Screening and isolation of the liamocin-producing yeast Aureobasidium melanogenum using xylose as the sole carbon source

Journal of Bioscience and Bioengineering VOL. xxx No. xxx, xxx, xxxx www.elsevier.com/locate/jbiosc

Screening and isolation of the liamocin-producing yeast Aureobasidium melanogenum using xylose as the sole carbon source Azusa Saika,1 Tokuma Fukuoka,1 Shuntaro Mikome,2 Yukishige Kondo,2 Hiroshi Habe,3 and Tomotake Morita1, * Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 5-2, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan,1 Department of Industrial Chemistry, Faculty of Engineering, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku, Tokyo 162-8601, Japan,2 and Environmental Management Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba, Ibaraki 305-8569, Japan3 Received 9 August 2019; accepted 17 October 2019 Available online xxx

Xylose, the main component of xylan, is the second most abundant sugar in nature after glucose. Consequently, xylose represents an attractive feedstock for the production of value-added compounds such as biosurfactants (BSs), which are produced by various bacteria and yeasts. In this study, we screened and isolated yeast strains that synthesize BSs using xylose as the sole carbon source. We applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to screen for BS-producing yeasts and isolated eight strains as the liamocin producers. Two of the eight strains, AS37 and SK25, were identified as Aureobasidium melanogenum, which is known as black yeasts, by based on 26S ribosomal RNA gene sequences. Both strains produced a wide variety of liamocin structures from not only xylose but also glucose and sucrose. According to the MALDI-TOF MS analysis, signals corresponding to sodium ion adducts of di-, tri-, tetra-, penta- and hexa-acylated C6-liamocins and di-, tri- and tetra-acylated C5-liamocins were detected. In addition, their mono-acetylated form was also detected. The dominant sugar component of liamocins produced by strains AS37 and SK25 is mannitol as estimated by HPLC analysis. This is the first report to describe the screening of liamocins-producing yeasts using xylose as the sole carbon source. Ó 2019, The Society for Biotechnology, Japan. All rights reserved. [Key words: Glycolipids; Biosurfactants; Xylose; Liamocins; Genus Aureobasidium]

Biosurfactants (BSs) produced by various bacteria and yeasts have received much attention because of their excellent interfacial activity, biodegradability, biocompatibility and environmentally friendly production process. To date, several types of BSs, such as glycolipids, lipopeptides, fatty acids and polymeric surfactants, have been identified (1e10). The glycolipid type of BSs including sophorolipids (SLs), mannosylerythritol lipids (MELs) and rhamnolipids have broad industrial uses due to their unique properties and high production (11e13). Thus, they have been commercialized for cosmetics, laundry detergent and personal care products in several countries over the past two decades. Various bacteria and yeasts produce BSs using purified carbon sources like glucose and plant oil and also crude carbon sources including wastes and by-products (1,14e19). The ability to use various feedstocks is advantageous for BS production. For industry, it is also important to consider the BS production cost, resource amount and feedstock availability. Xylan is a major component of hemicellulose, which is the second most abundant polysaccharide, comprising 25e35% of most plants (20). Xylose, the main component of xylan, is the second most abundant sugar in nature next to glucose (21). Therefore, xylose represents an attractive feedstock for production of value-added compounds such as BSs. However,

* Corresponding author. Tel.: þ81 29 861 4426; fax: þ81 29 861 4457. E-mail address: [email protected] (T. Morita).

compared with other sugars, xylose is a minor feedstock for BS production. Thus, our attention was focused on the search for yeasts that produce BSs using xylose as the sole carbon source. In our previous studies, various BS-producing yeast strains, especially MEL producers, were screened from natural sources using the drop collapse method and thin layer chromatography (TLC) analysis (18,22e25). TLC analysis is a very effective screening method for detecting well-known BSs such as MELs and SLs. However, novel BSs may be overlooked by TLC. In this study, we applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis to screen novel BS producers. MALDI-TOF MS is used to detect organic compounds with a wide range of mass-to-charge ratios. MALDI-TOF MS is an excellent high-throughput screening tool that requires only a small amount of sample and is relatively quick compared with other methods. MALDI-TOF MS has demonstrated utility in a variety of fields (26e30), and we predicted that it would greatly aid our search to identify novel BS producers from natural sources. In the present study, we screened and isolated BS-producing yeast strains using xylose as the sole carbon source. Various yeast strains show high BS productivity and is suitable for high-density culture (12,13). We thus focused on yeast as a producer of BS. Of the 229 yeast strains initially characterized as BS-producing strains based on the drop collapse method, the culture products of eight strains were subjected to MALDI-TOF MS analysis. The results indicated that the eight yeast strains produce liamocins, which is a

1389-1723/$ e see front matter Ó 2019, The Society for Biotechnology, Japan. All rights reserved. https://doi.org/10.1016/j.jbiosc.2019.10.010

Please cite this article as: Saika, A et al., Screening and isolation of the liamocin-producing yeast Aureobasidium melanogenum using xylose as the sole carbon source, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.10.010

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SAIKA ET AL.

J. BIOSCI. BIOENG.,

recently identified glycolipid type of BS (31,32). Two of the eight strains were identified based on 26S ribosomal RNA gene sequencing as Aureobasidium melanogenum. This is the first report to describe the screening of liamocin-producing yeasts using xylose as the sole carbon source.

Milford, MA, USA) with a SH1011 column (Shodex, Tokyo, Japan) at 60 C. The mobile phase was 5 mM H2SO4 and the flow rate 0.6 mL/min. The standards for the HPLC sugar component analysis were 1% mannitol, sorbitol, arabitol and xylitol. The differential refractive index detector (RI-8020) was used for sugar detection.

