographed

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as described above.

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2.8 Observation of the yeast cells and cell size measurement

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The yeast cells of A. melanogenum P16, all the disruptants 6-1, 6727, P2 and the transformants B61 and B76 obtained above were washed two times by centrifugation with

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a PBS buffer (pH 7.0). The washed yeast cells were observed under a phase microscope

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with a 100 × oil immersion objective. The images were recorded using a cellSens Standard software and photographed.

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At the same time, the surface area and circumference of each yeast cell were measured

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and calculated using a software provided by the Olympus Company. 2.9. Intracellular trehalose, total sugar and polysaccharide contents measurement

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The washed yeast cells of A. melanogenum P16, all the disruptants 6-1, 6727 and P2

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obtained above were disrupted with a high pressure cracker (CONSTANT SYSTEM LTD) and the cell-free extracts of them were prepared as described by Zhang et al. [14]. The disrupted cells were centrifuged at 12,000 × g and 4 °C for 10 min, and the polysaccharides in 10 mL of the supernatants were precipitated by adding 30 mL of 95% absolute ethanol. The precipitation was incubated at 4 °C for 10 h. The suspension was centrifuged at 10,000  g for 5 min, the precipitate was dried at 80 °C until its weight was constant and the amount (mg) of the precipitate per mg of cell dry weight was calculated. 12

ACCEPTED MANUSCRIPT The precipitate (10 mg) was dissolved in 0.9 mL of 50 mM sodium acetate and acetic acid buffer (pH 4.5), the solution was mixed with 0.1 mL of a commercial pullulanase (Sigma, USA) and the mixture was incubated for 15 min at 60 °C [10]. At the same time, the commercial pullulan and maltotriose were also treated using the same methods. The

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released reducing sugars was identified by ascending thin layer chromatography (Silica gel

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60, MERCK, Germany) with the solvent system of n-butanol–pyridine–water (6:4:3) and a

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detection reagent comprising 20.0 g/L diphenylamine in acetone–20.0 g/L aniline in acetone–850.0 g/L phosphoric acid (5:5:1, v/v/v) [15]. The pure sucrose, glucose and

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maltotriose were used as the standards. Meanwhile, the total sugar in the supernatants was

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measured using the methods described by Chi et al. [16] and Spiro, [17]. The yeast strains were aerobically cultivated in the pullulan production medium for 5

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days at 28 °C and 180 rpm. The yeast cells in the cultures were stained using an iodine

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solution (0.01 M I2, 0.03 M KI) for 1 min [18]. The iodine-stained yeast cells were observed under the phase microscope and photographed. Meanwhile, the yeast cells were

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treated at 95 °C and 0.25 M Na2CO3 for 4 h, the pH the cell suspension were adjusted to

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pH 5.2 by addition of 0.15 mL of 1.0 M acetic acid and 0.6 mL of 0.2 M Na-acetate and the cell suspensions were hydrolyzed at 57 °C for 10 h under constant agitation using a commercial amyloglucosidase (1.2 U/mL) (Aspergillus niger lyophilized powder, 30-60 units/mg protein, ≤0.02% glucose, Cas:A7420 MSDS, Sigma), which could specifically degrade glycogen into glucose. The released glucose was quantitatively measured using a glucose determination kit (Nanjing Jiancheng Bioeng Institute, Nanjing, China) and the amount (mM) of the glycogen per 108 cells was calculated. [19]. 13

ACCEPTED MANUSCRIPT At the same time, all the trehalose in the washed cells was extracted with 0.5 M ice-cold trichloroacetic acid (TCA) for three times according to the methods described by Chi et al., [16]. All the extracts were combined and the amount of trehalose in the extract was analyzed by using a high-performance exchange anionic chromatography (HPEAC) on a

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Dionex Bio-LC 500 system (Sunnyvale, CA, USA) using a Carbo Pac PA1

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anion-exchange column (4  250 mm) as described by Chi et al., [16]. The pure trehalose

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from Sigma was used as a standard. The amount (mg) of the trehalose per mg of cell dry

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weight was calculated

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3. Results

3.1. Cloning and characterization of the GT6 and GT7 genes in A. melanogenum P16

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Supplementary files 6 and 7 showed that an ORF of the GT6 gene had 1756 bp with

