Targeted transcriptomic study of the implication of central metabolic pathways in mannosylerythritol lipids biosynthesis in Pseudozyma antarctica T-34.

Pseudozyma antarctica is a nonpathogenic phyllosphere yeast known as an excellent producer of industrial lipases and mannosylerythritol lipids (MELs), which are multi-functional glycolipids. The fungus produces a much higher amount of MELs from vegetable oil than from glucose, whereas its close relative, Ustilago maydis UM521, produces a lower amount of MELs from vegetable oil. In the present study, we used previous gene expression profiles measured by DNA microarray analyses after culturing on two carbon sources, glucose and soybean oil, to further characterize MEL biosynthesis in P. antarctica T-34. A total of 264 genes were found with induction ratios and expression intensities under oily conditions with similar tendencies to those of MEL cluster genes. Of these, 93 were categorized as metabolic genes using the Eukaryotic Orthologous Groups classification. Within this metabolic category, amino acids, carbohydrates, inorganic ions, and secondary metabolite metabolism, as well as energy production and conversion, but not lipid metabolism, were enriched. Furthermore, genes involved in central metabolic pathways, such as glycolysis and the tricarboxylic acid cycle, were highly induced in P. antarctica T-34 under oily conditions, whereas they were suppressed in U. maydis UM521. These results suggest that the central metabolism of P. antarctica T-34 under oily conditions contributes to its excellent oil utilization and extracellular glycolipid production.

Introduction

Pseudozyma antarctica is a basidiomycetous yeast belonging to Ustilaginomycetes, which includes the corn smut fungus, Ustilago maydis [1,2], and is known to extracellularly produce not only lipases but also a biodegradable plastic-degrading enzyme that hydrolyzes polybutylene succinate and polybutylene succinate-co-adipate [3]. Pseudozyma antarctica T-34 was isolated in Tsukuba, Japan, as a producer of extracellular glycolipids, mannosylerythritol lipids (MELs; ), which consist of 4-O-β-D-mannopyranosyl-meso-erythritol as the hydrophilic moiety and fatty acids as the hydrophobic moiety [4]. MELs not only have high potential as eco-friendly biosurfactants due to their excellent surface activity, but also have attracted considerable recent interest because of their unique properties, including self-assembly, anti-tumor and cell differentiation induction activities, and moisturizing and hair-repairing properties [5, 6]. Further improvements to the mass production of MELs, and their applications to life science, nanotechnology, and environmental technology, have been investigated [7-10].

Biosynthetic pathway for mannosylerythritol lipids.

Emt1p: mannose/erythritol transferase; Mac1p and Mac2p: acyl-transferases; Mat1p: acetyl-transferase; Mmf1p: predicted MEL transporter.

Pseudozyma antarctica T-34 produces large amounts of MELs when grown in culture containing vegetable oil as the carbon source, and the production yield reaches 140 g/L using n-alkanes as the carbon source [11]. Pseudozyma aphidis is also an efficient producer of MELs with a yield of more than 165 g/L from vegetable oil as the main carbon source [12, 13]. It should be noted that a closely related fungus, Ustilago maydis UM521, produces lower amounts of MELs from vegetable oils than yeast strain of the genus Pseudozyma, including the strain T-34. While U. maydis DSM4500 was reported to produce extracellular glycolipids in the yield of 30 g/L from 45 g/L of sunflower oil as main carbon source, the glycolipids are the mixture of MELs and cellobiose lipids (CLs) [14]. The Pseudozyma strains therefore have considerable potential for large-scale industrial production of MELs using vegetable oil.

Recently, we reported the genome sequence of P. antarctica T-34 [15] and found that this yeast has an oleaginous nature based on genomic and transcriptomic analyses; a gene encoding an ATP/citrate lyase conserved in oleaginous strains found in the genome of strain T-34 [16]. Using gene set enrichment analysis, the gene sets related to fatty acid metabolism were significantly upregulated in the presence of vegetable oil, and the gene cluster for MEL biosynthesis was also highly expressed in P. antarctica T-34, regardless of whether the carbon source was glucose or soybean oil (). Genomic analysis showed that the P. antarctica T-34 genome was similar to that of U. maydis UM521 in chromosomal organization. However, the gene sets enriched were significantly different, suggesting different regulatory mechanisms. In addition, MEL production from vegetable oil by P. antarctica T-34 (30 g/L of MELs, mainly MEL-A) was more effective than that of U. maydis UM521 (slight amounts of glycolipids on TLC analysis), suggesting that P. antarctica T-34 may utilize vegetable oil for growth and glycolipid synthesis. However, the characteristics of oil utilization by P. antarctica T-34 remained unclear [16].

In the present study, we further analyzed transcriptomic data to improve our understanding of the molecular mechanisms of oil utilization and MEL production in P. antarctica T-34. The transcriptional analysis focused on genes that acted similarly to the MEL biosynthesis gene cluster, and were selected using “guilt-by-association” in the induction ratio. The gene expression intensities revealed that genes related to central metabolic pathways such as glycolysis and the tricarboxylic acid cycle (TCA) were upregulated in P. antarctica T-34 when compared to U. maydis UM521 under oily conditions. These results suggest that P. antarctica T-34 is adapted to aerobically produce larger amounts of MELs from vegetable oil by modification of its central metabolic system. Overall, insight into the oil utilization capacity of microorganisms will lead to more effective strategies for using feedstocks to produce functional bio-based materials.

