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Identification of the gene PtMAT1 encoding acetyltransferase from the diastereomer type of mannosylerythritol lipid-B producer Pseudozyma tsukubaensis
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e6, 2018 www.elsevier.com/locate/jbiosc
Identification of the gene PtMAT1 encoding acetyltransferase from the diastereomer type of mannosylerythritol lipid-B producer Pseudozyma tsukubaensis Azusa Saika,1 Yu Utashima,2 Hideaki Koike,3 Shuhei Yamamoto,2 Takahide Kishimoto,2 Tokuma Fukuoka,1 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 Toyobo Co., Ltd., Tsuruga Institute of Biotechnology, 10-24 Toyo-cho, Tsuruga, Fukui 914-8550, Japan,2 and Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6-9, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan3 Received 16 January 2018; accepted 29 May 2018 Available online xxx
Mannosylerythritol lipids (MELs) are biosurfactants produced from feedstocks by basidiomycetous yeasts. MELs exhibit different properties depending on their structures, such as the degree of acetylation or acylation and the chirality of the mannosylerythritol moiety. Pseudozyma tsukubaensis produces a diastereomer type of MEL-B (monoacetylated MEL); therefore, deletion of an acetyltransferase could yield a diastereomer type of MEL-D (deacetylated MEL), which has only been produced in in vitro reactions of lipase using MEL-B as a substrate. Here, we deleted the gene PtMAT1 in P. tsukubaensis 1E5 encoding an acetyltransferase related to MEL biosynthesis via targeted gene deletion and generated a producer of the diastereomer type of MEL-D. The uracil auxotrophic mutant of P. tsukubaensis 1E5 (PtURA5mutant) was used as a host strain for gene deletion. The gene PtMAT1 was replaced with a PtURA5 cassette by homologous recombination using uracil auxotrophy as a selectable marker. According to thin-layer chromatography and nuclear magnetic resonation spectroscopy, we identified the strain DPtMAT1 as a producer of the diastereomer type of MEL-D instead of MEL-B. Ó 2018, The Society for Biotechnology, Japan. All rights reserved. [Keywords: Acetyltransferase; Basidiomycetous yeast; Diastereomer type of mannosylerythritol lipid; Gene disruption; Pseudozyma tsukubaensis]
Biosurfactants (BSs) are a type of surfactant produced from renewable feedstock by various fungi and bacteria. BSs have attracted considerable interest because of their excellent interfacial activity, biodegradability, biocompatibility and mild production condition. Recently, some BSs are commercialized in several countries for cosmetics, laundry detergent and personal care. Many types of BSs have been reported, such as sophorolipids, mannosylerythritol lipids (MELs), and rhamnolipids (1e5), among which MELs have received much attention because of their high structural variation. The basic structure of MELs includes mannose and erythritol as a hydrophilic moiety and fatty acid chain and acetyl group as a hydrophobic moiety. MELs and their derivatives can be categorized based on four structural components: the degree of acetylation (MEL-A, MEL-B, MEL-C, and MEL-D) (6); the degree of acylation (mono-, di-, and tri-acylated MEL) (7,8); the chirality of the erythritol bond (conventional type and diastereomer type) (9); and the sugar alcohol type (mannosylmannitol lipid, mannosylribitol lipid, and mannosylarabitol lipid) (10,11). These MELs show various and unique properties, such as selfassembling, gene deliver, anti-tumor, anti-bacteria, anti-oxidation and damaged human skin and hair repair, depend on their structures (12e18). Thus, MELs are promising for cosmetics,
* Corresponding author. Tel.: þ81 29 861 4426; fax: þ81 29 861 4457. E-mail address: [email protected] (T. Morita).
