L-Asparaginase (L-asparagine amidohydrolase, ASNase) is an enzyme that hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. ASNase is clinically used to treat acute lymphoblastic leukemia (ALL) and lymphosarcoma (Darvishi et al., 2022). In food industry, this enzyme is used to mitigate formation of acrylamide in cooked foods which is a probable human carcinogen (Jia et al., 2021). The global market size of ASNase was approximately 778 million US dollars in 2023, and is projected to reach 3,582 million dollars in 2033 due to the importance in both pharmaceutical and food industries (http://market.us).

ASNases are found in plants, animals, and microorganisms including bacteria, actinomycetes, and fungi (Krishnapura et al., 2015). Among these various resources, microorganisms are preferred for large scale production of ASNase, because microbial processes are relatively easier for modification and optimization in a cost-effective manner. Bacteria including Escherichia coli and several species belonging to the genera Bacillus, Erwinia, Pseudomonas, and Streptomyces are known to produce ASNase. Moreover, some fungal species in the Aspergillus, Penicillium, and Trichoderma genera are also sources of ASNase (Muneer et al., 2020; Yu et al., 2023). Currently, the major sources of ASNase in the medicinal use are E. coli and Pectobacterium carotovorum (Erwinia carotovora) (Batool et al., 2016). These microbial ASNases are responsible for approximately one third of the global requirements for anticancer agents (Qeshmi et al., 2018). However, due to adverse effects of the currently used clinical ASNases including hypersensitivity, immonosuppression, and resistance development, it is imporant to explore alternative resources for novel ASNases (Cachumba et al., 2016).

Marine yeasts have been isolated from seawater, sand, mud flat, sea plants, and animals (Kwon et al., 2021; Yu et al., 2023). Because they have evolved to adapt to harsh environments such as high salinity, high pressure, and a wide range of temperatures, marine yeasts have the potential to produce unique and useful natural products (Giddings and Newman, 2022). For example, a marine-derived Saccharomyces cerevisiae strain C-19 is halo-tolerant and shows higher bioethanol production than the control type strain S. cerevisiae NBRC 10217 (Obra et al., 2012). Moreover, a novel serine protease inhibitor produced by a marine yeast strain Candida parapsilosis ABS1 exhibits anti-cancer activities (Sarkar and Rao, 2023). However, compared to the terrestrial counterparts, only little is known about marine yeasts for biotechnological applications (Zaky et al., 2014).

In this study, we aimed to provide basic information to discover novel microbial ASNase from marine yeasts. To address this, our laboratory isolated and identified yeasts from various marine resources. A total of twelve yeast species were obtained and investigated for their growth and ASNase activities.

Sample collection and fungal isolation

General information of yeast strains used in this study is described in Table 1. Sample collection and fungal isolation were performed as described previously (Yu et al., 2023) with a few modifications. Briefly, seawater was prepared by vacuum filtration using a 0.45 μm membrane filter. The filters were placed on yeast extract-peptone-dextrose (YPD; BD, USA) agar containing 3.5% NaCl, 0.01% (w/v) ampicillin, and 0.01% (w/v) streptomycin. After incubation at 20°C for 7 days, fungal colonies were transferred on fresh media for collection of pure colonies. Marine sediments were resuspended in phosphate buffered saline, and animal samples were homogenized using a blender. Then, the prepared samples were spread on the media described above. Marine algae were cut into pieces using sterile scissors and placed on the media. After obtaining pure yeast cultures, all strains were stored in 20% glycerol at -80°C, and deposited at the Microbial Marine Bio Bank (MMBB) of the National Marine Biodiversity Institute of Korea (MABIK) (Table 1). All yeast strains were cultured on YPD agar or potato dextrose agar (PDA; BD) at 25°C unless stated otherwise.