Materials and methods

Microorganisms

Pseudozyma antarctica (formerly Candida antarctica) T-34 used in this study was isolated as a MEL producer using soybean oil as the sole carbon source [4]. Ustilago maydis DSM14603 (UM521) was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany. Stock cultures were cultivated for 3 days at 25°C in YM plate medium containing 1% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt extract, and 3.0% agar. The cultures were stored at 4°C and renewed every 2 weeks.

Culture conditions

Seed cultures were prepared by inoculating cells grown on slants into test tubes containing YM medium at 25°C on a rotary shaker (250 rpm) for 2 days. Seed cultures (1 mL) were transferred to test tubes containing 20 mL of an experimental medium (0.03% MgSO4, 0.03% KH2PO4, 0.1% yeast extract, pH 6.0) containing different carbon sources, and were then incubated as above for 5 days.

Isolation and detection of glycolipids

After cultivation, the whole culture, including glycolipids, was extracted using an equal volume of ethyl acetate. The ethyl acetate extracts were analyzed using thin layer chromatography (TLC). TLC was performed using chloroform-methanol-7 N ammonium hydroxide (65:15:2, vol/vol/vol) as the solvent system. Although not a quantitative method, the MEL production was evaluated by TLC analysis in order to make a simple visual comparison of differences in MEL production depend on the producers and substrates. Visualization was performed by spraying 0.3% anthrone-sulfate reagents on the TLC plate and heating it at 90°C for 5 min [4]. Purified MEL-A, MEL-B, and MEL-C, which were prepared from soybean oil by P. antarctica T-34, were used in the following experiments as standards.

Estimation of cell growth

To estimate the effect of cell growth on different substrates, MELs and residual hydrophobic substrates were removed from culture broths by ethyl acetate. Then, the culture broth was washed twice with methanol to remove residual ethyl acetate. Cells were suspended in water of the same volume as the culture broth. The suspensions were centrifuged at 6,000 × g, and the pellets were weighed. All measurements were calculated from at least three independent experiments.

Enzyme assay

The cells were harvested by centrifugation, washed, and suspended in 50 mM sodium acetate buffer (pH 5.0) containing 2 mM phenylmethylsulfonyl fluoride. The cells were disrupted in a 2 mL microtube with 0.5 mm glass beads using the BEADS CRUSHER μT-12 (TAITEC, Tokyo, Japan). The resulting homogenate was centrifuged at 20,000 × g for 20 min to remove the glass beads and cell debris, and the supernatant was used as the crude enzyme solution.

Isocitrate dehydrogenase was measured in 96-well plates containing 0.2 mL of a reaction mixture consisting of 40 mM Tris-HCl (pH 7.0), 1 mM tri-sodium-isocitrate, 4 mM MgCl2, and 0.5 mM NAD+. Reactions were started by adding the crude enzyme solution (5 μg), and the absorbance at 340 nm was read every 1 min for up to 20 min using Synagy2 (BioTek Instruments, Winooski, VT, USA). A control lacking tri-sodium-isocitrate was also run. Enzymatic activity was determined by measuring the maximum rate of NADH production [17]. Protein concentrations were determined using a BCA protein assay kit (Pierce, Rockford, IL, USA) with bovine serum albumin as a standard. All measurements were calculated from at least three independent experiments.

Statistical analysis of DNA microarray data

Two values of the expression level and induction of each gene by each experiment using a two-color platform were obtained from previous microarray data [16]. The Seris accession number in the Gene Expression Omnibus for the gene expression data was GSE47171. In the data, duplicated measurements were performed for each sample using the dye-swap method. Data were normalized by setting the baseline measurement per spot and per segment of the chip via intensity dependent (LOESS) normalization using the marray module of the R package [18] and Bioconductor [19]. Duplicated data files generated by the dye-swap experiment for an individual test were merged using the limma module. The logarithmic induction ratio (M-value) was calculated using the following equation: and the average of the logarithmic signal intensities (A-value) was calculated using the following equation: where Cy5 and Cy3 are normalized intensities for each transcript under the respective conditions using soybean oil or glucose as the sole carbon source [20]. The P. antarctica T-34 genes showing upregulated transcriptional expression in the presence of vegetable oil as compared to glucose were selected as those upregulated under oily conditions.

Results

Pseudozyma antarctica genes expressing under oily conditions

To demonstrate the transcriptomic regulatory characteristics of P. antarctica T-34 under oily conditions that result in the production of MELs, we focused on genes that were differentially regulated between P. antarctica T-34 and U. maydis UM521, using the DNA microarray data. Previously, the microarray data were derived from the transcriptomes of each strain cultured in medium containing 5% soybean oil or 10% glucose as the sole carbon source for 3 days. During this cultivation, larger amounts of MELs (30 g/L of MELs, mainly MEL-A) were produced from soybean oil by P. antarctica T-34, whereas U. maydis UM521 produced only small quantities of MELs on TLC analysis, and considerable soybean oil remained in the culture [16]. MEL production by P. antarctica T-34 from glucose (5 g/L of MELs, mainly MEL-A and mono-acylated type of MELs) was lower than that from soybean oil. Transcriptional expression analysis was previously performed using a two-color microarray platform, and the relative abundances of each transcript were compared between cells grown in the presence of soybean oil or glucose as the sole carbon source. Values, the logarithmic induction ratio (M-value), average of the logarithmic signal intensities (A-value) and probability values (p-value), were obtained after processing the raw DNA microarray data [16].