pharmaceutical, agriculture, food and environmental application, and manipulation of the structure of MEL is important for expanding its industrial applications. Most yeast species in the genus Pseudozyma (e.g., P. antarctica [currently designated as Moesziomyces antarcticus], P. parantarctica [currently designated as M. parantarcticus], P. aphidis [currently designated as M. aphidis], and P. rugulosa [currently designated as M. rugulosus]), produce a MEL mixture consisting of MEL-A (>70% of all produced MELs; di-acetylated), MEL-B (mono-acetylated), and MEL-C (mono-acetylated) (3,19e23). However, P. tsukubaensis, P. graminicola, P. hubeiensis, P. siamensis, and P. shanxiensis predominantly produce MEL-B or MEL-C (24e26). Meanwhile, there are no reports of natural MEL producers that dominantly or selectively produce MEL-D (deacetylated). Acetyltransferase deletion strains of Ustilago maydis and P. hubeiensis, conventional types of MEL producers, have been shown to produce the conventional type of MEL-D (27,28). Fukuoka et al. (29) reported that MEL-D was produced via deacetylation reaction of lipases from MEL-B as a substrate in vitro. MEL-D has advantageous properties, such as higher critical aggregation concentration, higher hydration ability, and self-assembly into a lamellar structure at a remarkably wide range of concentrations. In addition, compared with the conventional type of MEL-D, the diastereomer type of MEL-D shows higher hydrophilicity and hydration ability (6). Therefore, the diastereomer type of MEL-D is
1389-1723/$ e see front matter Ó 2018, The Society for Biotechnology, Japan. All rights reserved. https://doi.org/10.1016/j.jbiosc.2018.05.025
Please cite this article in press as: Saika, A., et al., Identification of the gene PtMAT1 encoding acetyltransferase from the diastereomer type of mannosylerythritol lipid-B producer Pseudozyma tsukubaensis, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.05.025
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expected to be useful in aqueous applications that would expand the utility of MELs in industrial applications. However, no diastereomer type of MEL-D producer has been reported. P. tsukubaensis is the only effective producer of a diastereomer type of MEL. The P. tsukubaensis strain 1E5 produces over 70 g L1 of MEL-B selectively from olive oil as a carbon source (26). Fig. 1 shows the biosynthesis pathway of MELs in P. tsukubaensis. Plant oil is hydrolyzed into glycerol and fatty acids by lipase catalysis, which are introduced into the cell and converted into guanosine diphosphate (GDP)-mannose and erythritol as MEL precursors. Subsequently, GDP-mannose is transferred to erythritol via PtEMT1p-encoding glycosyltransferase, yielding mannosylerythritol. PtMAC1p- and PtMAC2p-encoding acyltransferases catalyze the transition of medium-chain acyl-CoA into mannose. Finally, acetyl-CoA is transferred to mannose at the C-6 0 position by PtMAT1p-encoding acetyltransferase. Recently, whole-genome sequencing of strain 1E5 was performed, and the gene cluster related to MEL biosynthesis was identified (30). We performed the functional characterization of PtMAC2p in P. tsukubaensis 1E5, and found that PtMAC2p catalyzed acylation at the C-3 0 position of mannose in MEL (30). In addition, the putative acetyltransferase gene (PtMAT1) of P. tsukubaensis 1E5 was obtained by whole-genome sequencing. PtMAT1 is likely related to MEL acetylation; therefore, we hypothesized that a PtMAT1-deleted strain would produce the diastereomer type of MEL-D. Here, we identified the function of PtMAT1p in P. tsukubaensis 1E5 via gene deletion. Targeted gene deletion was performed by homologous recombination with uracil auxotrophy as a selective marker. Strain DPtMAT1 produced MEL-D selectively, and its complemented strain DPtMAT1 harboring pUC_UARSneo-PtMAT1 restored MEL acetylation. Therefore, we concluded that PtMAT1p catalyzes the transfer reaction of acetyl-CoA to the mannose moiety. MATERIALS AND METHODS Strains and plasmids P. tsukubaensis 1E5 (JCM16987) and its uracilauxotrophic mutant strain PtURA5-mutant were used as the host strains for MEL production (26,30). The plasmids pUC-PtMAT1-URA5 and pUC_UARSneo-PtMAT1 used for PtMAT1 deletion and complementation were constructed as described below. These plasmids were introduced into the strains via electroporation as