RESULTS MATERIALS AND METHODS Screening of BS-producing strains One hundred and sixty samples from natural sources, such as leaves, flowers and bark, were collected to screen for BSproducing strains. The collected samples were added to a test tube containing a screening medium (1 g/L yeast extract, 0.4 g/L Na2SO4, 1 g/L NH4NO3, 0.5 g/L KH2PO4 and 0.5 g/L MgSO4$7H2O) supplemented with 10% (w/v) xylose as the sole carbon source. Two antibiotics (100 mg /mL streptomycin and 50 mg /mL chloramphenicol) were added to the medium to prevent bacterial growth. Samples were cultured overnight at 28 C with shaking at 200 rpm. The cultures were then inoculated in a new test tube containing the screening medium supplemented with 10% (w/v) xylose and cultured at room temperature with shaking at 180 rpm. The cultures were spread on screening medium plates supplemented with 10% (w/v) xylose and incubated for 2e3 d at 28 C. The isolated colonies were inoculated in 96-well plates containing 0.5 mL screening medium supplemented with 20% (w/v) xylose and cultured for 3 d at 28 C with shaking at 1600 rpm on a deep-well shaker (M BR-022UP, TAITEC, Tokyo, Japan). Several hundred colonies were selected based on the surface activity of the cultures by the drop collapse method using 20 mL culture on Parafilm M (3  v6 cm, American National Can, Chicago, IL, USA) (33). Selected strains were cultured in test tubes containing 3 mL screening medium supplemented with 10% (w/v) xylose for 7 d at 28 C with shaking at 250 rpm, and the surface activity of the cultures was estimated based on the drop collapse method. Production of BSs using xylose as the sole carbon source BS-producing yeast strains were cultured in 3 mL screening medium containing 10% (w/v) xylose for 1 d at 28 C with shaking at 250 rpm. An aliquot of this seed culture (3 mL) was inoculated into 30 mL screening medium containing 10% (w/v) xylose and cultured for 14 d at 28 C with shaking at 250 rpm. The products containing BSs were extracted with 15 mL ethyl acetate from the whole culture. TLC analysis The extracted culture products were detected using TLC (33) with the appropriate modifications. This method is suitable for glycolipid detection. The extracted samples were analyzed by TLC using chloroform, methanol and 12% NH4OH at a 65:15:2 (v:v:v) ratio as the eluent. Glycolipids were detected on the TLC plate by spraying the plate with 2% anthrone-sulfate reagent and heating at 95 C for 5 min. Purified MEL-B produced by Pseudozyma tsukubaensis was used as a reference (23). MALDI-TOF MS analysis The extracted culture products were resuspended in 1 mg/mL ethyl acetate, and the molecular weight of the extracted BSs was measured by MALDI-TOF MS (JMS-S3000 SpiralTOF, JEOL Ltd., Tokyo, Japan) using 2,4,6-trihydroxyacetophenone as the matrix. Identification of strains based on 26S ribosomal RNA gene sequences To identify BS-producing yeast strains, large-subunit (26S) ribosomal RNA (rRNA) gene sequencing and phylogenetic analysis were performed at TechnoSuruga Lab Laboratory Co., Ltd. (Shizuoka, Japan). Each strain was cultured in YM medium (5 g/L yeast extract, 5 g/L soy peptide, 3 g/L NaNO3 and 30 g/L glycerol) at 25 C, and genomic DNA was extracted by the method described in Marmur, 1961 (34) with suitable modifications. The 26S rRNA gene was amplified using PrimeSTAR HS DNA polymerase (Takara Bio, Shiga, Japan). Sequencing was performed using the ABI PRISM 3130 xl Genetic Analyzer System (Applied Biosystems, Foster City, CA, USA). The 16S rRNA gene sequence was compared with related sequences retrieved from DB-FU 10.0 (Techno Suruga Lab Laboratory Co., Ltd., Shizuoka, Japan), DDBJ, EMBL and GenBank. Neighbor-joining trees were constructed using the neighbor-joining method (35) with the Kimura 2-parameter (36) and bootstrap analysis based on 1000 replicates (37). Liamocin production using different carbon sources BS-producing yeast strains were cultured in 25 mL YM medium at 28 C for 2 d with shaking at 250 rpm. An aliquot of this seed culture (2 mL) was inoculated into 30 mL medium supplemented with sucrose (Medium A; 0.6 g/L peptone, 0.4 g/L yeast extract, 1 g/L NaCl, 5 g/L K2HPO4 and 0.4 g/L MgSO4$7H2O, pH 6.5) (38) containing 5% (w/v) sugar (sucrose or glucose or xylose) with or without 5% (w/v) mannitol and cultured for 14 d at 28 C with shaking at 250 rpm. Products in the whole culture were extracted with 15 mL ethyl acetate, and the weight was determined after evaporation of the solvent. The BS-containing yields are reported as the mean dry weight of the ethyl acetate extraction products. Sugar component analysis To estimate the sugar components of liamocins, acid hydrolysis was performed by mixing approximately 30 mg extracted liamocins  with 600 mL 2N HCl for 3 h at 100 C. After the reaction was quenched, the lipophilic compounds were extracted and removed by n-hexane. The aqueous layer was harvested and subjected to HPLC analysis using the Waters 2695 HPLC system (Waters,