As described previously [15], in P. antarctica T-34, the MEL biosynthesis gene cluster was identified () as being expressed under both conditions, although the production level was higher in the presence of excess vegetable oil. The gene expression intensity (A-value) and induction ratio (M-value) of the five genes were significantly higher in P. antarctica T-34 than in U. maydis UM521 [16]. In the present study, we further analyzed the data and visualized the distribution of gene expression and the induction ratio by vegetable oil as a two-dimensional histogram, which revealed that U. maydis UM521 genes were widely scattered compared with those of P. antarctica T-34 (). To estimate the transcriptional regulatory characteristics of both strains under oily conditions, genes showing expression patterns similar to the MEL biosynthesis genes shown in were selected and analyzed. The 264 genes with an A-value greater than 14 in P. antarctica and an M-value less than 0 for the ortholog in U. maydis are listed in . As expected, the four genes for MEL biosynthesis, PaEMT1 (19c00001), PaMAC1 (19d00003), PaMMF1 (19d00004), and PaMAT1 (19c00002), were included in the list.

Gene clusters of mannosylerythritol lipid (MEL) biosynthesis and gene expression profiles.

The MEL biosynthesis gene cluster in U. maydis UM521 and P. antarctica T-34 (A). Two-dimensional histogram showing the gene expression distribution (A-value, ordinate) and gene transcription induction ratio (M-value, abscissa) in the presence of vegetable oil and glucose in P. antarctica T-34 (B) and U. maydis UM521 (C). The number of genes is represented by the gray shading of each hexagonal cell. This hexplot was drawn using the hexbin module in R [21]. The genes responsible for MEL biosynthesis are overlaid on the hexplot in pink.

Based on the Eukaryotic Orthologous Groups classification, 179 of the 264 genes were assigned to each category. 93 of the genes were assigned to the metabolism category, corresponding to 35.2% of the 264 genes, whereas metabolism related genes comprised only 17.7% of the total coding sequence (CDS) (1,157 genes). However, the proportions of genes categorized as cellular processes (15.9%), information storage and processing (6.1%), and poorly characterized (10.6%) were lower than those of the total CDS ().

Within the metabolism category, the proportions of genes classified as amino acid, carbohydrate, inorganic ions, and secondary metabolite metabolism, as well as energy production and conversion, were higher among the listed 264 genes than among the total CDS, whereas the proportions involved in lipid transport and metabolism were almost identical between the listed genes (3.8%) and total CDS (3.3%). The listed genes responsible for metabolism, with the exception of lipid metabolism, were therefore expressed along with MEL biosynthesis genes under oily conditions, suggesting that P. antarctica T-34 efficiently generated energy from vegetable oil via respiratory metabolism. In addition, the genes related to glycolysis and the TCA cycle, which are part of the central metabolic pathway, may relate to MEL production, because the transcriptional regulatory characteristics are similar to MEL biosynthesis genes.

Metabolic genes involved in MEL production

As detailed above, the genes related to metabolism, e.g., beta-oxidation, fatty acid synthesis, glycolysis/gluconeogenesis, and TCA cycle, were predominantly expressed during MEL production. The genes involved in beta-oxidation and fatty acid synthesis were extracted from the Saccharomyces Genome Database, and the orthologs were selected using a homology search (S2 and S3 Tables). The genes involved in peroxisomal and mitochondrial beta-oxidation were induced in both P. antarctica T-34 and U. maydis UM521, whereas the induction ratios of the genes for fatty acid synthesis were suppressed in both strains (). These results suggest that both strains degraded vegetable oil via the beta-oxidation pathway, and that fatty acid synthesis was required in the presence of glucose because fatty acid must be synthesized from glucose to supply cellular demands and for MEL biosynthesis. In addition, M-value average of MEL biosynthesis genes were −0.499 and −1.38 in P. antarctica T-34 and U. maydis UM521, respectively. This result indicates that MEL biosynthesis pathway in U. maydis UM521 is suppressed compared to that in P. antarctica T-34.

Induction of genes responsible for oil degradation and conversion and primary metabolism.

The M-value averages of the genes responsible for MEL biosynthesis, mitochondrial beta-oxidation, peroxisomal beta-oxidation, fatty acid synthesis, glycolysis, tricarboxylic acid cycle, pentose phosphate pathway, and malate/pyruvate cycle are shown in orange (U. maydis UM521) and blue (P. antarctica T-34).

In addition, a gene encoding an extracellular lipase (12c00005), which is required for effective oil degradation [22], was expressed (A-value: 12.0) and strongly induced (M-value: 1.70) in P. antarctica T-34. However, the orthologous gene (um03410.1, accession #: Q4P903) was slightly suppressed in U. maydis UM521 (M-value: −0.28), and the expression intensity (A-value: 9.98) was significantly lower. This gene was likely a major factor in the more effective utilization of vegetable oil in P. antarctica T-34 than in U. maydis UM521.

The expression profiles of the genes responsible for glycolysis and the TCA cycle differed between P. antarctica T-34 and U. maydis UM521 (). Notably, the average logarithmic induction ratio (M-value) of most genes in the central metabolic pathway, such as those involved in glycolysis and the TCA cycle, in P. antarctica T-34 was positive, whereas that in U. maydis UM521 was negative. We thus further analyzed the detailed expression levels of glycolysis and TCA cycle genes.