Table 1

General information of 12 marine-derived yeasts

Strain name	Deposit number	Collection site	Isolation source	Identification	GenBank Accssion Numbera
					ITS	LSU
21SA1-Ul15	MABIK 00001374	Sinan-gun, Jeollanam-do (34°51'42.3"N, 126°19'18.6"E)	Sea algae (Ulva linza)	Rhodotorula babjevae	PQ336029	PQ336041
BC-Cla8	MABIK 00001375	Seosan-si, Chungcheongnam-do (36°58'03.7"N, 126°20'11.0"E)	Clam (Lajonkairia lajonkairii)	Debaryomyces hansenii	PQ336026	PQ336038
BC-Cla9	MABIK 00001376	Seosan-si, Chungcheongnam-do (36°58'03.7"N, 126°20'11.0"E)	Clam (Lajonkairia lajonkairii)	Filobasidium magnum	PQ336030	PQ336042
BP-Ms2	MABIK 00001268	Goseong-gun, Gangwon-do (38°14'56.4"N, 128°34'05.8"E)	Sea algae (Myelophycus simplex)	Cystobasidium sp.	PQ336031	PQ336043
GH-Ll4	MABIK 00001120	Sacheon-si, Gyeongsangnam-do (34°55'44.8"N, 128°03'23.0"E)	Common periwinkle (Littorina littorea)	Metschnikowia bicuspidata	PQ336028	PQ336040
HW-W2	MABIK 00001257	Jeju-si, Jeju island (33°33'26.99"N, 126°48'41.86"E)	Sea water	Papiliotrema fonsecae	OR691666	OR714786
HW-W3	MABIK 00001262	Jeju-si, Jeju island (33°33'26.99"N, 126°48'41.86"E)	Sea water	Yarrowia lipolytica	OR691672	OR714792
KG-W2	MABIK 00001377	Taean-gun, Chungcheongnam-do (35°35'15.7"N, 126°17'25.0"E)	Sea water	Rhodotorula mucilaginosa	PQ336032	PQ336044
MPS3-W9	MABIK 00001290	the Southern Sea (34°5'35.0"N, 127°39'45.0"E)	Sea water	Hortaea werneckii	PQ336027	PQ336039
PH-Gra1	MABIK 00001378	Pohang-si, Gyeongsangnam-do (36.13′35.5″N, 129.35′23.23″E)	Sea algae (Grateloupia sp.)	Sporidiobolus pararoseus	MK994014	MK994015
US-Sd1	MABIK 00001260	Seosan-si, Chungcheongnam-do (36°36'51.5"N, 126°17'14.6"E)	Sediment	Vishniacozyma carnescens	OR691669	OR714789
US-Sd3	MABIK 00001254	Seosan-si, Chungcheongnam-do (36°36'51.5"N, 126°17'14.6"E)	Sediment	Aureobasidium sp.	OR691662	OR714782

^a Gene sequences: ITS, internal transcribed spacer regions; LSU, D1/D2 domains of the nuclear large subunit rRNA gene.

DNA extraction and polymerase chain reaction (PCR)

Genomic DNA (gDNA) extraction and PCR were performed as described previously (Yu et al., 2023). Yeast cells were cultured in YPD broth at 25°C, 150 rpm for 3 days, and collected by centrifugation at 5,000 rpm for 5 min. Then, gDNA was isolated using the previously established phenol:chloroform method (Chung et al., 2019).

For molecular identification of yeasts, sequences of two genetic markers, ribosomal internal transcribed spacer (ITS) and the partial D1/D2 domain of a large subunit (LSU, 28S) were analyzed. PCR was conducted using the primers ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') to amplify the ITS regions (Gardes and Bruns, 1993). To perform PCR for the LSU regions, LROR (5'-ACCCGCTGAACTTAAGC-3') and LR5 (5'-TCCTGAGGGAAACTTCG-3') were used as primers (Vilgalys and Hester, 1990). The PCR products were purified using a PCR purification kit (Qiagen, Germany) and sequenced by Macrogen Inc. (Korea).

Phylogenetic analysis

The obtained sequences of ITS and D1/D2 domain of each strain were used as a query for a BLASTN search in the GenBank database. Alignment and adjustment of the closely related sequences were conducted using MEGA X software (Tamura et al., 2011). Phylogenetic trees were constructed using the neighbor-joining (NJ) and the maximum-likelihood (ML) method with 1,000 bootstrap replicates in MEGA X. Kimura-2 parameter and general time reversible models were applied for the NJ and ML methods, respectively.

Examination of yeast growth on various NaCl concentrations

For spot assays, exponentially growing yeast cultures in YPD broth were normalized to OD₆₀₀ = 1.0, and serially diluted 10-fold. Five microliter of the diluted spore suspension was spotted on YPD agar supplemented with 0%, (w/v) 3%, 7%, 11%, 15%, and 20% NaCl. Following incubation at 25°C for 72 h, yeast growth was observed with naked eyes and imaged. This experiment was performed in triplicate.