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four introns, encoding a protein with 513 amino acids, PI of 6.15 and a molecular weight of 59.8 kDa while an ORF of the GT7 gene had 1271 bp with one intron, encoding a

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protein with 405 amino acids, PI of 6.28 and a molecular weight of 47.1 kDa. In the

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promoter of the GT6 gene, there were one TATA box, one CAAT box and one CCAAT box, but no conserved sequences 5’-HGATAR-3’ and 5′-SYGGRG-3′ (Supplementary file 6) while in the promoter of the GT7 gene, there were two CAAT boxes, one conserved sequence 5’-HGATAR-3’ and one conserved sequence 5′-SYGGRG-3′ (Supplementary file 7). The proteins deduced from the cloned GT6 gene and GT7 gene had the conserved sequences YNMCHFWSNFEI and YSTCHFWSNFEI of the Ktr mannosyltransferase 14

ACCEPTED MANUSCRIPT family, respectively (Supplementary files. 6 and 7). This meant that the proteins deduced from the cloned GT6 gene and GT7 gene belonged to the Ktr mannosyltransferase family. After phylogenetic analysis of both the proteins deduced from the GT6 gene and the GT7 gene in A. melanogenum P16, the results in Fig. 1 indicated that both the proteins

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indeed belonged to the members of the mannosyltransferase. However, they were not

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clustered together in the phylogenetic tree, meaning that these two mannosyltransferases

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may have different catalytic activities.

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3.2. Knockout of the GT6 and GT7 genes

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Fig. 1

In order to confirm if the two proteins are implicated with pullulan biosynthesis of A.

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melanogenum P16, the GT6 gene alone and the GT7 gene alone were removed,

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respectively, and both the GT6 gene and GT7 gene were simultaneously deleted as described in Materials and methods. The data in Fig. 2 revealed that a disruptant 6-1

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deficient in the GT6 gene and a disruptant 6727 without the GT7 gene produced much less pullulan than its wild type strain P16. Especially, it can be clearly seen from the data in Fig.

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2 that the values of the expolysaccharide/cell dry weight of the disruptant 6-1 and the disruptant 6727 were much lower than those of its wild type strain P16. Fig. 2 indicated that a disruptant P2 in which both the GT7 gene and the GT7 gene were simultaneously knocked out also yielded much less pullulan than its wild type strain P16, but did not further reduce pullulan titer compared to the disruptants 6-1 and 6717. This meant that disruption of the GT6 gene and the GT7 gene negatively affected pullulan biosynthesis.

15

ACCEPTED MANUSCRIPT Fig. 2

3.3. Evidence for the damaged cell wall integrity of the disruptants It could be clearly seen from the data in Fig. 3 that when the wild type strain P16 was

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grown on the YPD plate, its colonies were pinkish, the colony surface was smooth and

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slimy due to abundant exopolysaccharide formation and margin of the colony was

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composed of arachnoid and thick undulating hyphae. All these were the characteristics of A. melanogenum [20]. However, the colonies of both the disruptants 6-1 and 6727 and the

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disruptant P2 were not pinkish, the colony surface was rough and not slimy, but

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significantly wrinkled and thick undulating hyphae of the colony margin disappeared (Fig. 3). This meant that the removal of the GT6 and GT7 genes rendered the colony

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morphology of the disruptants to be greatly changed compared to that of their wild type

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strain P16.

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 Fig. 3

In order to know if the changes in colony morphology of the disruptants are related to

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the changes in cell wall and cell morphology, the yeast cells were observed under the microscope and photographed. It can be very clearly seen from the photos in Fig. 4 that all the cells of the disruptants were swollen, become irregular and were not the yeast-like cells compared to those of their wild type strain P16 were. For example, the area (29.42 μm2) and circumference (19.54 μm) of the cells of the yeast strain P16 was much lower than those (102.72 μm2 and 35.96 μm), (89.21 μm2 and 33.54 μm) and (115.04 μm2 and 38.02

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ACCEPTED MANUSCRIPT μm) of the cells of the disruptant 6-1, the disruptant 6727 and the disruptant P2, respectively (data not shown). These may be due to the defect in the cell wall integrity of the disruptants because of the genes deletion. In order to get the evidence to show the defect in the cell wall, the disruptant 6-1, the

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disruptant 6727, the disruptant P2 and their parent strain P16 were grown on the YPD