Glycolysis and TCA cycle genes

As shown in , the M-values of all genes related to the TCA cycle in P. antarctica T-34 were positive, indicating that these genes were induced in the presence of vegetable oil, whereas the M-values in U. maydis UM521 close to zero. The genes encoding isocitrate lyase (22c00252, M-value: 0.78; A-value: 13.2) and malate synthase (14d00059, M-value: 1.3; A-value: 15.0) as the glyoxylate shunt, and malic enzyme (7c00096, M-value: 0.43; A-value: 14.8) and phosphoenolpyruvate carboxykinase (2d00021, M-value: 1.1; A-value: 15.3) as the anaplerotic reaction, were highly expressed under oily conditions in P. antarctica T-34. These results indicate that the glyoxylate shunt and the anaplerotic reaction assisting gluconeogenesis for sugar synthesis in P. antarctica T-34 were active under oily conditions. However, the M-values of these genes were also positive in U. maydis UM521, whereas the citrate synthase catalyzing primary reactions of the TCA cycle were strongly repressed (M-value: −0.88). These results suggest that the glyoxylate shunt and the anaplerotic reaction were inactive in metabolic flow, although each gene was expressed under oily conditions in U. maydis UM521. Moreover, most genes related to glycolysis were highly expressed even under oily conditions in P. antarctica T-34, whereas those in U. maydis UM521 were repressed.

M-values in the central metabolic pathway of P. antarctica T-34 and U. maydis UM521.

Comparison of the expression profiles of genes encoding enzymes that participate in glycolysis and the tricarboxylic acid cycle under oily conditions in P. antarctica T-34 (A) and U. maydis UM521 (B). To facilitate the comparison of the two microorganisms, portions of the metabolic pathways of Saccharomyces cerevisiae are presented, and the behaviors of genes encoding the enzymes that catalyze each step were estimated. GUT: glycerol-3-phosphate dehydrogenase; TPI: triose phosphate isomerase; FBA: fructose 1,6-bisphosphate aldolase; TDH: glyceraldehyde-3-phosphate dehydrogenase; PGK: 3-phosphoglycerate kinase; GPM: phosphoglycerate mutase; ENO: enolase; PYK: pyruvate kinase; PDA: pyruvate dehydrogenase; PDC: pyruvate decarboxylase; ACS: acetyl-coA synthetase; ADH: alcohol dehydrogenase; ALD: aldehyde dehydrogenase; PCK: phosphoenolpyruvate carboxykinase; MAE: malic enzyme; CIT: citrate synthase; ACO: aconitase; IDH: isocitrate dehydrogenase; KGD: α-ketoglutarate dehydrogenase; LSC: succinyl-CoA ligase; SDH: succinate dehydrogenase; FUM: fumarate hydratase; MDH: malate dehydrogenase; ICL: isocitrate lyase; MAS: malate synthase. Red and green boxes represent those genes whose expression levels were increased and decreased, respectively, under oily conditions.

Activities of the TCA cycle enzymes in P. antarctica and U. maydis

Among TCA cycle gene expression, the M-value of the gene encoding isocitrate dehydrogenase, which is a rate-limiting enzyme of the cycle, in P. antarctica T-34 was positive, whereas that in U. maydis UM521 was negative. We thus measured the enzyme activity to further estimate the function of the TCA cycle based on the enzymatic activities in P. antarctica T-34 and U. maydis UM521 under oily conditions. Both strains were cultured in medium containing 5% soybean oil or 10% glucose as the sole carbon source for 5 days. Glycolipids were then extracted using an equal amount of ethyl acetate, and MEL production was confirmed by TLC analysis using the anthrone staining method. P. antarctica T-34 produced larger amounts of MELs, mainly MEL-A, with soybean oil but lower quantities from glucose. During cultivation, almost all of the soybean oil was consumed by P. antarctica T-34 (). In contrast, U. maydis UM521 produced lower amounts of MELs, and larger amounts of soybean oil remained in the medium. U. maydis UM521 grew slowly under the oily conditions compared with P. antarctica T-34 (). These results of MEL production characteristics of both of strains are identical to previous study [16].

Comparison of mannosylerythritol lipid (MEL) production, cell growth, and isocitrate dehydrogenase (NAD+) activity of P. antarctica T-34 and U. maydis UM521 in the presence of glucose or soybean oil as the sole carbon source.

(A) MELs were extracted from the culture after 3 and 5 days using an equal amount of ethyl acetate, and the organic solvent fractions (with glucose: 10 μL, with soybean oil: 2.5 μL) were spotted on a TLC plate. A typical sample of three independent experiments was shown in each lane. Spots were visualized using the anthrone reagent. Purified MEL-A, MEL-B, and MEL-C were used as standards. (B) The weight of wet cells was measured to estimate growth. (C) The activity of isocitrate dehydrogenase (NAD+) was measured as described in the Materials and methods section. Error bars show standard deviations.

Isocitrate dehydrogenase activity was induced in the T-34 strain of P. antarctica under oily conditions, whereas no activity was detected in the presence of glucose (). In U. maydis UM521, the activity was low in the presence of glucose, and no activity was noted under oily conditions ().

Discussion

Transcriptomic analyses of associated genes and pathways provided new insight into the capacity of P. antarctica T-34 to produce functional bio-based materials from oil biomass. Surprisingly, the genes responsible for central metabolic pathways such as glycolysis, the TCA cycle, the glyoxylate shunt, and the anaplerotic reaction were highly expressed in P. antarctica T-34, regardless of whether the carbon source was glucose or soybean oil, whereas these genes were not induced in U. maydis UM521, which could be the reason for poor growth and lower MEL production under oily conditions. The characteristics of P. antarctica T-34 that enabled it to efficiently metabolize vegetable oil via the central metabolic pathway facilitated the production of large amounts of extracellular MELs. In the future, P. antarctica will be increasingly used as a platform for new biomaterial production processes, such as producing functional lipids from oily biomass.