described by Saika et al. (30). The DNA Data Bank of Japan (DDBJ) accession numbers of PtMAT1p and PtURA5p from P. tsukubaensis 1E5 are LC274623 and LC274622, respectively. Sequence analysis The draft genome sequence of P. tsukubaensis 1E5 was analyzed using the framework of the Adaptable and Seamless Technology Transfer Program through Target-driven R&D (30). The BLAST program was used to determine sequence similarity by searching the database available on the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignments were displayed using ClustalW. Phylogenetic analysis was performed using the neighbor-joining method (31) with the program MEGA7 (32) and bootstrap analysis based on 1000 replicates (33). Plasmid construction The pUC-PtMAT1-URA5 plasmid (Fig. 2A) was constructed as follows. A PtMAT1 fragment (upstream of PtMAT1 [2 kb]-PtMAT1downstream of PtMAT1 [2 kb]; 5.7 kb) was amplified by PCR using the genomic DNA of P. tsukubaensis 1E5 as a template and the following set of oligonucleotide primers: 50 -CTCTAGAGGATCCCCTGTCTACTCGCTCGACTTT-30 (forward) and 50 TCGAGCTCGGTACCCCAAGCACTCCTGCAAGCA-30 (reverse). The 5.7-kb PtMAC2 fragment was inserted into SmaI-digested pUC18 using the In-Fusion Cloning Kit (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions, yielding pUC_PtMAT1_2K. The PtURA5 fragment (promoter [1 kb]-PtURA5-terminator [0.5 kb]; 2.3 kb) was amplified by PCR using P. tsukubaensis 1E5 genomic DNA as a template and the following set of oligonucleotide primers: 50 -TTTCAAGTG(forward) and 50 -GTAGCTAAAGTAATAAGTATTCCGAAGGTCATGGTGTTCC-30 ACAAGCCAGATCAAGTTCGTC-30 (reverse). The linearized vector was prepared by inverse PCR using pUC_PtMAT1_2K as a template and the following set of oligonucleotide primers: 50 -TATTACTTTAGCTACCCATCTTCTTG-30 (forward) and 50 AATACTCACTTGAAAGTGCACGCATC-30 (reverse). Then, it was ligated with the PtURA5 fragment using the In-Fusion Cloning Kit according to the manufacturer’s instructions, yielding pUC-PtMAT1-URA5. The plasmid pUC_UARSneo-PtMAT1 (Fig. 2B) was constructed as follows. A PtMAT1 fragment containing promoter and terminator sequences was amplified by PCR using the genomic DNA of P. tsukubaensis 1E5 as the template and the following set of oligonucleotide primers: 50 -CTCTAGAGGATCCCCTCAGGCGCTCCACACTTT-30 (forward) and 50 -TCGAGCTCGGTACCCTCAAGAAGGTCTATGCCGA-30 (reverse). The 3.2-kb PtMAT1 fragment was inserted into linearized SmaI-digested pUC_UARSneo (34) using the In-Fusion Cloning Kit according to the manufacturer’s instructions, yielding the plasmid pUC_UARSneo-PtMAT1. PtMAT1 gene deletion To obtain a diastereomer type of MEL-D-producing strain, the PtMAT1 gene was deleted by homologous recombination in the uracilauxotrophic strain of the PtURA5-mutant as described by Saika et al. (30). The DNA fragment for PtMAT1 deletion, termed PtMAT1D::URA5, was prepared by KpnI and XbaI digestion of pUC-PtMAT1-URA5. PtMAT1 deletion was confirmed with the colony PCR method using two sets of oligonucleotide primers: 50 -CTCCTCGTCGACTTGTCCTC-30 (forward) and 50 -TTGAGCGACTCCTTGATGTG-30 (reverse) (ColonyPCR_B); and 50 -TTGCGTCGGGAGGACTGGCTGG-30 (forward) and 50 TGCATGCCTGAACCTGGTCG-30 (reverse) (ColonyPCR_C). The resulting colonies were selected on a yeast nitrogen base without amino acid (YNB) plate (YNB-ura) (1.7 g L1 YNB, 5 g L1 (NH4)2SO4, 20 g L1 glucose, and 20 g L1 agar) to estimate the ability to recover uracil synthesis.
FIG. 1. Biosynthetic pathway of the diastereomer type of mono-acetylated mannosylerythritol lipid (MEL-B) produced in Pseudozyma tsukubaensis from plant oil. PtEMT1p, erythritol/mannose transferase; PtMAC1p and PtMAC2p, acyltransferases; PtMAT1p, acetyltransferase; and PtMMF1p, putative transporter.
Please cite this article in press as: Saika, A., et al., Identification of the gene PtMAT1 encoding acetyltransferase from the diastereomer type of mannosylerythritol lipid-B producer Pseudozyma tsukubaensis, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.05.025
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FIG. 2. The plasmids used in this study. (A) pUC_PtMAT1-URA5; (B) pUC_UARSneo-PtMAT1. PtMAT1 and PtURA5 are derived from P. tsukubaensis 1E5. URA5-P, promoter region of PtURA5; and URA5-T, terminator region of PtURA5.