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plates with 0.6 M KCl, an osmotic stabilizer [21]. The results Fig. 5 showed that the cell

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and colony morphologies of the disruptant 6-1, the disruptant 6727, the disruptant P2 grown on the plates were totally restored in the presence of the osmotic stabilizer. In the

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presence of 0.6 M KCl, pullulan biosynthesis and cell growth of the disruptant 6-1 and the

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disruptant 6727 were also restored (Fig. 6). However, pullulan biosynthesis and cell growth of the disruptant P2 were unable to be restored in the presence of 0.6 M KCl (Fig. 6). The

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reasons for this are still unknown. May be the cell wall integrity of the double disruptant

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P2 was more severely damaged than that of the single disruptants. This meant that the cell

 Fig. 4

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 Fig. 5

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wall integrity of the disruptants 6-1and 6727 was indeed negatively damaged.

 Fig. 6

It has been confirmed that Congo red can specifically bind to β-1,3 glucan in the yeast cell wall, resulting in inhibition of normal cell wall assemble, disturbance of cell wall stability and stop of cell growth [22] while the fungal cells deficient in cell wall integrity were highly sensitive to H₂ O₂ [23]. Therefore, effects of different concentrations of

17

ACCEPTED MANUSCRIPT Congo red and H₂ O₂ on the yeast cell growth of the wild type strain P16, the disruptant 6-1, the disruptant 6727 and the disruptant P2 on the YPD plates were examined. The results in Fig. 7 demonstrated that in the presence of different concentrations of Congo red, the colony sizes of all the disruptants were smaller than those of their wild type strain P16.

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Especially, cell growth of all the disruptants was highly sensitive to different

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concentrations of H₂ O₂ compared to that of their wild type strain P16 (Fig. 7).

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Furthermore, the disruptant cells could absorb more Aniline blue than the wild type strain P16 (Supplementary file 8).

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All these results mentioned above demonstrated that the cell wall integrity of all the

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disruptants was damaged after the GT6 and GT7 genes were removed, resulting in the changes in colony morphology (Fig. 3) and in cell morphology (Fig. 4) and the reduced

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pullulan biosynthesis (Fig. 2) in the disruptants.

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Fig. 7

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3.4. Intracellular polysaccharide, total sugar and trehalose content analysis

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It can be seen from the data in Fig. 4 that all the yeast cells of the disruptants were swollen and the cell sizes were much larger than those of their wild type strain P16 (data not shown). In order to know why all the yeast cells of the disruptants were swollen and the cell sizes were much larger than those of their wild type strain P16, the washed cells were broken, the total sugars in the cell free extracts were measured and the polysaccharides in the cell free extracts were precipitated with absolute ethanol and the amount of the precipitated polysaccharide was determined as described in Materials and 18

ACCEPTED MANUSCRIPT methods. The results in Fig. 8A showed that the amounts of the total sugar and polysaccharide in the disruptants were much higher than those of the total sugar and polysaccharide in their wild type strain P16. This meant that more intracellular sugar and polysaccharide were accumulated in the cells of all the disruptants than those

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accumulated in the cells of their wild type strain P16 were, resulting in the swollen cells

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of the disruptants (Fig. 4). This suggested that due to defects in cell wall integrity (Figs. 5,

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6 and 7) more polysaccharides and total sugar were accumulated in the cells of the disruptants than in those of their wild type strain P16, leading to the reduced pullulan

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production (Fig. 2). It has been well known that the yeast cells can accumulate glycogen

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and trehalose in their cells [7]. Therefore, the accumulated total sugar may contain trehalose and the precipitated polysaccharide may be glycogen or pullulan. In order to

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confirm this, the extracted polysaccharides were hydrolyzed by using the pullulanase as

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described in Materials and methods. On contrary to our hypothesis, the results in Fig. 9 showed that the extracted polysaccharides could not be hydrolyzed into maltotriose, the

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basic subunits of pullulan by the commercial pullulanase, demonstrating that the

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extracted polysaccharides did not contain pullulan. However, commercial pullulan could be hydrolyzed into maltotriose by the commercial pullulanase (Lane 14 in Fig. 9), but maltotriose could not be hydrolyzed by the commercial pullulanase, either (data not shown).