Screening of BS-producing strains using xylose as the sole carbon source To isolate BS producers, 160 environmental samples, including leaves, flowers, sepals, soils and bark, were collected and used as the source material for screening. Each sample was cultured and spread on agar plates, and 2608 colonies were selected. Subsequently, 565 colonies were cultured in 96-well deep plates using xylose, and the drop collapse method was used to determine the interfacial activity of the cultures. Next, 229 colonies were identified as BS-producing strains, and each was cultured in a test tube for 7 d using xylose as the sole carbon source. Finally, we selected eight BS-producing yeast strains: AS37, AS38, AS39, NK18, NK29, SK24, SK25 and TM18. The culture of these eight yeast strains caused a significant collapse of the droplet (Fig. S1). Since no spots corresponding to MELs were detected by TLC analysis (Fig. S2), these eight strains are presumed to belong to different types of BS producers. Oil spots were detected in the AS37, AS38, AS39 and NK29 strains, indicating that these four strains produce oil (Fig. S2). On the screening medium plate containing xylose as the sole carbon source, the colors of each colony were blackish yellow (for AS37, AS38, AS39, NK29 and SK25), white (NK18), black (SK24) and pinkish white (TM18). The colors of each culture, displayed in Fig. S1, were yellow (for AS37, AS38 and AS39), greyish light yellow (NK18), light yellow (NK29 and SK25) and white (SK24 and TM18). MALDI-TOF MS analysis of BSs extracted from the screened strains The eight selected strains were cultured in screening medium containing 10% (w/v) xylose. The BS-containing products were extracted from whole cultures using 15 mL ethyl acetate and analyzed by MALDI-TOF MS. The entire MS spectrum is shown in Fig. S3. Because the analyzed samples were crude extracts, many signals were detected in each sample. However, the same m/z values were detected in every sample at m/z ¼ 577, 763 and 949, which correspond to the sodium ion adducts of di-, tri- and tetra-acylated C6-liamocins (32,39), and at m/z ¼ 547, 733 and 919, which correspond to the sodium ion adducts of di-, tri- and tetra-acylated C5-liamocins (40). The m/z values and exact masses of the liamocins are summarized in Table 1 and S1, TABLE 1. MALDI-TOF MS analysis of liamocins produced by screened strains. Strains

Exact mass AS37 AS38 AS39 NK18 NK29 SK24 SK25 TM18

Predict sugar-alcohol component C6 C5 C6 C5 C6 C5 C6 C5 C6 C5 C6 C5 C6 C5 C6 C5 C6 C5

Molecular weight of liamocins [MþNa]þ Di-acylated

Tri-acylated

Tetra-acylated

577.32 547.31 577.3136 547.3042 577.3144 547.3056 577.3144 e 577.3205 547.3107 577.3243 547.3142 577.3158 547.3053 577.3222 e 577.3236 547.3130

763.45 733.44 763.4346 733.4242 763.4327 733.4228 763.4371 e 763.4412 733.4315 763.4479 733.4373 763.4404 733.4294 763.4466 733.4358 763.4493 733.4385

949.57 919.56 949.5582 919.5467 949.5555 919.5440 949.5615 e 949.5667 919.5560 949.5740 919.5634 949.5635 919.5530 949.5772 919.5613 949.5738 919.5635

Please cite this article as: Saika, A et al., Screening and isolation of the liamocin-producing yeast Aureobasidium melanogenum using xylose as the sole carbon source, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.10.010

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respectively. The expected structures for the extracted liamocins are displayed in Fig. 1. Liamocins were recently identified BSs produced by the genus Aureobasidium and consist of a C6 or C5 sugar-alcohol as a hydrophilic moiety and 3,5-dihydroxy decanoic

(A) CH2OH HO HO H H

H H OH OH CH2O O

di-acylated tri-acylated

OH O O

OH O O

tetra-acylated penta-acylated

OH O O

OH O O

hexa-acylated

OH O O

(B)

OH OH

CH2OH HO

H

H

OH

H

OH CH2O O

di-acylated tri-acylated

OH O O

OH O O

tetra-acylated penta-acylated

OH O O

OH O O

hexa-acylated

OH O O

OH OH

FIG. 1. The structure of liamocins. (A) C6-liamocins and (B) C5-liamocins.

3

acid as a hydrophobic moiety (31,32). Two of the eight strains, AS37 (isolated from leaf collected in Kanagawa, Japan) and SK25 (isolated from flower collected in Ibaraki, Japan), showed good reproducibility and were selected for further investigation. Identification of liamocin-producing yeast strains Two liamocin-producing strains, AS37 and SK25, were subjected to 26S rDNA D1/D2 domain sequencing and phylogenetic analyses (Fig. 2). Fig. 2A shows the AS37 and SK25 morphologies on YM plates and microscopic images. Phylogenetic analysis revealed that both strains were A. melanogenum (Fig. 2B, Tables S2 and S3). Members of the genus Aureobasidium are known as black yeasts, because many strains produce fungal melanin (31). Black yeasts produce various bio-based materials, such as xylanase, poly(b-malic acid), pullulan and liamocins (31,32,41e43). Leathers et al. (38) demonstrated the production of liamocins by several Aureobasidium pullulans strains using different medium formulations with sucrose or glucose as the carbon source. According to this result, Medium A was the most suitable for liamocins production. Thus, we attempted to produce liamocins from the AS37 and SK25 strains using xylose, glucose and sucrose as different carbon sources. Production of liamocins using different carbon sources Two A. melanogenum strains, AS37 and SK25, were cultured in Medium A containing 5% (w/v) sucrose, glucose or xylose and with or without 5% (w/v) mannitol. Mannitol is known

(A) a

b

c

d

(B) FJ150943.1_Selenophoma mahoniae CBS388.92 FJ150946.1_Kabatiella lini CBS125.21

92

82

AY016359.1_Discosphaerina fagi CBS171.93

85 71

FJ150942.1_Aureobasidium pullulans CBS584.75 JN712557.1_Aureobasidium proteae CPC2824 FJ150913.1_Aureobasidium subglaciale CBS123387

41

SK25

99 98 98

FJ150926.1_Aureobasidium melanogenum CBS105.22 AS37 JN712555.1_Aureobasidium leucospermi CPC15180

72

FJ150937.1_Aureobasidium namibiae CBS147.97

EU167576.1_Aureobasidium caulivorum CBS242.64 JX462674.1_Aureobasidium thailandense NRRL58539 FJ150970.1_Pringsheimia smilacis CBS 873.71 63

GQ303324.1_Selenophoma australiensis CBS124776 0.0050

FIG. 2. Morphology and phylogenetic relationships of AS37 and SK25. (A) AS37 and SK25 morphologies on YM plates and microscopic images. (a, b) AS37 and (c, d) SK25. (B) Molecular phylogenetic trees constructed using the 26S rDNA D1/D2 domain sequences of AS37 and SK25.