Previously, we reported that the genome sequence of P. antarctica T-34 was closely related to that of U. maydis UM521, based on the results of synteny analysis [16]. However, gene set enrichment analysis of the transcriptome revealed that gene expression differed markedly between P. antarctica T-34 and U. maydis UM521 in the presence of glucose. The present analysis focused on the expression of genes required for cell growth under oily conditions, which prompted us to further investigate the prerequisites for extracellular production of MELs utilizing large quantities of vegetable oil.

In P. antarctica T-34, fatty acids derived from vegetable oil by enzymatic degradation were processed via chain-shortening pathways such as beta-oxidation, and the intermediates were directly transferred into mannosylerythritol, resulting in production of MELs [23]. However, U. maydis UM521 did not metabolize vegetable oil, resulting in poor growth and low MEL productivity () identical to our previous report [16]. This phenomenon was supported by the present transcriptomic analysis, showing that the expression levels of most genes of central metabolic pathways were reduced under oily conditions in U. maydis UM521.

However, the genes encoding enzymes in the TCA cycle (citrate synthase, aconitase, succinate dehydrogenase, fumarase, and malate dehydrogenase), glyoxylate shunt (isocitrate lyase and malate synthase), and anaplerotic reaction (malic enzyme and phosphoenolpyruvate carboxykinase) were highly expressed in the presence of vegetable oil in P. antarctica T-34 (). A similar process has been reported in Escherichia coli when cultured with acetate or fatty acid as a sole carbon source [24, 25]. In addition, 13C-metabolic flux analysis revealed that the glyoxylate shunt was activated in Yarrowia lipolytica for growth and lipids production when cultured with acetic acid, which is kind of volatile fatty acid, as sole carbon source [26]. Importantly, the glyoxylate shunt and anaplerotic reaction contribute to carbon assimilation from acetyl-CoA and effective carbon delivery into the gluconeogenesis pathway, respectively. Additionally, enhancement of these primary metabolic activities may lead to improved sugar synthesis for cell growth and MEL production from vegetable oil. Therefore, although the direction of the carbon flow was unclear in the results of transcriptomic analysis, most reactions in the glycolytic process are reversible, and it is probable that the enhanced expression of glycolysis genes reflected the activation of gluconeogenesis under oily conditions. Further metabolomics studies are needed to reveal the carbon flow in the primary metabolic pathways under oily conditions. Although P. antarctica T-34 and U. maydis UM521 are closely related species, characteristics of growth and MEL production, including the activities of central metabolic pathways, differ between them. We hypothesize that these differences originated in specific growth environments and lifestyles. The genus Pseudozyma is often isolated from plant surfaces in nature [4, 27, 28], and utilizes cuticles composed of water insoluble fatty acid esters covering leaves with their lipases and esterases [29]. Genus Pseudozyma may therefore utilize the secretion of biosurfactants such as MELs to uptake emulsified cuticles as a carbon source. However, U. maydis UM521 is a maize pathogen, and can utilize starch-derived sugar, which is abundant in the growing environment. Our transcriptomic analysis suggested that sufficient primary metabolic activity was required for growth and MEL production under oily conditions, in addition to expression of the genes responsible for MEL biosynthesis and oil degradation in P. antarctica. These results indicate that U. maydis, unlike P. antarctica, adapted to utilize sugars as the main carbon source.

Moreover, 111 of the 264 genes in had no clear assignment, and were listed as “function unknown,” “general function predicted only”, or “no description.” We further categorized these genes based on the Pfam database. The gene functions of 88 of the genes were predicted, but the remaining 23 genes were uncharacterized. Fifty-three of the P. antarctica T-34 genes nonorthologous to U. maydis UM521 are also listed in S4 . Genes contributing to oil utilization may be hidden among them. Further studies related to these genes will help identify fundamental differences in the metabolic systems between nonpathogenic P. antarctica T-34 and plant-pathogenic U. maydis UM521.

Various vegetable oils have been used as main carbon sources for MEL production, due to high productivities, e.g., P. aphidis produced 165 g/L of MELs (the main product is MEL-A) from soybean oil and glucose, and P. regulosa produced 142 g/L of MELs (the main product is MEL-A) from soybean oil and erythritol, P. tsukubaensis produced 73.1 g/L of MELs (the main product is MEL-B) from soybean oil. While U. maydis also produced MELs from sunflower oil, the productivity was lower than that of the genus Pseudozyma due to the products were a mixture of MELs and CLs. On the other hand, soluble carbon sources such as glycerol, glucose, and sucrose were examined for MEL production, e.g., P. antarctica produced mono-acylated type of MEL as the main product from glucose [10], and U. maydis produced 32.1 g/L of a glycolipid mixture of MELs and CLs from glycerol [30]. Therefore, vegetable oils are sufficient carbon sources for the MEL production. Recently, we accomplished with improvement of MEL production from olive oil by increase of the expression of a gene encoding lipase in P. tsukubaensis [22].

The present transcriptomic and biochemical analyses focused on gene expression in P. antarctica T-34, a highly oil-assimilating and glycolipid-producing yeast, in the presence of vegetable oil. Further genetic study will facilitate not only the development of useful industrial strains as MEL producer by genetic modification, but also improve our understanding of phytopathological mechanisms and environmental adaptation of these basidiomycetous genera.