Screening of ASNase and GLNase activities in marine yeasts

ASNase- and/or glutaminase (GLNase)-producing yeasts were screened as described previously (Yu et al., 2022). Yeast strains were cultured on modified Czapek-Dox (MCD) agar: 2.0 g glucose, 10.0 g L-asparagine monohydrate (Sigma-Aldrich, USA), 1.52 g KH₂PO₄, 0.52 g KCl, 0.52 g MgSO₄•7H₂O, 0.05% (w/v) CuNO₃•3H₂O, 0.05% ZnSO₄•7H₂O, 0.05% FeSO₄•7H₂O, and 15.0 g agar for 1 L media. The final pH was adjusted to 6.0 using 1 M NaOH. A 2% (w/v) stock solution of two individual pH indicators, bromothymol blue (BTB) and phenol red (PR), were prepared in ethanol and added to MCD agar at final concentration of 0.005% (v/v). L-Glutamine and NaNO₃ was added to MCD instead of L-asparagine for examination of GLNase activity and as a control medium, respectively. This experiment was performed in triplicate.

The ASNase and GLNase activities were determined by the formation of blue (from BTB) or pink (from PR) zone that surrounded the colony. The enzymatic index (EI) was calculated as follows (Ashok et al., 2019):

EI = [diameter of the pink(or blue) zone / diameter of the colony]

Statistical analysis

The EI data were analyzed using the Prism software version 5 (GraphPad, USA). One-way analysis of variance (ANOVA) followed by the Tukey’s multiple comparison test was conducted and the significance level was 5%.

Identification of marine yeasts

A total of 12 marine yeast strains were isolated from 7 types of marine resources collected from 9 distinct locations in Republic of Korea (Table 1). Marine resources included seawater, sediment, marine alga (Ulva linza, Myelophycus simplex, and Grateloupia sp.), and marine animals (Lajonkairia lajonkairii and Littorina littorea).

When cultured on PDA and YPD at 25°C for 5 days, the strains 21SA1-Ul15, BP-Ms2, KG-W2, and PH-Gra1 formed orange to pink colonies (Fig. 1). MPS3-W9 and US-Sd3 produced dark yellow and beige colonies on YPD, respectively, whereas they produced dark brown and black colonies on PDA. In addition, the colonies of BC-Cla8, BC-Cla9, GH-Ll4, HW-W2, HW-W3, and US-Sd1 were white to cream colored.

Fig. 1. Growth of marine yeasts on PDA and YPD. Yeast strains were streaked and incubated on PDA and YPD at 25ºC for 5 days.

Neighbor-joining and maximum-likelihood phylogenetic analyses using the sequences of ITS and D1/D2 domain of LSU regions showed that yeast isolates belonged to 2 phyla (Ascomycota and Basidiomycota), 6 classes, 8 orders, 9 families, 11 genera, and 12 species (Figs. 2 and 3). In phylogenetic analyses on the basis of the concatenated sequences of the ITS regions and D1/D2 domains, the trees were shown to be similar. The marine yeasts represented 10 known species and 2 candidates of novel species distributed in four main fungal lineages: Ascomycota, Saccharomycotina (3 strains), Ascomycota, Pezizomycotina (2 strains), Basidiomycota, Agaricomycotina (3 strains), Basidiomycota, Pucciniomycotina (4 strains). The phylogenetic trees of the marine yeast strains are shown in Fig. 2 (Ascomycota) and Fig. 3 (Basidiomycota).

Fig. 2. The phylogenetic tree of ascomycetous yeasts (Phylum Ascomycota). The tree was constructed based on the concatenated sequences of the ITS regions and the D1/D2 domains of LSU rRNA genes. The tree backbone was built with the neighbor-joining (NJ) method. Numbers at the nodes indicate percentages of bootstrap values of NJ and maximum likelihood (ML) methods from 1,000 replicates. Bootstrap values lower than 70 were not shown.