Please cite this article as: Saika, A et al., Screening and isolation of the liamocin-producing yeast Aureobasidium melanogenum using xylose as the sole carbon source, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.10.010

SAIKA ET AL.

J. BIOSCI. BIOENG., compared with AS37. Liamocin productivity was four times higher in SK25 than AS37 (1.5e2.0 g oil/g DCW), except when using sucrose plus mannitol.

as a sugar component of liamocins (32). Liamocins were extracted with ethyl acetate from whole cultures and weighed after solvent evaporation. Whole extract weight was used as a proxy for liamocins weight, since liamocins represent the dominant components of whole extracts (Fig. 3). The liamocins content was measured in g/L oil according to Manitchotpisit et al. (31). The dry cell weights, liamocins yields and productivities are listed in Table 2. The growth of AS37 cells was fairly constant regardless of the carbon source. The dry weight of SK25 cells was one-third to one-half that of AS37 cells in every medium evaluated, except for sucrose plus mannitol. Liamocins production by AS37 when consuming xylose, glucose or sucrose as the carbon source was 4.2  0.6, 5.0  0.3 or 5.6  0.6 g/L oil, respectively, and increased slightly when using mannitol plus glucose or sucrose (7.2  1.5 or 7.8  0.6, respectively). Based on these results, sucrose is a good carbon source for liamocin production by AS37. Although the growth was greater, liamocin production was less, in AS37 than SK25 cells. The liamocin productivity of AS37 remained low (0.3e0.6 g oil/g dry cell weight, DCW). SK25 produced a higher quantity of liamocins compared with AS37 when using xylose, glucose or sucrose (7.0e9.9 g/L oil), but not mannitol. Xylose and glucose are most suitable for liamocin production in SK25

(A)

a: deacetylated C5-liamocins; b: deacetylated C6-liamocins; c: mono-acetylated C5-liamocins; d: mono-acetylated C6-liamocins

700

547.3070

a

2.0 1.6 1.2

c

d

a 733.4312

b

619.3278

2.8 2.4

Intensity

b

105)

577.3174 589.3170

(

a

d+56

500

600

700

800

1321.7617

1363.7743

1177.6447

949.5225 961.4943

1000

b

0.4 900

1177.6945

1100

1200

b

d 1363.8431

d

1321.8254

b 1135.6808

d

991.5576

900

d+28

0.8 0.0

991.5258

919.5066

a

949.5502 961.5096

d+28 d+56

d c

1300

1400

m/z

1400

m/z

1400

m/z

b c

800

Xylose

d

1300

c

d

1000

1100

b

d

1200

b

d 1363.8226

600

1200

b

1321.8171

500

d

1135.6880

0.5

b

1100

991.5755

1.0

hexa-acylated

d

1000

919.5334

1.5

733.4119

547.3059

a

c

763.4451 775.4428

Intensity

2.0

a

619.3181

c

2.5

0.0

d

577.3126 589.3111

3.0

900

d

763.4269 775.4208

b

penta-acylated

c

949.5680 961.5501

733.3911

800

b

105)

a d+28 d+56

700

Sucrose (

b

805.4394

600

c

805.4583

500

a

619.2971

577.2906 589.2892

a 547.2827

Intensity

c

1.2 0.8 0.4 0.0

d

763.4052 775.3999

b

2.8 2.4 2.0 1.6

d

b

105)

919.5542

Glucose

tetra-acylated

1135.6340

tri-acylated

805.4173

di-acylated

(

MALDI-TOF MS analysis of liamocins from AS37 and SK25 Fig. 3 shows the results of MALDI-TOF MS analysis of liamocins extracted from AS37 and SK25 cultured with xylose, glucose and sucrose. The MS spectra obtained from AS37 and SK25 cultured with xylose or glucose or sucrose plus mannitol are displayed in Fig. S4, because these results showed the same tendency with using a single carbon source. In both strains, the MS spectra were almost the same regardless of the carbon source. Signals corresponding to sodium ion adducts of di-, tri-, tetra-, penta- and hexa-acylated C6-liamocins (m/z ¼ 577, 763, 949, 1135 and 1321) were detected in all samples. Signals representing a 42 Da mass different from C6-liamocins (m/z ¼ 619, 805, 991 and 1177) were also detected. The spectra of some samples also included a signal at m/z ¼ 1363. Because the 42 Da difference corresponds to an acetyl group, these signals were assigned to mono-acetylated liamocins. Normally, liamocins consist of 3,5dihydroxy decanoic acids as a hydrophobic moiety (Fig. 1). However, signals at m/z ¼ 833 (805 þ 28) and 861 (805 þ 56)

1177.6981

4

1300

FIG. 3. MALDI-TOF MS spectra of liamocins from AS37 and SK25. (A) AS37 and (B) SK25 deacetylated C5-liamocins (a), deacetylated C6-liamocins (b), mono-acetylated C5-liamocins (c) and mono-acetylated C6-liamocins (d).