The list of 264 genes extracted by guilt-by-association.

(XLSX)

Click here for additional data file.

The list of the genes related to lipogenesis pathway in P. antarctica T-34.

(XLSX)

Click here for additional data file.

The list of the genes related to lipogenesis pathway in U. maydis UM521.

(XLSX)

Click here for additional data file.

The list of the non-ortholog genes expressing and inducing under oily conditions in P. antarctica T-34.

(XLSX)

Click here for additional data file.

6 Sep 2019

PONE-D-19-22493

A novel aspect of the production of mannosylerythritol lipids in Pseudozyma antarctica T-34 based on gene expression of central metabolic pathways

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

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Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Linguistic comments

• Line 65: “large amounts of vegetable oil”. Change it to “vegetable oil”.

• Line 153: “a large quantities of MELs.” Change it to “MELs” or define large quantities of MELs

• Line 197: Remove “surprisingly”

• Line 251: “were almost negative”. Change it to “close to zero”.

• Line 289: “enzymes”. Change it to “gene expressions”.

• Line 314: “strongly induced”. Change it to “induced”.

• Line 338 & 352: “large quantities”; Change it to “larger quantities”.

• General: When adding production numbers, they should be mentioned in volumetric productivities for improved comparison in literature.

Research comments

• There is no mentioning of any statistical significance of the genes or the selection procedure of the genes illustrated in figure 2 B&C. This part needs extended clarification.

• Line 177-181: What is the scientific background of this selection procedure for “similar genes expressions to the MEL biosynthesis genes”? Cut-offs of A-values > 14 and M < 0 for U. Maydis orthologs seem to be defined arbitrary. One of the MEL production genes has even a M>0. Revise the selection procedure or mention the scientific/statistical incentives for the cut-offs.

• There is no mention of exact replicate numbers for all the experiments performed.

• Line 310: Why was wet cell weight used and dry cell weight. Change in wet cell weight can be caused by changes in substrate concentrations or evaporation.

Conclusion comments

• Line 170: Can you compare A-values between two different organisms?

• Line 211-212: State with literature or experimental evidence why TCA and glycolysis are related to production of MELs.

• Line 265-267: It’s only a slight indication. There are more levels of regulation (post-transcriptional, translational, post-translational,…). This statement needs either metabolomic data or needs to be removed.

• Line 326: change “resulting in” to “which could be the reason for”.

• Discussion: Add comparisons with literature concerning the central metabolism of lipophilic fungi.

• Discussion: Add comparisons with literature concerning the carbon sources used for MEL production and their respective production numbers.

Figure comments

• Figure 1: Figure description doesn’t match the figure. Discrepant abbreviations and full names of enzymes.

• Figure 1: rarely does vegetable oil solely consist of C18 fatty acids. It should be made clear that the fed vegetable oil predominantly consists of C18 fatty acids.

• Figure 2 B&C: Density is not clear. Use a continuous colour scale.

• Figure 2: “The genes responsible for MEL biosynthesis are overlaid on the hexplot”. Mention they are coloured in pink.

• Figure 5 B: Can it be that the higher production is cause by higher biomass? Calculate specific productivities (g MEL/ (g Biomass*h).

• Figure 5 B & C: Define the error flags. Are they standard deviations, confidence intervals,…

Extra information/reference necessary

• Line 58-60: “Pseudozyma antarctica T-34 produces large amounts of MELs when grown in culture containing vegetable oil as the carbon source, and the production yield reaches 140 g/L using n-alkanes as the carbon source”. Define “large amounts” and state with relevant research.

• Line 63: Define “lower amounts” and state with relevant research

• Line 75: Define “more effective”.

• Line 124 & 127: “Tri-isocitrate”. Do you mean tri-sodium-isocitrate?

• Line 137: URL is redundant information

• Line 144: Add the type of normalization that was used. In case of normalization with certain household genes, mention the genes used.

• Line 158: “(more than 30 g/L)”. Mention the exact amount that was measured

• Line 159: amount of MELs produced by U. maydis should be defined

• Line 161: MEL production should be mentioned.

• Line 167: “As described previously”. Add reference..

• Line 344: Quantify “large quantities” of MELs.

Reviewer #2: In the manuscript entitled „A novel aspect of the production of mannosylerythreitol lipids in Pseudozyma antacrtica T-34 based on gene expression of central metabolic pathways“ the authors Wada et al. have analysed micro array data of two fungi (P. antarctica T-34 and Ustilago maydis 521) and two growth conditions (glucose and soybean oil).

P. antarctica T-34 has produced under soy bean oil much more MELs than under glucose whereas in U. maydis the MEL production under soybean oil growth conditions is heavily repressed.

The authors have used the KOG nomenclature to categorize all genes and have identified genes of the primary metabolism to be upregulated in soybean oil conditions in P. antarctica. In contrast, most of these genes are down-regulated in U. maydis.

Finally, the authors demonstrate that under soybean oil conditions isocitrate dehydrogenase expression is increased and this coincides with increased activity.

The current work is interesting and will allow further manipulation of P. antarctica T-34 to increase MEL biosynthesis.

But there are some points necessary to improve the manuscript, the tables and the figures:

The title starts with “A novel aspect..” Please specify or change the title.

Lane 20: industrial lipases, mannosyl…

Lane 85: please complete the sentence: “…adapted to aerobically…”

Lane 110: please specify the concentration of the NH4OH-solution used as solvent system

Lane 150: The title “Pseudozyma antarctica genes are expressed…” is confusing. What kind of genes do you mean? Not all genes are expressed with high intensity, are they?