Fig. 3. The phylogenetic tree of basidiomycetous yeasts (Phylum Basidiomycota). The tree was constructed based on the concatenated sequences of the ITS regions and the D1/D2 domains of LSU rRNA genes. The tree backbone was built with NJ method. Numbers at the nodes indicate percentages of bootstrap values of NJ and ML methods from 1,000 replicates. Bootstrap values lower than 70 were not shown.

The 5 combined ITS and D1/D2 domain of LSU sequences were assigned in Ascomycota to 5 genera (Fig. 2). MPS3-W3 was identified as Hortaea werneckii, providing high level bootstrap support (100%). Although US-Sd3 was placed in the Aureobasidium clade, it was distinct from close reference strains. Based on the similarity of their ITS and LSU sequences, US-Sd3 was not assigned to a distinct species: A. namibiae CBS 147.97^T (97.13% identity in ITS regions and 99.46% in D1/D2 domains), A. leucospermi CBS 130593^T (97.78% identity in ITS and 98.73% in D1/D2), and A. subglaciale CBS 123387^T (98.95% identity in ITS and 98.56% in D1/D2) (Yu et al., 2023).

A BLASTN search showed a 100% similarity between BC-Cla8 and Debaryomyces hansenii JCM 1990^T and CBS 11132. It was closely related to D. vindobonesis CBS 11666^T (99.53% identity in ITS regions and 100% in D1/D2 domains) and D. fabryi Y-17914^T (99.83% identity in ITS and 99.66% in D1/D2). Thus, we suggest that BC-Cla8 represents D. hansenii. GH-Ll4 and HW-W3 made monophyletic groups with Metschnikowia bicuspidata (bootstrap value: NJ, 100%; ML, 95%) and Yarrowia lipolytica (bootstrap value: 100%), respectively.

The 7 concatenated sequences were placed in Basidiomycota and assigned to 6 genera: Papiliotrema, Vishniacozyma, Cystobasidium, Filobasidium, Rhodotorula, and Sporidiobolus (Fig. 3). As HW-W2 was placed in a monophyletic clade with Papiliotrema fonsecae (bootstrap value: 100%), this strain was identified as P. fonsecae. It was closely related to P. fonsecae EXF-4087^T, KF921, and ZM13F84 with a 100% bootstrap value. US-Sd1 was located in the Vishniacozyma clade. BC-Cla9 belonged to the genus Filobasidium, and was closely related to F. magnum 140^T (100%) and CBS 5591 (99.68% identity in ITS and 100% in D1/D2). BP-Ms2 was located the genus Cytobasidium and closely related to C. psychroaquaticum CBS 11769^T (97.9% and 98.91% identity in ITS and D1/D2, respectively). Cytobasidium slooffiae, C. minutum, and C. pinicola were placed as a sister group to the group including BP-Ms2 and C. psychroaquaticum. This strain was not assigned to the close reference strains and thus regarded as a new candidate species.

As PH-Gra1 was in a monophyletic clade with Sporidiobolus pararoseus and showed a 100% similarities to S. pararoseus CBS 4216 and 491^T (100%, respectively), it was identified as S. pararoseus. The isolates PH-Gra1, 21SA1-Ul15, and KG-W2 were grouped in the genus Rhodotorula clade. In the S. pararoseus section, PH-Gra1 formed a clade with 82% (NJ) and 71% (ML) degree of confidence. 21SA1-Ul15 belonged to the Rhodotorula babjevae clade with 81% (NJ) and 70% (ML) degree of confidence. In addition, its sequence identities were 99.66% in ITS and 99.83% D1/D2, respectively, to R. babjevae CBS 7808^T. Therefore, 21SA1-Ul15 was identified as R. babjevae. KG-W2 was grouped with the Rhodotorula mucilaginosa clade (bootstrap value: NJ, 100%; ML, 99%) and showed high degree of similarity to R. mucilaginosa 316^T (100% identity in ITS and 99.89% in D1/D2). As a result, it was identified as R. mucilaginosa.

Effect of NaCl concentration on growth of marine yeasts

Growth of yeast strains were examined on YPD agar supplemented with 0% (w/v), 3%, 7%, 11%, 15%, and 20% NaCl (Fig. 4). Although all strains were able to growth at 0% to 7% NaCl, they showed the maximum growth (the biggest colony size) in the absence of NaCl (0%). The growth of KG-W2 and PH-Gra1 decreased drastically between 0% and 3% NaCl, and no growth was observed in 21SA1-Ul15, BC-Cla9, BP-Ms2, PH-Gra1, and US-Sd3 at 11% NaCl. At 15% NaCl, marginal growth was observed in BC-Cla8, GH-Ll4, and MPS3-W9, suggesting these strains were more tolerant to NaCl than other 9 strains. None of the strains showed detectable growth at 20% NaCl.