Please cite this article as: Saika, A et al., Screening and isolation of the liamocin-producing yeast Aureobasidium melanogenum using xylose as the sole carbon source, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.10.010

VOL. xxx, xxxx

LIAMOCIN-PRODUCING YEAST STRAIN

(B)

a: deacetylated C5-liamocins; b: deacetylated C6-liamocins; c: mono-acetylated C5-liamocins; d: mono-acetylated C6-liamocins tri-acylated

800

d

500

600

700

800

a

0.8 0.4 500

600

800

a d+28 d+56

919.5477

c

d 805.4467

733.4225 700

1135.6823

1177.6993

1321.8326 1300

1400

m/z

b

1100

1400

m/z

1400

m/z

d

1200

b

1300

b

763.4373 775.4322

a

619.3222

a

1.2

577.3143

1.6

547.3050

Intensity

2.0

0.0

d

b

2.4

1200

c

1000

b

105)

949.5662 961.5400 991.5789

900

Xylose (

1100

d

d+28 d+56

733.4265

a

b

d c

619.3289

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

Intensity

b

b

105)

577.3219

(

1000

900

d

1000

1100

b

d

1200

b 1321.8180

Sucrose

900

b

1321.8416

700

1177.7105

600

1177.6952

500

d

1135.6980

0.0

d+56

b

1135.6842

0.4

d+28

991.5724

0.8

919.5425

1.2

d

a

919.5508

a

c

805.4528

1.6

763.4359 775.4285

2.0

763.4119 775.4363

Intensity

2.4

c

d

733.4192

577.3125

b

619.3259

105)

hexa-acylated

b

d

949.5775 961.5376 991.5797

b

penta-acylated

949.5686 961.5565

Glucose

tetra-acylated

805.4453

di-acylated

(

5

1300

FIG. 3. (continued).

correspond to mono-acetylated C6-liamocins with tri-acyl groups containing 3,5-dihydroxy dodecanoic acid or 3,5-dihydroxy tetradecanoic acid and were clearly detected in every sample. Signals corresponding to sodium ion adducts of di-, tri- and tetraacylated C5-liamocins (m/z ¼ 547, 733 and 919) and its monoacetylated form (m/z ¼ 589, 775 and 961) were also detected. These results indicate that AS37 and SK25 strains produce a wide variety of liamocin derivatives.

corresponding to the C5-sugar alcohol were detected by HPLC analysis without samples described above. The MALDI-TOF MS analysis suggested that the production of C5-liamocins may less than that of C6-liamocins; thus, the C5-sugar alcohol concentration in the sample may be below the detection limit.

Analysis of liamocins sugar components To evaluate the sugar components of liamocins produced by AS37 and SK25, the extracted liamocins were subjected to HPLC analysis (Fig. 4). The peak at 13.6 min corresponded to mannitol and was detected as the main peak in all samples. Thus, mannitol is the dominant sugar component of AS37 and SK25 liamocins. When xylose with or without mannitol was used as the carbon source, a very weak peak at 14.6 min corresponding to xylitol was detected (Fig. 4A and B, closed arrow). A weak peak at 14.4 min corresponding to arabitol was detected in the sample from AS37 cultured with sucrose plus mannitol (Fig. 4A, open arrow). Collectively, these findings indicate that the liamocin structure depends on the carbon source. Whereas C5-liamocins were detected in all samples by MALDI-TOF MS analysis (Fig. 3), no peaks

We isolated and identified two strains of A. melanogenum, AS37 and SK25, that produce glycolipid type of BSs using xylose as the sole carbon source. Both AS37 and SK25 produce a variety of liamocin structures from xylose, glucose and sucrose. According to the MALDI-TOF MS and HPLC analyses, the liamocins produced by both strains consist mainly of mannitol and 3,5-dihydroxy decanoic acids, whereas C5-sugar alcohols such as arabitol and xylitol and long acyl-chains such as 3,5-dihydroxy dodecanoic acid and 3,5dihydroxy tetradecanoic acid are minor components. According to the TLC analysis (Fig. S2), clear spots similar to the MEL standard were not detected in the cultures of any of the tested strains using anthrone-sulfate reagent. Thus, the eight strains screened in this study may have been overlooked if we relied solely on TLC analysis. The crude extracts yielded many signals detected

DISCUSSION

Please cite this article as: Saika, A et al., Screening and isolation of the liamocin-producing yeast Aureobasidium melanogenum using xylose as the sole carbon source, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.10.010

6

SAIKA ET AL.

J. BIOSCI. BIOENG., TABLE 2. AS37 and SK25 production of liamocins using different carbon sources.

Strain

Carbon source

AS37

Xylose Glucose Sucrose Xylose þ mannitol Glucose þ mannitol Sucrose þ mannitol Xylose Glucose Sucrose Xylose þ mannitol Glucose þ mannitol Sucrose þ mannitol

SK25

Dry cell weight (g/L) 13.2 12.2 13.3 14.8 13.5 13.9 4.6 5.8 7.7 5.7 4.2 10.0

           

Liamocins (g oil/L)

1.1 0.2 1.0 0.7 0.8 2.0 1.3 0.7 1.7 0.6 1.0 1.8

4.2 5.0 5.6 4.1 7.2 7.8 7.8 9.9 7.0 8.5 8.0 7.5

           

0.6 0.3 0.6 0.3 1.5 0.6 0.8 0.4 1.4 0.7 0.3 1.1

Liamocins productivity (g oil/g-DCW) 0.3 0.4 0.4 0.3 0.5 0.6 1.8 1.7 1.0 1.5 2.0 0.8

           