Table 1: I guess that you have ordered the table according to the Alphabet of the description. I would prefer to have then the description in the first column (not in the second). For the metabolism section “Energy production…” should be reordered. The row “Total CDS” should be changed with the row “The genes categorized…”. You have mentioned in the entire table only the KOG classified genes (93+42+16+28=179)

Lane 241: remove “Consequently,”

Lane 258 spelling: Antarctica

Lane 297: “large amounts of MELs”: can you specify, which variants of MELs are produced?

Lane 311: …isocitrate dehydrogenase (NAD+)…” In the figure you use MaxV. Please clearify.

Lane 317: “…no activity was noted under oily conditions.

Figure 1: According to the legend (Lanes 54-56) the proteins are assigned differently (Emt1p not EMT1 and so on). The proteins involved in the step from mono-acylation to di-acylation are the acyl-transferases MAC1 and MAC2 (Mac1p/Mac2p). In your scheme acylation of C3 (by Mac2p) precedes the acylation of C2 (by Mac1p). Please comment on that in the introduction.

Figure 3: “vegetable oil over glucose”

Figure 4: Great work!!

Figure 5:

A: Please add MEL-A, -B, -C; the curly bracket for MELs is huge. Are you sure, that the slow migrating bands are MELs?

Why you mention “Olive oil” and not “Soybean oil”?

B: again “Olive oil”

C: again “Olive oil”. I propose to use different colors as in 5B, because you performed a different experiment.

Lane 339-344: I think you can remove the sentence: “Hence, ….quantities of MELs”. To my opinion it repeats the sentence before.

Lane 383: “..will help to identify…”

Supplemental tables:

As for table 1: The sorting criteria must be in the first column.

S1: Start with KOG, KOG number, Pa gene…

S2 and S3: please use either upper- or lowercase names for the enzyme description and resort according to Alphabet, then EC-number…

Legend: …*3 BBH means bi-directional best hit. Plural means the homology…” Please change all “pulural” to plural in column Type of S2

S4: please sort the gene numbers: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and so on

Start with: 1c00036, 1d00040, 2c00011 and so on

The description column uses cryptic abbreviations e.g. row 9: Aldedh ? Please use for all domain abbreviations the full form.

Reviewer #3: The manuscript "A novel aspect of the production of mannosylerythritol lipids in Pseudozyma antarctica

T-34 based on gene expression of central metabolic pathways" gives new insights on the genetic and metabolic regulations controlling mannosylerythritol lipids (MEL) biosynthesis in two yeasts.

The manuscript is providing a new piece o knowledge ans is sound for publications, yet after considering the following comments:

1- Lines 344-345: The authors mention that the Ustilago maydis had poor growth when using vegetable oils as carbon source, which is not the case with Pseudozyma antarctica. I strongly suggest the absence of lipases and esterases in U. maydis which explain their poor growth and low MEL production. Therefore, I suggest the authors screen the genome of U. maydis for lipases and esterases and if absent, to use this as the explanation of the poor growth and low MEL production. Also, does U. maydis grow on alkanes or fatty acids or glycerol separately?

2- Line 67-68: Although authors are referring to reference 15, yet it would be easier for the reader to know briefly how the genomic and transcriptomic analyses led to the conclusion that P. antarctica is oleaginous.

3- Lines 198-199: hard to read. it is better to say "metabolism related genes" rather than "metabolism genes"

4- Line 253: glyocylate to glyoxylate.

5- it is strongly recommended to show the expression fold changes of MEL biosynthetic genes and transport in a single separate figure similar to figure 3.

6- English style and grammer needs some improvement for a more easy reading.

Reviewer #4: The results embody this manuscript is a continuation of their previous studies, and has merit. This manuscript can be published with some minor modifications. The suggested changes are as follows:

Full Title: Production characteristics of mannosyl.....

Short Title: Metabolic transcriptomics.....

Abstract L34-36: These results suggest that central metabolism of.......

L85-86: These results ... ....... ...... modification of its central metabolic system.

L386-391: Based on the transcriptomic characterization and biochemical changes - the authors concluding statements are far and wide or out of focus.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Ahmad M. Abdel-Mawgoud

Reviewer #4: No

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1 Oct 2019

We would like to thank you and 4 reviewers for careful reading our manuscript and for giving useful comments. According to the reviewers’ comments, we have revised the manuscript, tables, and figures. We enclosed the revised manuscript and our replies to the comments by the reviewers together with a marked-up manuscript without figure.

We hope that you will consider this revised version suitable for publication in the PLOS ONE.

Submitted filename: Responses.docx

Click here for additional data file.

16 Oct 2019

PONE-D-19-22493R1

Production characteristics of mannosylerythritol lipids in Pseudozyma antarctica T-34 based on gene expression of central metabolic pathways

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

**********

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments on the revice

Most comments were resolved still some aren’t.

1)

There are claims about higher productivities and higher growth but these are not supported with the right data.

MEL production is measured by TLC which is a qualitative measurement technique and not a quantitative one.

I have the feeling that the author cannot or won't quantify there MEL's with an adequate technique (e.g. HPLC-ELSD).

Biomass growth is measured with cell wet weight after MeOH extraction. There are so many biasses on this technique (substrate, lipid extraction by MeOH, evaporation, cell size, lipid content,....).