It has been well known that the yeast cells that accumulate a large amount of intracellular glycogen can be stained by the iodine solution (0.01 M I2, 0.03 M KI) and 19

ACCEPTED MANUSCRIPT the stained glycogen will be brown [18]. So, the cultivated yeast cells of all the disruptants and their wild type strain P16 were stained using the iodine solution. It was found that that the yeast cells of all the disruptants were stained to be strongly brown whereas the yeast cells of the wild type strain P16 were stained to be only weakly brown

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(Fig. 8B). At the same time, the glycogen released from the whole cells was specifically

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hydrolyzed using the commercial amyloglucosidase and released glucose was measured

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as described in Materials and methods. It can be clearly seen from the results in Fig. 8C that the amount of glycogen in all the disruptants was higher than that of glycogen in

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their wild type strain P16. All the results demonstrated that the yeast cells of all the

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disruptants accumulated much more glycogen than those of their wild type strain P16, resulting in the decreased pullulan biosynthesis in the disruptants.

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In contrast, analysis of the trehalose extracted from the yeast cells revealed that all

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the disruptants stored much less trehalose than their wild type strain P16 (Fig. 8A).  Fig. 8

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 Fig. 9

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3.5. Complementation of the GT6 and GT7 genes in the disruptants 6-1 and the disruptant 6727 After the GT6 and GT7 genes were complemented in the disruptants 6-1 and the disruptant 6727, respectively, pullulan biosynthesis (Fig. 2), colony morphology (Fig. 3) and cell morphology (Fig. 4) in the transformants B61 and B76 obtained were totally restored. Furthermore, like their wild type strain P16, the cell growth of the transformants

20

ACCEPTED MANUSCRIPT B61 and B76 were not sensitive to the inhibition by Congo red and H2O2 (Fig. 7), suggesting that cell wall integrity of the transformants B61 and B76 was also restored. Meanwhile, the amount of the accumulated glycogen and trehalose in the transformants B61 and B76 was also the same as that of the accumulated glycogen and trehalose in the

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yeast strain P16 (Fig. 8).

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4. Discussion

In the promoter of the GT6 gene, there were no conserved sequences 5’-HGATAR-3’

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and 5′-SYGGRG-3′, suggesting that expression of the GT6 gene was not influenced by

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both high concentrations of nitrogen source and glucose [11]. However, in the promoter of the GT7 gene, there were one conserved sequence 5’-HGATAR-3’ and one conserved

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sequence 5′-SYGGRG-3′, indicating that expression of the GT7 gene was influenced by

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both high concentrations of nitrogen source and glucose [11]. It has been reported that all the Ktrps have the conserved sequence

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423-YNLCHFWSNFEI-434 and the invariant glutamate at residue 433 of the 'NFEI' site is

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a good candidate to be the active site nucleophile [2]. Indeed, the proteins deduced from both the cloned GT6 gene and the GT7 gene had one such conserved sequence CHFWSNFEI of the Ktr mannosyltransferase family (Supplementary files 6 and 7). This meant that the proteins encoded by the GT6 gene and the GT7 gene belonged to the members of the Ktr mannosyltransferase family and glyco-transf-15 superfamily (Fig. 1). This

superfamily

mainly

includes

α1,2

mannosyltransferase

responsible

for

N-glycosylation and O-glycosylation of oligosaccharides and the glycoprotein in the Golgi 21

ACCEPTED MANUSCRIPT complex of the yeast cells. It has been well documented that the genes coding for the mannosyltransferases are responsible for the synthesis of linear α1,6, α1,3 and α1,2 mannan which can be connected to various proteins, enzymes, oligosaccharides and lipids [24]. However, the protein deduced from the cloned GT6 gene had a transmembrane

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domain and its three-dimension structure was similar to that of α-1, 2 mannosyltransferase

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Kre2p/Mnt1p of S. cerevisiae (the supplementary 9), but the protein deduced from the

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cloned GT7 gene had no such transmembrane domain and its three-dimension structure was similar to that of a complex of a mannosyltransferase Ktr4p and GDP of

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Saccharomyces cerevisiae (the supplementary 9). This suggested that the proteins deduced

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from the cloned GT6 gene and GT7 gene were significantly different. Fig. 1 showed that the proteins encoded by the GT6 gene and the GT7 gene indeed

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belonged to α1,2 mannosyltransferase although they were not clustered in the same branch.