Transformation of plasmid pUC_UARSneo-PtMAT1 The expression plasmid pUC_UARSneo-PtMAT1 was introduced into the chromosome of strain DPtMAT1 by non-homologous recombination using electroporation according to Saika et al. (34). Following electroporation, the resulting colonies were grown at 25 C for 3 days on YM plates (3 g L1 yeast extract, 3 g L1 malt extract, 5 g L1 peptone, and 10 g L1 glucose) containing 500 mg mL1 G418. Three different clones for each recombinant strain were selected and used in subsequent experiments. MEL production The gene-deleted and gene-complemented strains of P. tsukubaensis 1E5 were cultivated in YM medium containing 50 g L1 glycerol at 25 C for 2 days with shaking at 250 rpm. An aliquot of the seed culture (1 mL) was inoculated in 20 mL of MEL production medium (5 g L1 yeast extract, 3 g L1 NaNO3, 0.3 g L1 KH2PO4, and 0.3 g L1 MgSO4$7H2O) containing 3% (w/v) olive oil and 2% (w/v) glycerol, and cultivated at 25 C for 4 days with shaking at 250 rpm. MELs produced in the culture were extracted with an equal volume of ethyl acetate. Thin-layer chromatography analysis MELs were detected using thin-layer chromatography (TLC) (35). The extracted MELs were analyzed by TLC using chloroform, methanol, and 12% NH4OH in a 65:15:2 (v:v:v) ratio as an eluent. MELs were detected on the TLC plate by spraying the plate with 2% anthronesulfate reagent and heating at 150 C for 5 min. Purified MEL mixture produced by P. antarctica was used as a reference. Structural analysis The structure of purified MEL was characterized by H nuclear magnetic resonance spectroscopy (NMR) with a Bruker AVANCE 400 (400 MHz) at 30 C in a CDCl3 solution (diastereomer type of MEL-B) or CDCl3 containing 10 % (v/v) methanol-d4 (diastereomer type of MEL-D). Tetramethylsilane was used as an internal chemical shift standard. 1
RESULTS Amino acid sequence analysis of PtMAT1p from P. tsukubaensis 1E5 Based on BLASTP analyses, the putative acetyltransferase gene (PtMAT1) was found in the draft genome of P. tsukubaensis 1E5. An amino acid sequence analysis showed that PtMAT1p shared approximately 40e60% of its identity with 13 homologous proteins from 10 strains belonging to the genera Ustilago, Melanopsichium, Pseudozyma, and Sporisorium (Table S1). There is no putatively conserved domain found. Among the 10 strains, U. maydis, P. antarctica, P. aphidis, P. hubeiensis, and S. scitamineum are well-known MEL producers. We aligned the deduced amino acid sequences of 15 proteins (13 homologous proteins and PtMAT1p from P. tsukubaensis NBRC1940 and 1E5) using ClustalW, and created a phylogenetic tree (Figs. 3 and S1). The phylogenetic analysis placed PtMAT1p from P. tsukubaensis in a separate clade from those of the other MEL producers. PtMAT1 deletion in the PtURA5-mutant An acetyltransferase (PtMAT1)-deleted stain of the PtURA5-mutant (uracil auxotrophic mutant of P. tsukubaensis 1E5) was generated by homologous recombination. In total, 84 candidate strains were obtained by plate selection, and deletion of PtMAT1 was evaluated by colony
PCR (Fig. 4). Primer set B (Fig. 4B) amplified the 1.0-kb inner region of PtMAT1. There was no corresponding band in strain DPtMAT1 (Fig. 4B, lanes 2e6), but a 1.0-kb band was apparent in the PtURA5-mutant (Fig. 4B, lane 1). In addition, 2.1 kb in length corresponding to the fragment PtMAT1D::URA5 was detected in strain DPtMAT1 (Fig. 4C, lanes 2e6). From the colony PCR results, five strains were selected as PtMAT1 deletion strains (DPtMAT1 No. 1e5). Uracil biosynthesis was restored in the PtMAT1 deletion strains, as they could grow without uracil supplementation in the medium (Fig. S2). Based on these observations, PtMAT1 deletion was successful in the PtURA5mutant of P. tsukubaensis. MEL biosynthesis of strain DPtMAT1 Five strains of DPtMAT1 were cultivated in MEL production medium containing 3%
(w/v) olive oil for 4 days at 25 C, and the MEL structure was evaluated by TLC analysis (Fig. 5A). The strain 1E5 (parent strain of PtURA5-mutant) was used as a control strain. The produced MELs were extracted with ethyl acetate and detected with TLC using anthrone-sulfate reagent. In the TLC analysis, only two strains produced MEL (DPtMAT1 No. 2 and No. 4). MEL-B produced by strain 1E5 was detected in the upper position on the TLC plate, whereas the spot from the extract of strain DPtMAT1 was detected in a lower position. Because MEL-D has no acetyl groups, it shows higher hydrophilicity than MEL-B. The TLC analysis strongly suggested that strain DPtMAT1 produced MEL-D due to PtMAT1 deletion.