0.1 0.1 0.1 0.1 0.1 0.1 0.7 0.3 0.4 0.2 0.4 0.1

Each strain was cultured in medium A containing 5% (w/v) sugar (sucrose or glucose or xylose) with or without 5% (w/v) mannitol, and cultivated at 28 C for 14 d with shaking at 250 rpm. The results are the average of three independent cultivations.

by MALDI-TOF MS, but it was possible to detect those corresponding to liamocins in the screened strains. In addition, this method allows assessment of the acetylation level and acyl chain length without any column purification. From these results, MALDITOF MS represents an effective and reliable tool for screening BS producers from natural sources due to its high-throughput nature and ability to detect structural details from crude samples. Liamocins were described in 2011 as heavy oils produced by A. pullulans using sucrose as a carbon source (31). Since then, sucrose has been commonly used as a carbon source for liamocin production (32,38,44e46). Here, we describe the first investigation of whether xylose acts as a carbon source for liamocin production. Xylose is an attractive feedstock abundant in nature like glucose and sucrose, however, its use in microbial production of chemicals is limited. Using xylose as a feedstock for liamocin production may contribute to the acceleration of the xylose utilization for valueadded compounds production. Both A. melanogenum strains screened in this study produced liamocins from xylose (Table 2). The SK25 strain produced liamocins at higher yields when using xylose compared with sucrose as the carbon source. Leathers et al. (38) reported that the A. pullulans strain NRRL50384 produced the highest level of liamocins (8.0  0.3 g/L oil) from sucrose, which was comparable with the level (7.8  0.8 g/L oil) produced by SK25 from xylose. Additionally, liamocin production from xylose in strain SK25 was high (1.8  0.7 g oil/g DCW), suggesting that strain SK25 holds promise as a liamocin producer using xylose as the sole carbon source. Recently, Tang et al. (45) demonstrated that over-

(A)

(B)

expression of the pyruvate carboxylase gene PYC1 enhanced production of liamocins by A. melanogenum M39, and this mutant strain produced 43.04  1.2 g/L liamocins within 156 h. In addition, Saur et al. (46) indicated that a manual shift in pH to 3.5 during the production phase enhanced the synthesis of liamocins by A. pullulans NRRL62031. Therefore, additional modifications to the culture conditions and improvements in liamocin production by gene engineering may further enhance the production of liamocins from xylose. In the present study, producers of well-known BSs (i.e., SLs and MELs) were not detected in natural sources. NH4NO3 was used as a nitrogen source in the screening medium. However, in our previous reports, some MEL producers such as Pseudozyma parantarctica, Pseudozyma ruglosa and Ustilago scitaminea did not produce MEL in medium containing NH4NO3 as a nitrogen source (47e49). This suggests that NH4NO3 is not a suitable nitrogen source for detecting MEL producers and highlights the possibility of identifying new targets by modifying certain medium components, such as carbon and nitrogen sources. Further study may contribute to find a promising xylose-utilizing BS producer, and accelerate the xylose utilization for production of value-added compounds. Supplementary data to this article can be found online at https://doi.org/10.1016/j.jbiosc.2019.10.010. ACKNOWLEDGMENTS This work was partially funded by the Advanced Low Carbon Technology Research and Development Program of Japan Science and Technology Agency (JST-ALCA). We thank Dr. Nagatoshi Koumura, Mr. Shohei Kawamoto and Ms. Wujisiguleng for their technical assistance. The authors declare no other competing interests.

Sucrose + Mannitol Glucose + Mannitol Xylose + Mannitol MV

Sucrose Glucose Xylose Xylitol STD Arabitol STD Sorbitol STD Mannitol STD 12

14

16

18

12

14

16

18

Retention time (min)

FIG. 4. Sugar component analysis by HPLC. (A) AS37 and (B) SK25. Closed arrow, putative xylitol peak; open arrow, putative arabitol peak.

References 1. Kitamoto, D., Haneishi, K., Nakahara, T., and Tabuchi, T.: Production of mannosylerythritol lipids by Candida antarctica from vegetable oils, Agric. Biol. Chem., 54, 37e40 (1990). 2. Maget-Dana, R. and Ptak, M.: Interfacial properties of surfactin, J. Colloid Interface Sci., 153, 285e291 (1992). 3. Matsuyama, T., Kaneda, K., Nakagawa, Y., Isa, K., Hara-Hotta, H., and Yano, I.: A novel extracellular cyclic lipopeptide which promotes flagellumdependent and -independent spreading growth of Serratia marcescens, J. Bacteriol., 174, 1769e1776 (1992). 4. Ban, T., Sato, T., and Yen, T. F.: Interfacial activity of n-alkylamines from microbially produced spiculisporic acid, J. Pet. Sci. Eng., 21, 223e238 (1998). 5. Lang, S. and Philp, J. C.: Surface-active lipids in rhodococci, Antonie Van Leeuwenhoek, 74, 59e70 (1998). 6. Rosenberg, E. and Ron, E. Z.: High- and low-molecular-mass microbial surfactants, Appl. Microbiol. Biotechnol., 54, 154e162 (1999). 7. Maier, R. M. and Soberón-chávez, G.: Pseudomonas aeruginosa rhamnolipids: biosynthesis and potential applications, Appl. Microbiol. Biotechnol., 54, 625e633 (2000).