Fig. 4. Growth of yeast strains under various NaCl concentrations. Each strain was serially diluted and spotted on YPD agar supplemented with 0%, 3%, 7%, 11%, and 15% NaCl. The plates were incubated at 25ºC for 3 days.

Asparaginase and glutaminase activities of marine yeasts

All yeast strains grew readily on MCD agar supplemented with L-asparagine, L-glutamine, or NaNO₃. The enzymatic activities were evaluated by the presence of pink (PR) or blue (BTB) zone on MCD, indicating formation of NH₄ (Fig. 5). Out of 12 yeast strains, 3 strains including 21SA1-Ul15 (R. babjevae), PH-Gra1 (S. pararoseus), and US-Sd3 (Aureobasidium sp.) showed ASNase activity.

Fig. 5. Screening of ASNase-producing marine yeasts. Yeast strains were inoculated on modified Czapek-Dox agar with different nitrogen sources (asparagine, glutamine, and NaNO₃) and indicators (BTB = bromothymol blue, PR = phenol red). On the table, ‘+’ and ‘–’ indicate ‘activity of ASNase or GLNase was detected’ and ‘not detected’, respectively.

Overall, the ASNase EI values calculated using a BTB indicator were approximately 1.6~2.5-fold higher than those calculated using a PR indicator (Fig. 6), although the yeast colony sizes were not affected by the pH indicators. Based on the EI values, the highest ASNase activity was observed in 21SA1-Ul15 (EI value = 7.38 ± 0.13 using BTB, 4.46 ± 0.08 using PR). When BTB was used, US-Sd3 showed the lowest ASNase activity among 3 strains (EI value = 5.43 ± 0.16). However, when PR was used instead of BTB, ASNase activity of US-Sd3 was not significantly different from that of PH-Gra1 (P > 0.05).

Fig. 6. Evaluation of ASNase activity in marine yeasts. The enzymatic index (EI) values were calculated based on the results presented in Fig. 5. This experiment was performed in triplicate. ‘*’ and ‘ns’ indicate statistically significant (P < 0.05) and non-significant (P > 0.05), respectively.

Both PH-Gra1 and US-Sd3 strains showed GLNase activities, although the activities were substantially lower than their ASNase activities (Fig. 5). The blue/pink zones observed in 21SA1-Ul15 cultured in glutamine plates were not considered as GLNase activity, because the area of the blue/pink zones were not significant compared to that of NaNO₃ plates (control). This result suggests that R. babjevae produced ASNase free of GLNase.

Generally, identification of yeasts using molecular analysis is conducted based on both the ITS regions and D1/D2 domains (Nutaratat et al., 2022). According to the dataset of culturable yeast barcodes for identification, the threshold to regard as a different species from its close reference strains is less than 98.31% (Ascomycota) and 98.61% (Basidiomycota) in ITS regions. For LSU regions, the taxonomic threshold to distinguish yeast species is less than 99.41% (Ascomycota) and 99.51% (Basidiomycota) (Vu et al., 2016). In addition, analysis of the ITS sequence is considered to be more effective than LSU for species discrimination in Ascomycota, whereas LSU sequences perform better than ITS in Basidiomycota (Vu et al., 2016).

In this study, sequences of ITS and D1/D2 domain of 12 strains were compared with closely related species. The sequence similarities of BP-Ms2 to the closely related species were lower than the threshold values in ITS and LSU sequences. The sequence similarity of US-Sd3 ITS was lower than 98.31% when compared to that of the close reference strains. As a result, BP-Ms2 (Cystobasidium sp., Basidiomycota) and US-Sd3 (Aureobasidium sp., Ascomycota) were not able to be identified to the species level, suggesting these strains are putative novel species.