FIG. 3. Phylogenetic relationships among P. tsukubaensis 1E5 and homologous strains based on the MAT1p amino acid sequence.
Please cite this article in press as: Saika, A., et al., Identification of the gene PtMAT1 encoding acetyltransferase from the diastereomer type of mannosylerythritol lipid-B producer Pseudozyma tsukubaensis, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.05.025
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J. BIOSCI. BIOENG., harboring pUC_UARSneo-PtMAT1 was in the same position as that of the MEL-B standard. In contrast, the mobility of MEL produced by strain DPtMAT1 harboring the empty vector was unchanged. Based on the change in mobility, PtMAT1 expression restores MEL acetylation. Structural analysis of MELs The structure of MEL produced by strain DPtMAT1 was identified by NMR analysis, and the signal pattern was compared with the diastereomer type of MEL-B produced by the strain 1E5 (Fig. 6). Each signal was assigned as described previously (29). The 1H NMR spectrum of MEL produced by strain 1E5 and DPtMAT1 were similar; however, the signal assigned to the acetyl group (2.13 ppm) in MEL-B produced by strain 1E5 was not apparent for the MEL produced by strain DPtMAT1. In addition, the signal assigned to H-60 was shifted to a higher magnetic field in strain DPtMAT1. The signal assigned to H4a and H-4b showed that the MEL extracted from strain DPtMAT1 was a diastereomer; therefore, PtMAT1 deletion did not affect the chirality of the mannosylerythritol moiety. According to the NMR analysis, strain DPtMAT1 produced the diastereomer type of MEL-D by PtMAT1 deletion. DISCUSSION
FIG. 4. PCR analysis of parental strain 1E5 and strain DPtMAT1. (A) Primer sets were used in the PCR amplification to assess PtMAT1 deletion. Agarose gel electrophoresis of amplified DNA fragments was performed to confirm gene disruption. Gene fragments were amplified using primer set ColonyPCR_B (B) and primer set ColonyPCR_C (C). Lane M, DNA marker; lane 1, PtURA5-mutant5; lanes 2e6, strains DPtMAT1 No. 1, No. 2, No. 3, No. 4, and No. 5.
Gene complementation of strain DPtMAT1 using pUC_URASneo-PtMAT1 To obtain further evidence of the function of PtMAT1p, PtMAT1 underwent complementation using the expression plasmid pUC_UARSneo-PtMAT1. The empty vector pUC_UARSneo was used as a negative control. A plasmid harboring PtMAT1 from P. tsukubaensis 1E5 was introduced into strain DPtMAT1 No. 2, which was cultivated in MEL production medium containing 3% (w/v) olive oil for 4 days at 25 C. Fig. 5B shows the TLC pattern of the complementary strain of DPtMAT1. The corresponding spot of MEL produced by strain DPtMAT1
In our previous studies, the diastereomer type of MEL-B produced by P. tsukubaensis showed higher water-holding capacity and greater self-assembling properties across a wide range of concentrations and temperatures compared to the conventional type of MELs (6,36). Therefore, the diastereomer type of MEL has a greater potential for use in aqueous solutions. We recently demonstrated the expansion of the diastereomer type of MEL structures using gene engineering methods. We focused on PtEMT1p, which is crucial in determining the sugar conformation of MEL, and succeeded in producing the diastereomer type of MEL-A using the recombinant strain of P. antarctica expressing PtEMT1 from P. tsukubaensis (37). In addition, the mono-acylated diastereomer type of MEL-D producer was generated by targeted gene-disruption of PtMAC2 from P. tsukubaensis as a host strain (30). As demonstrated by these results, modification of the MEL biosynthesis pathway by gene engineering techniques enables the tailor-made production of the diastereomer type of MELs.