Please cite this article as: Saika, A et al., Screening and isolation of the liamocin-producing yeast Aureobasidium melanogenum using xylose as the sole carbon source, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.10.010

VOL. xxx, xxxx 8. Ogawa, S. and Ota, Y.: Influence of exogenous natural oils on the u-1 and u-2 hydroxy fatty acid moiety of sophorose lipid produced by Candida bombicola, Biosci. Biotechnol. Biochem., 64, 2466e2468 (2000). 9. Rau, U., Hammen, S., Heckmann, R., Wray, V., and Lang, S.: Sophorolipids: a source for novel compounds, Ind. Crops Prod., 13, 85e92 (2001). 10. Lang, S.: Biological amphiphiles (microbial biosurfactants), Curr. Opin. Colloid Interface Sci., 7, 12e20 (2002). 11. Zhu, L., Yang, X., Xue, C., Chen, Y., Qu, L., and Lu, W.: Enhanced rhamnolipids production by Pseudomonas aeruginosa based on a pH stage-controlled fedbatch fermentation process, Bioresour. Technol., 117, 208e213 (2012). 12. Saika, A., Koike, H., Fukuoka, T., and Morita, T.: Tailor-made mannosylerythritol lipids: current state and perspectives, Appl. Microbiol. Biotechnol., 102, 6877e6884 (2018). 13. Jezierska, S., Claus, S., and Van Bogaert, I.: Yeast glycolipid biosurfactants, FEBS Lett., 592, 1312e1329 (2018). 14. Bednarski, W., Adamczak, M., Tomasik, J., and Płaszczyk, M.: Application of oil refinery waste in the biosynthesis of glycolipids by yeast, Bioresour. Technol., 95, 15e18 (2004). 15. Rau, U., Nguyen, L. A., Schulz, S., Wray, V., Nimtz, M., Roeper, H., Koch, H., and Lang, S.: Formation and analysis of mannosylerythritol lipids secreted by Pseudozyma aphidis, Appl. Microbiol. Biotechnol., 66, 551e559 (2005). 16. Fukuoka, T., Morita, T., Konishi, M., Imura, T., Sakai, H., and Kitamoto, D.: Structural characterization and surface-active properties of a new glycolipid biosurfactant, mono-acylated mannosylerythritol lipid, produced from glucose by Pseudozyma antarctica, Appl. Microbiol. Biotechnol., 76, 801e810 (2007). 17. Takahashi, M., Morita, T., Wada, K., Hirose, N., Fukuoka, T., Imura, T., and Kitamoto, D.: Production of sophorolipid glycolipid biosurfactants from sugarcane molasses using Starmerella bombicola NBRC 10243, J. Oleo Sci., 60, 267e273 (2011). 18. Morita, T., Fukuoka, T., Imutra, T., Hirose, N., and Kitamoto, D.: Isolation and screening of glycolipid biosurfactant producers from sugarcane, Biosci. Biotechnol. Biochem., 76 (2012) 120251-1-4. 19. Maddikeri, G. L., Gogate, P. R., and Pandit, A. B.: Improved synthesis of sophorolipids from waste cooking oil using fed batch approach in the presence of ultrasound, Chem. Eng. J., 263, 479e487 (2015). 20. Deutschmann, R. and Dekker, R. F. H.: From plant biomass to bio-based chemicals: latest developments in xylan research, Biotechnol. Adv., 30, 1627e1640 (2012). 21. Ishihara, M., Matsunaga, M., Hayashi, N., and Tisler, V.: Utilization of D-xylose as carbon source for production of bacterial cellulose, Enzyme Microb. Technol., 31, 986e991 (2002). 22. Konishi, M., Morita, T., Fukuoka, T., Imura, T., Kakugawa, K., and Kitamoto, D.: Production of different types of mannosylerythritol lipids as biosurfactants by newly isolated yeast strains belonging to the genus Pseudozyma, Appl. Microbiol. Biotechnol., 75, 21e531 (2007). 23. Morita, T., Takashima, M., Fukuoka, T., Konishi, M., Imura, T., and Kitamoto, D.: Isolation of basidiomycetous yeast Pseudozyma tsukubaensis and production of glycolipid biosurfactant, a diastereomer type of mannosylerythritol lipid-B, Appl. Microbiol. Biotechnol., 88, 679e688 (2010). 24. Morita, T., Ogura, Y., Takashima, M., Hirose, N., Fukuoka, T., Imura, T., Kondo, Y., and Kitamoto, D.: Isolation of Pseudozyma churashimaensis sp. nov., a novel ustilaginomycetous yeast species as a producer of glycolipid biosurfactants, mannosylerythritol lipids, J. Biosci. Bioeng., 112, 137e144 (2011). 25. Konishi, M., Maruoka, N., Furuta, Y., Morita, T., Fukuoka, T., Imura, T., and Kitamoto, D.: Biosurfactant-producing yeasts widely inhabit various vegetables and fruits, Biosci. Biotechnol. Biochem., 78, 516e523 (2014). 26. Westphal, Y., Schols, H. A., Voragen, A. G. J., and Gruppen, H.: MALDI-TOF MS and CE-LIF fingerprinting of plant cell wall polysaccharide digests as a screening tool for Arabidopsis cell wall mutants, J. Agric. Food Chem., 58, 4644e4652 (2010). 27. Gottardo, R., Chiarini, A., Prà, I. D., Seri, C., Rimondo, C., Serpelloni, G., Armato, U., and Tagliaro, F.: Direct screening of herbal blends for new synthetic cannabinoids by MALDI-TOF MS, Mass Spectrom., 47, 141e146 (2012). 28. Tani, A., Sahin, N., Matsuyama, Y., Enomoto, T., Nishimura, N., Yokota, A., and Kimbara, K.: High-throughput identification and screening of novel Methylobacterium species using whole-cell MALDI-TOF/MS analysis, PLoS One, 7, e40784 (2012). 29. Su, X., Zhou, H. Y., Chen, F. C., Gao, B. X., Liu, Z. W., Zhang, Y. H., Liu, F., Li, Z. R., and Gao, Z. X.: Modified SBA-15 matrices for high-throughput

LIAMOCIN-PRODUCING YEAST STRAIN

30.