Although all strains showed their maximum growth on YPD in the absence of NaCl, their tolerance to high NaCl concentrations varied. The most NaCl-tolerant strains were BC-Cla8 (D. hansenii), GH-Ll4 (M. bicuspidate), and MPS3- W9 (H. werneckii). These species have been previously isolated from hypersaline habitats (Butinar et al., 2005), and D. hansenii and H. werneckii are generally considered as halotolerant yeasts (Prista et al., 1997; Kogej et al., 2005). Moreover, M. bicuspidata isolated from brine shrimp shows growth at 0–18% NaCl (Moore and Strom, 2002). Although these 3 strains did not produce ASNases, their halotolerance could be applied for other biotechnological industries (Margesin and Schinner, 2001).

21SA1-Ul15 (R. babjevae), PH-Gra1 (S. pararoseus), and US-Sd3 (Aureobasidium sp.) were identified as ANSase- producing yeasts. ASNase activities have not been previously reported in R. babjevae and S. pararoseus. Both species are basidiomycetous yeasts belonging to the same family sporidiobolaceae. In the genus Aureobasidium (ascomycetous yeast), A. pullulans ASNase has been elucidated to reduce asparagine content in food (Francesco, 2022).

It is notable that 21SA1-Ul15 (R. babjevae) showed GLNase-free ASNase activity. The adverse effects of commercial ASNase in treating cancer patients are due to the GLNase side activity of ASNase (Narta et al., 2007). Moreover, it has been suggested that bacterial ASNases are more immunogenic than eukaryotic congeners (Freire et al., 2021). Thus, there has been an increasing number of studies to discover GLNase-free ASNases in fungi (Ashok et al., 2019; Freire et al., 2021; Arumugam et al., 2022). To date, a few yeast (S. cerevisiae and Yarrowia lipolytica)-derived ASNases have been suggested as candidates for a new pharmaceutical ASNase (Girao et al., 2016; Darvishi et al., 2019). To serve an additional candidate for a new clinical ASNase, purification, culture optimization, and examination of anti-tumor activity will be needed for future studies of R. babjevae ASNase.

아스파라긴분해효소(Asparaginase, ASNase)는 아스파라긴을 아스파라긴산으로 가수분해 시키는 효소이다. 급성 림프구성 백혈병 환자를 치료하는 항암제로 사용되고, 가열된 음식에 생성되는 발암 물질인 아크릴아미드 저감에도 사용된다. 본 연구에서는 다양한 해양환경으로부터 분리한 효모 균주들의 ASNase 활성을 선별하였다. 형태학 및 계통학적 분석 결과, 확보된 균주들은 2문, 6강, 8목, 9과, 11속, 12종의 해양효모로 동정되었다. 이 중 BP-Ms2 (Cystobasidium sp.)와 US-Sd3 (Aureobasidium sp.)은 잠재적인 신종으로 밝혀졌다. BC-Cla8 (Debaryomyces hansenii), GH-L4 (Metschnikowia bicuspidata), MPS3-W9 (Hortaea werneckii)는 내염성(halotolerant)을 띄며, yeast peptone dextrose (YPD)에 15% 염화나트륨을 첨가한 배지에서 생장할 수 있었다. 12개 균주 중 21SA1-Ul15 (Rhodotorula babjevae), PH-Gra1 (Sporidiobolus pararoseus), US-Sd3 (Aureobasidium sp.) 세 균주는 ASNase 활성을 나타냈으며, 이 중 21SA1-Ul15에서 가장 높은 활성이 관찰되었다. 특히 21SA1-Ul15 균주의 경우 글루타민분해효소(Glutaminase, GLNase)의 활성이 관찰되지 않았다. GLNase는 ANSase에 부수적으로 존재하는 효소 활성으로, ASNase로 치료받는 환자에게서 발생하는 부작용의 원인인 것으로 알려져 있다. 이 결과는 GLNase 활성이 없는 ASNase를 생산하는 R. babjevae에 대한 최초의 보고이다. 본 연구에서 확보된 데이터는 향후 신규 ASNase 개발을 위한 기초자료로 활용될 수 있을 것으로 사료된다. 나아가 21SA1-Ul15의 ASNase는 부작용이 적은 신규 치료용 ASNase의 후보 역할을 할 수 있을 것으로 기대된다.

This study was supported by the National Marine Biodiversity Institute of Korea (MABIK) under an in-house research program (2024M00600).

The authors declare no conflict of interest.