FIG. 5. Effect of PtMAT1 deletion on MEL production. The MEL production of the strain 1E5 (parent strain of PtURA5-mutant5), strain DPtMAT1, and PtMAT1-complemented strain was determined using MEL production medium containing and 3% olive oil at 25 C for 4 days. The produced MEL was extracted with ethyl acetate and visualized using anthrone reagent. (A) Lanes 1e5, strains DPtMAT1 No. 1, No. 2, No. 3, No. 4, and No. 5; lane 6, strain 1E5; (B) lane 1, strain DPtMAT1 No. 2 harboring pUC_UARSneo; lane 2, strain DPtMAT1 harboring pUC_UARSneo-PtMAT1; STD, MEL mixture of MEL-A, MEL-B, and MEL-C.
Please cite this article in press as: Saika, A., et al., Identification of the gene PtMAT1 encoding acetyltransferase from the diastereomer type of mannosylerythritol lipid-B producer Pseudozyma tsukubaensis, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.05.025
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FIG. 6. Partial 1H NMR spectra. (A) Diastereomer type of MEL-B synthesized by strain 1E5, and (B) diastereomer type of MEL-D synthesized by strain DPtMAT1.
In the phylogenetic analysis (Fig. 3), PtMAT1p from P. tsukubaensis NBRC1940 and 1E5 were considered as a separate clade from the other MEL producers. U. maydis, S. scitamineum, P. antarctica, P. aphidis, and P. hubeiensis produce MEL mixtures containing MEL-A (acetylated at the C-40 and C-60 positions of mannose), MEL-B (acetylated at the C-60 position of mannose), and MEL-C (acetylated at the C-40 position of mannose). In other words, MAT1p from these five strains shows relaxed regioselectivity. Conversely, PtMAT1p from P. tsukubaensis only catalyzes the transfer of acetyl-CoA to the C-60 position of the mannosyl moiety, because P. tsukubaensis selectively produces MEL-B. From these results, the protein structure and substrate-binding mechanism of PtMAT1p may differ from other MEL producers. Although 84 strains lacking uracil auxotrophy were obtained on the YNB-ura plate, only five were PtMAT1-deleted strains (Fig. 4), suggesting that non-homologous recombination occurred at a high frequency. In addition, according to the TLC analysis, strains DPtMAT1 No. 1, No. 3, and No. 5 lacked MEL productivity (Fig. 5A). Non-homologous recombination occurred simultaneously during PtMAT1 deletion; therefore, these three strains may be affected by undesired gene deletion via nonhomologous recombination. A previous study demonstrated that Ku70 deletion led to improved targeted gene deletion efficiency in Saccharomyces macrospora (38). Therefore, further modification of the gene deletion method, such as Ku70 deletion, could improve the gene deletion technique in yeast strains of the genus Pseudozyma.
In the TLC analysis (Fig. 5A), the MEL productivity of the strains
DPtMAT1 No. 2 and No. 4 decreased compared with the parent strain. Non-homologous recombination with the fragment PtMAT1D::URA5 was considered to be the cause of the decrease in MEL production. In another study, the MAT1-deleted strain of P. hubeiensis maintained about 70% of MEL productivity (91.6/ 129 g L1 ¼ MAT1-deleted strain/parent strain) (28). P. tsukubaensis can produce over 70 g L1 of the diastereomer type of MEL-B; therefore, strain DPtMAT1 has the potential to produce abundant levels of the diastereomer type of MEL-D. In addition, because antibiotics selection is not required during cultivation, strain DPtMAT1 may be suitable for industrial-scale production. We demonstrated that lipase expression in P. tsukubaensis strain 1E5 led to an increase in diastereomer type of MEL-B production using olive oil as a carbon source (34). The overexpression of the lipase gene in strain DPtMAT1 may efficiently increase productivity of the diastereomer type of MEL-D. In conclusion, we studied PtMAT1 in P. tsukubaensis and successfully generated the PtMAT1 deletion strain via homologous recombination. Based on TLC and NMR analyses, PtMAT1p catalyzes the transfer reaction of the acetyl group to the mannose moiety of MEL, and strain DPtMAT1 produces the diastereomer type of MEL-D (i.e., non-acetylated MEL). These results can be used to promote the development of tailor-made MEL production, and support the expansion of industrial applications of MELs. Supplementary data related to this article can be found at https://doi.org/10.1016/j.jbiosc.2018.05.025.