31.

32.

33.

34. 35. 36.

37. 38.

39.

40.

41.

42.

43. 44.

45.

46.

47.

48.

49.

7

screening of melamine in milk samples by MALDI-TOF MS, Int. J. Mass. Spectrom., 338, 39e44 (2013). Pandey, A., Jain, R., Sharma, A., Dhakar, K., Kaira, G. S., Rahi, P., Dhyani, A., Pandey, N., Adhikari, P., and Shouche, Y. S.: 16S rRNA gene sequencing and MALDI-TOF mass spectrometry based comparative assessment and bioprospection of psychrotolerant bacteria isolated from high altitudes under mountain ecosystem, SN Appl. Sci., 1, 278 (2019). Manitchotpisit, P., Price, N. P. J., Leathers, T. D., and Punnapayak, H.: Heavy oils produced by Aureobasidium pullulans, Biotechnol. Lett., 33, 1151e1157 (2011). Price, N. P. J., Manitchotpisit, P., Vermillion, K. E., Nowman, M.,J., and Leathers, T. D.: Structural characterization of novel extracellular liamocins (mannitol oils) produced by Aureobasidium pullulans strain NRRL 50380, Carbohydr. Res., 370, 24e32 (2013). Morita, T., Konishi, M., Fukuoka, T., Imura, T., and Kitamoto, D.: Physiological differences in the formation of the glycolipid biosurfactants, mannosylerythritol lipids, between Pseudozyma antarctica and Pseudozyma aphidis, Appl. Microbiol. Biotechnol., 74, 307e315 (2007). Marmur, J.: A procedure for the isolation of deoxyribonucleic acid from microorganisms, J. Mol. Biol., 3, 208e218 (1961). Saitou, N. and Nei, M.: The neighbor-joining method: a new method for reconstructing phylogenetic trees, Mol. Biol. Evol., 4, 406e425 (1987). Kimura, M.: A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences, J. Mol. Evol., 16, 111e120 (1980). Felsenstein, J.: Confidence limits on phylogenies: an approach using the bootstrap, Evolution, 39, 783e791 (1985). Leathers, T. D., Price, N. P. J., Bischoff, K. M., Manitchorpisit, P., and Skory, C. D.: Production of novel types of antibacterial liamocins by diverse strains of Aureobasidium pullulans grown on different culture media, Biotechol. Lett., 37, 2075e2081 (2015). Bischoff, K., Leathers, T. D., Price, N. P. J., and Manitchotpisit, P.: Liamocin oil from Aureobasidium pullulans has antibacterial activity with specificity for species of Streptococcus, J. Antibiot., 68, 642e645 (2015). Price, N. P. J., Bischoff, K., Leathers, T. D., Cossé, A. A., and Manitchotpisit, P.: Polyols, not sugars, determine the structural diversity of anti-streptococcal liamocins produced by Aureobasidium pullulans strain NRRL 50380, J. Antibiot., 70, 136e141 (2017). Leathers, T. D.: Colour variants of Aureobasidium pullulans overproduce xylanase with extremely high specific activity, Appl. Environ. Microbiol., 52, 1026e1030 (1986). Nagata, N., Nakahara, T., and Tabuchi, T.: Fermentative production of poly (bL-malic acid), a polyelectrolytic biopolyester, by Aureobasidium sp. Biosci. Biotechnol. Biochem., 57, 638e642 (1993). Singh, R. S., Saini, G. K., and Kennedy, J. F.: Pullulan: microbial sources, production and applications, Carbohydr. Polym., 73, 515e531 (2008). Brumano, L. P., Antunes, F. A. F., Souto, S. G., dos Santos, J. C., Venus, J., Schneider, R., and da Silva, S. S.: Biosurfactant production by Aureobasidium pullulans in stirred tank bioreactor: new approach to understand the influence of important variables in the process, Bioresour. Technol., 243, 264e272 (2017). Tang, R. R., Chi, Z., Jiang, H., Liu, G. L., Xue, S. J., Hu, Z., and Chi, Z. M.: Overexpression of a pyruvate carboxylase gene enhances extracellular liamocin and intracellular lipid biosynthesis by Aureobasidium melanogenum M39, Process Biochem., 69, 64e74 (2018). Saur, K. M., Brumhard, O., Scholz, K., Hayen, H., and Tiso, T.: A pH shift induces high-titer liamocin production in Aureobasidium pullulans, Appl. Microbiol. Biotechnol., 103, 4741e4752 (2019). Morita, T., Konishi, M., Fukuoka, T., Imura, T., and Kitamoto, D.: Discovery of Pseudozyma rugulosa NBRC10877 as a novel producer of glycolipid biosurfactants, mannosylerythritol lipids, based on rDNA sequence, Appl. Microbiol. Biotechnol., 73, 305e313 (2006). Morita, T., Konishi, M., Fukuoka, T., Imura, T., Sakai, H., and Kitamoto, D.: Efficient production of di- and tri-acylated mannosylerythritol lipids as glycolipid biosurfactants by Pseudozyma parantarctica JCM11752T, J. Oleo Sci., 57, 557e565 (2008). Morita, T., Ishibashi, Y., Fukuoka, T., Imura, T., Sakai, H., Abe, M., and Kitamoto, D.: Production of glycolipid biosurfactants, mannosylerythritol lipids, by a smut fungus, Ustilago scitaminea NBRC 32730, Biosci. Biotechnol. Biochem., 73, 788e792 (2009).

Please cite this article as: Saika, A et al., Screening and isolation of the liamocin-producing yeast Aureobasidium melanogenum using xylose as the sole carbon source, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.10.010