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Even though the genes involved in yeast mannan synthesis have been identified, the respective function of the encoded proteins in the biosynthesis of mannans is not fully

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understood. The N-mannan chains are usually considered as a “coating” component of the

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yeast cell wall while the O- and N-mannosylation pathways in S. cerevisiae and Candida albicans are required for cell wall integrity, dimorphism, adhesion, virulence and recognition by the host innate immune system. For example, C. albicans N-mannans are the structures composed of a core oligosaccharide (Man9GlcNAc2) synthesized in the endoplasmic reticulum (ER) and is further modified in the Golgi complex with a α1,6-mannose polymer [25], which is further modified with lateral mannose branches that in S. cerevisiae and C. albicans are synthesized by the α1,2-mannosyltransferases Mnn2 22

ACCEPTED MANUSCRIPT and Mnn5, adding the first and second mannose residues to the branches, respectively [24]. This suggested that both the proteins deduced from the GT6 gene and the GT7 gene from A. melanogenum P16 may be involved in addition of the first and second mannose residues to the mannose branches.

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Removal of the GT6 gene alone or the GT7 gene alone and both the two genes resulted

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in obvious decline in pullulan biosynthesis (Fig. 2). So far, only deletion of a pullulan

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synthase gene which functions are still unknown and the UGT1 gene encoding a glucosyltransferase in Aureobasidium spp. has led to the decrease in pullulan biosynthesis

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[5-6] because the whole pullulan biosynthetic pathway in Aureobasidium spp. is still

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unclear [4,6]. It has been reported that Kre2p, Ktr1p and Ktr3p may form a protein subfamily in S. cerevisiae and in a ktr1 ktr3 kre2 triple mutant the third mannose was

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missing, and a severely reduced level of mannose was observed [3]. In a strain where the

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KTR1 and KTR3 genes are disrupted, the Kre2p is able to add both the second and third α-1, 2-mannose residues in the linear α-1, 2-mannose carbohydrate chains [27]. Indeed, it has

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been known that the OCH1 gene in S. cerevisiae encodes a α1, 6-mannosyltransferase

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functioning in the initiation of the mannose outer chain addition to the ER-form core oligosaccharide while the MNN1 gene was responsible for this addition of the terminal α1, 3-mannose residues on both N- and O-linked oligosaccharides. The mnn1 och1 double mutant exhibited defects in cell cytokinesis, showed a slower growth rate, and became temperature-sensitive. Meanwhile, the mnn1 och1 mutant tended to aggregate, which was probably due to the glycosylation defect [27]. This meant that deletions of the GT6 gene and the GT7 gene may lead to defects in cell wall integrity (Figs. 5, 6 and 7, 23

ACCEPTED MANUSCRIPT Supplementary file 8) and changes in colony and cell morphology (Fig. 3, 4 and 8) so that pullulan biosynthesis was negatively influenced (Fig. 2). Therefore, this is the first time to get the evidence that deletion of the GT6 gene and GT7 gene could result in defects in cell wall integrity and the reduced pullulan biosynthesis (Fig. 2).

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The knocked out GT6 gene or GT7 gene or both the two genes caused significant

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changes in colony morphology (Fig. 3), cell morphology (swollen cells) (Fig. 4), cell

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volume (data not shown) and cell wall integrity (sensitivity to Congo red and H2O2) (Fig. 7) compared to those in colony morphology (Fig. 3), cell morphology (swollen cells) (Fig. 4),

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cell volume (data not shown) and cell wall integrity (sensitivity to Congo red and H2O2)

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(Fig. 7) of their parent strain P16. At the same time, when the disruptants were grown on the YPD plates with 0.6 M KCl, colony and cell morphologies of the disruptants were

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totally restored (Fig. 5) and when the disruptants were cultivated on the pullulan

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production medium with 0.6 M KCl, pullulan production and cell growth by the disruptants 6-1 and 6727 were also recovered (Fig. 6). It has been observed that the cell

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morphologies of the mnn1 och1 mutant and the mnn1 mutant were different from

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those of their wild-type cells of S. cerevisiae because of the mannosylation defects [27]. This may also happen in the cells of the disruptant 6-1, the disruptant 6727 and the disruptant P2 (Figs 3 and 4). It has been reported that deletion of one member and many members of the Ktr family can cause the damage of cell wall in S. cerevisiae, Schizosaccharomyces pombe, Aspergillus spp. and C. albicans and sensitivity of the disruptants to cell wall disturbing compounds such as Congo red and H2O2 [22-23, 28]. Therefore, it was true that all the disruptants obtained in this study were sensitivity to 24