So I suggest, either you analyse your production parameters (in g/(L*h)). If this is no option than you will have to remove your claims of higher production or state that a TLC analysis based on visual comparison can only give a small hint for higher concentration.

Biomass should be measured with another technique than cell wet weight like e.g. cell dry weight after removal of product by ethyl acetate washing. (don't use MeOH, ethyl acetate will evaporate).

If there is a difference in biomass than should the productivity be linked to the biomass as well (specific productivity). This will probably result in interesting findings.

2)

Figure 2 B&C: As described in your article, “Genome and transcriptome analysis of the asidiomycetous yeast Pseudozyma Antarctica producing extracellular glycolipids, mannosylerythritol lipids”, there were gene transcriptions who were significantly different (p-value <0.005). Can you state that you only used these ones for further analysis when you were using the selection criterium M<0. This will improve your research by confirming that you lowered the false positive rate.

3)

Line 372 & 387: change large amounts to larger amounts or just to “presence of vegetable oil”.

4)

381: precious report should be changed to previous

5)

figure 5 A: the µl of sample should be under the respective lane for clarity.

Reviewer #2: Dears Authors,

Most of my concerns have been addressed in your revised version. But there are still two points which have not been answered satisfactory.

Figure 1: There is still a mistake in the biosynthesis pathway. Please look at Hewald et al. (2006) or Deinzer et al. (2019). Mat1 acetylates C4 and C6 residues of the mannose and not as depicted C2. I propose to cite these articles as basis for your figure. Do you have any hints that Mac2 acylates first then Mac1, or it is still done in a cooperative manner? Please change the figure accordingly to the literature.

The concentration (molarity) of NH4OH-solution is still missing.

Reviewer #3: The manuscript looks much better and is sound for publication in its current status. Yet I suggest the title be changed from :

Production characteristics of mannosylerythritol lipids in Pseudozyma antarctica T-34

based on gene expression of central metabolic pathways

to

Targeted transcriptomic study of the implication of central metabolic pathways in mannosylerythritol lipids biosynthesis in Pseudozyma antarctica T-34

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Ahmad Abdel-Mawgoud Saleh

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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27 Nov 2019

We would like to thank you and 3 reviewers again for careful reading our manuscript and for giving useful comments. According to the reviewers’ comments, we have revised the manuscript tables and figures. We enclosed the revised manuscript and our replies to the comments by the reviewers together with a marked-up manuscript.

We hope that you will consider this revised version suitable for publication in the PLOS ONE.

Submitted filename: response_R.docx

Click here for additional data file.

17 Dec 2019

Targeted transcriptomic study of the implication of central metabolic pathways in mannosylerythritol lipids biosynthesis in Pseudozyma antarctica T-34

PONE-D-19-22493R2

Dear Dr. Tomotake Morita,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Marie-Joelle Virolle, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

**********

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: (No Response)

**********

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: (No Response)

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6. Review Comments to the Author

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Reviewer #2: All points of my second review have been changed satisfactory. One Proposal: Citation of Hewald et al., 2006, in which the MEL biosynthesis pathway was described.

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Reviewer #2: No

23 Dec 2019

PONE-D-19-22493R2

Targeted transcriptomic study of the implication of central metabolic pathways in mannosylerythritol lipids biosynthesis in Pseudozyma antarctica T-34

Dear Dr. Morita:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Table 1

KOG classification of 264 genes extracted by guilt-by-association.

Category	Description	The number of genes	Ratio (%)	The number of genes “picked”	Ratio (%)
Metabolism
C	Energy production and conversion	206	3.1	20	7.6
E	Amino acid transport and metabolism	204	3.1	18	6.8
F	Nucleotide transport and metabolism	63	1.0	1	0.4
G	Carbohydrate transport and metabolism	172	2.6	19	7.2
H	Coenzyme transport and metabolism	78	1.2	4	1.5
I	Lipid transport and metabolism	219	3.3	10	3.8
P	Inorganic ion transport and metabolism	91	1.4	9	3.4
Q	Secondary metabolites biosynthesis, transport and catabolism	124	1.9	12	4.5
	Subtotal	1157	17.7	93	35.2
Cellular processes
D	Cell cycle control, cell division, chromosome partitioning	139	2.1	0	0.0
M	Cell wall/membrane/envelope biogenesis	48	0.7	3	1.1
N	Cell motility	3	0.0	0	0.0
O	Posttranslational modification, protein turnover, chaperones	373	5.7	21	8.0
T	Signal transduction mechanisms	29	4.4	2	0.8
U	Intracellular trafficking, secretion, and vesicular transport	256	3.9	9	3.4
V	Defense mechanisms	31	0.5	2	0.8
W	Extracellular structure	4	0.1	0	0.0
Y	Nuclear structure	24	0.4	0	0.0
Z	Cytoskeleton	95	1.4	5	1.9
	Subtotal	1,264	19.3	42	15.9
Information storage and processing
A	RNA processing and modification	203	3.1	2	0.8
B	Chromatin structure and dynamics	78	1.2	1	0.4
J	Translation, ribosomal structure and biogenesis	287	4.4	11	4.2
K	Transcription	216	3.3	0	0.0
L	Replication, recombination and repair	164	2.5	2	0.8
	Subtotal	948	14.5	16	6.1
Poorly characterized
R	General function prediction only	588	9.0	23	8.7
S	Function unknown	244	3.7	5	19
	Subtotal	832	12.7	28	10.6
The genes categorized with KOG classification		4,201	64.1	179	67.8
Total CDS		6,555	100.0	264	100.0