ACCEPTED MANUSCRIPT Congo red and H2O2 because of the defect in cell wall integrity (Fig. 7). At the same time, it was found that total sugar and polysaccharides in the yeast cells of the disruptants were much higher than those in their parent strain P16 (Fig. 8A). But the amount of the trehalose in in the yeast cells of the disruptants was much less than that of

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the trehalose in their parent strain P16 was (Fig. 8A) and the polysaccharides in the cells

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were not pullulan (Fig. 9). Finally, it was interesting to note that the yeast cells of the

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disruptants contained much more glycogen than those of their parent strain P16 were (Fig. 8B and C). Therefore, the defect in cell wall integrity (Figs. 3, 4 and 7) led to changes in

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colony and cell morphology, forming the swollen cells of the disruptants because of the

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deletion of the GT6 gene alone or the GT7 gene or both the two genes (Figs. 4 and 8B) and the enhanced accumulation of glycogen in the swollen cells (Fig. 8B and C). Finally, this

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resulted in the reduced pullulan biosynthesis (Fig. 2).

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It has been well known that during biosynthesis of glycogen, trehalose, cell wall β-glucan, and glycosylation of proteins, UDP-glucose is a common donor of glucose [7].

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Therefore, more UDP-glucose was channeled to glycogen biosynthesis (Fig. 8B and C) due

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to reduction of pullulan biosynthesis in the disruptants (Fig. 2). In general, glycogen is regarded as an energy and carbon store for the yeast cells [29]. From the results mentioned above, the glycogen in the yeast cells of the disruptants may have another function of an osmotic protectant when the cell wall integrity was defected (Figs. 3-8). Whether or not the glycogen has such a function remains to be demonstrated. However, the reasons for more glycogen and less pullulan in the disruptants than in their wild type strain P16 are still completely unknown. In S. cerevisiae, a phosphorylated UDPG-pyrophosphorylase (Ugp) 25

ACCEPTED MANUSCRIPT will reach to periplasm where the formed UDP-glucose will be used as donor for biosynthesis of β-glucan while the dephosphorylated UDPG-pyrophosphorylase (Ugp) will reach to cytoplasm where the formed UDP-glucose will be used as donor for biosynthesis of glycogen [30]. According to this finding, we think that in the disruptants, most of the

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demannosylated UDPG-pyrophosphorylase which may be ones of the mannoproteins in the

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periplasm would stay in the cytoplasm and take part in glycogen biosynthesis, leading to

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less pullulan biosynthesis and more glycogen biosynthesis (Figs. 2 and 8B and C) while in their wild type strain P16, the mannosylated UDPG-pyrophosphorylase would go to the

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periplasm and offer UDP-glucose for pullulan biosynthesis, resulting a large amount of the

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produced pullulan in the culture (Fig. 2) and a small amount of the accumulated glycogen in the cells (Fig. 8B and C). However, so far, we did not have any evidence to support this

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hypothesis. All these are being investigated in this laboratory and this hypothesis awaits to

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be examined.

Complementation of the GT6 gene in the disruptant 6-1 and the GT7 gene in the

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disruptant 6727 restored colony and cell morphology (Figs. 3 and 4), sensitivity to the

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Congo red and H2O2 (Fig. 7), glycogen biosynthesis (Fig. 8C) and trehalose biosynthesis (Fig. 8A), but pullulan production of the transformants B61 and B76 was enhanced (Fig. 2). These again demonstrated that the GT6 and GT7 genes were indeed implicated with the cell wall integrity, pullulan biosynthesis, colony morphology, cell morphology and glycogen and trehalose accumulation of A. melanogenum P16. This is the first report that the GT6 and GT7 genes could play an important role in pullulan biosynthesis, glycogen accumulation, colony morphology, and cell morphology of A. melanogenum P16. 26

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Funding and acknowledgements This study was financially supported by National Natural Science Foundation of China

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(Grant No. 31770061). The authors declare no conflict of interests and compliance with

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ethical standards

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Legends: Fig. 1. The phylogenetic tree of the mannosyltransferases from different bacteria, filamentous fungi and yeasts. The underlined mannosyltransferases are Gt6 and

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Gt7 from A. melanogenum P16 used in this study.

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Fig. 2. EPS titer, cell dry weight and EPS/cell dry weight by different disruptants,

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transformants and their wild type strain P16 (n=3, x ±S), The data were compared with those from the wild type strain P16, * P<0.05 meant difference; ** P<0.01 meant

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significant difference.

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Fig. 3. Colony morphologies of the wild type strain P16, the disruptant 6-1, the disruptant 6727, the disruptant P2, the transformant B61 and the transformant B76 grown on the YPD

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plates.

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Fig. 4. The yeast cells of the wild type strain P16, the disruptant 6-1, the disruptant 6727; the different P2, the transformant B61 and the transformant B76 under the phase

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microscope.

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Fig. 5. The colony and cell morphology of the wild type strain P16, the disruptant 6-1, the disruptant 6727, the disruptant P2, the transformant B61 and the transformant B76 grown on the presence of 0.6 M KCl. Fig. 6. Pullulan production and cell growth of the wild type strain P16, the disruptant 6-1, the disruptant 6727, the disruptant P2, the transformant B61 and the transformant B76 in the presence of 0.6 M KCl. Fig. 7. Effects of different concentrations of Congo red and H2O2 on the yeast cell growth 32

ACCEPTED MANUSCRIPT of the wild type strain P16, the disruptant 6-1, the disruptant 6727, the disruptant P2, the transformant B61 and the transformant B76 on the YPD plates. Fig. 8. Intracellular polysaccharide, total sugar and trehalose (A), the iodine-stained yeast cells (B) and the glucose released from intracellular glycogen (C) in the wild type

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strain P16, different disruptants and transformants. (n=3, x ±S). * P < 0.05, the data

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were different from those of the strain P16; ** P < 0.01, the data were significantly

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different from those of the strain P16. The yeast strains were aerobically cultivated in the pullulan production medium for 5 days. The yeast cells were stained using

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the iodine solution (0.01 M I2, 0.03 M KI) for 1 min. The iodine-stained yeast cells

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were observed under the microscope.

Fig. 9. The TLC analysis the intracellular polysaccharides. Lane 1, Glucose Lane 2,

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Intracellular polysaccharide from the yeast strain P16 + pullulanase; Lane 3, The

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intracellular polysaccharides from the yeast strain P16 + inactivated pullulanase; Lane 4, The intracellular polysaccharides from the yeast strain 6-1 + pullulanase; Lane 5, The

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intracellular polysaccharides from the yeast strain 6-1 + inactivated pullulanase; Lane 6.

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The intracellular polysaccharides from the yeast strain 6727 + pullulanase; Lane 7. The intracellular polysaccharides from the yeast strain 6727 + inactivated pullulanase; Lane 8, The intracellular polysaccharides from the yeast strain P2 + pullulanase; Lane 9, The intracellular polysaccharides from the yeast strain P2 + inactivated pullulanase; Lane 10. The intracellular polysaccharides from the yeast strain B61 + pullulanase; Lane 11, The intracellular polysaccharides from the yeast strain B61 + inactivated pullulanase; Lane 12, The intracellular polysaccharides from the yeast strain B76 + pullulanase; Lane 13, The 33

ACCEPTED MANUSCRIPT intracellular polysaccharides from the yeast strain B76 + inactivated pullulanase;the commercial pullulan + pullulanase; Lane 14, Commercial pullulan + pullulanase; Lane 15, the commercial pullulan + inactivated pullulanase; Lane 16, Maltotriose; Lane 17, Inactivated pullulanase; Lane 18, Intracellular polysaccharide from the yeast strain P16;

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Lane 19, Intracellular polysaccharide from the yeast strain 6-1; Lane 20, Intracellular

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polysaccharide from the yeast strain 6727; Lane 21, Intracellular polysaccharide from the

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yeast strain P2; Lane 22, Commercial pullulan

34

ACCEPTED MANUSCRIPT Highlights . The proteins deduced from both the GT6 and GT7 genes contained the conserved sequence CHFWSNFEI of a Ktr mannosyltransferase family; . The removal of each gene and both the two genes caused a reduced pullulan

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. The changes were related to defects in cell wall integrity;

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biosynthesis and increased glycogen accumulation;

35

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9