Please cite this article in press as: Saika, A., et al., Identification of the gene PtMAT1 encoding acetyltransferase from the diastereomer type of mannosylerythritol lipid-B producer Pseudozyma tsukubaensis, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.05.025
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J. BIOSCI. BIOENG., ACKNOWLEDGMENTS
This work was supported by the Japan Science and Technology Agency under the Adaptable and Seamless Technology Transfer Program through Target-driven R&D (AS2621413N). YU, SY, and TK are employees of Toyobo Co., Ltd.; however, Toyobo Co., Ltd. did not have any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. AS, HK, TF, SY, TK, and TM are the inventors of a submitted patent related to this study (JP2017-008703). The authors have declared that no other competing interests exist. This article does not contain any studies with human participants or animals performed by any of the authors. References 1. 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). 2. Rau, U., Hammen, S., Hechmann, R., Wray, V., and Lang, S.: Sophorolipids: a source for novel compounds, Ind. Crop. Prod., 13, 85e92 (2001). 3. 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). 4. Kitamoto, D., Akiba, S., Hioki, C., and Tabuchi, T.: Extracellular accumulation of mannosylerythritol lipids by a strain Candida antarctica, Agric. Biol. Chem., 54, 31e36 (1990). 5. Maier, R. M. and Soberón-chávez, G.: Pseudomonas aeruginosa rhamnolipids: biosynthesis and potential applications, Appl. Microbiol. Biotechnol., 54, 625e633 (2000). 6. Fukuoka, T., Yanagihara, T., Imura, T., Morita, T., Sakai, H., Abe, M., and Kitamoto, D.: The diastereomers of mannosylerythritol lipids have different interfacial properties and aqueous phase behavior, reflecting the erythritol configuration, Carbohydr. Res., 351, 81e86 (2012). 7. 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). 8. Fukuoka, T., Morita, T., Konishi, M., Imura, T., and Kitamoto, D.: Characterization of new glycolipid biosurfactants, tri-acylated mannosylerythritol lipids, produced by Pseudozyma yeasts, Biotechnol. Lett., 29, 1111e1118 (2007). 9. Fukuoka, T., Morita, T., Konishi, M., Imura, T., and Kitamoto, D.: A basidiomycetous yeast, Pseudozyma tsukubaensis, efficiently produces a novel glycolipid biosurfactant. The identification of a new diastereomer of mannosylerythritol lipid-B, Carbohydr. Res., 343, 555e560 (2008). 10. Morita, T., Fukuoka, T., Imura, T., and Kitamoto, D.: Formation of the two novel glycolipid biosurfactants, mannosylribitol lipid and mannosylarabitol lipid, by Pseudozyma parantarctica JCM 11752T, Appl. Microbiol. Biotechnol., 96, 931e938 (2012). 11. Morita, T., Fukuoka, T., Konishi, M., Imura, T., Yamamoto, S., Kitagawa, M., Sogabe, A., and Kitamoto, D.: Production of a novel glycolipid biosurfactant, mannosylmannitol lipid, by Pseudozyma parantarctica and its interfacial properties, Appl. Microbiol. Biotechnol., 83, 1017e1025 (2009). 12. Kitamoto, D., Morita, T., Fukuoka, T., Konishi, M., and Imura, T.: Selfassembling properties of glycolipid biosurfactants and their potential applications, Curr. Opin. Colloid Interface Sci., 14, 315e328 (2009). 13. Kitamoto, D., Isoda, H., and Nakahara, T.: Functions and potential applications of glycolipid biosurfactants d from energy-saving materials to gene delivery carriers, J. Biosci. Bioeng., 94, 187e201 (2002). 14. Zhao, X., Murata, T., Ohno, S., Day, N., Song, J., Nomura, N., Nakahara, T., and Yokoyama, K. K.: Protein kinase C_ plays a critical role in mannosylerythritol lipid-induced differentiation of melanoma B16 cells, J. Biol. Chem., 276, 39903e39910 (2001). 15. Kitamoto, D., Yanagishita, S., Shinbo, T., Nakane, T., Kamisawa, C., and Nakahara, T.: Surface active properties and antimicrobial activities of mannosylerythritol lipids as biosurfactants produced by Candida antarctica, J. Biotechnol., 29, 91e96 (1993). 16. Takahashi, M., Morita, T., Fukuoka, T., Imura, T., and Kitamoto, D.: Glycolipid biosurfactants, mannosylerythritol lipids, show antioxidant and protective effects against H2O2-induced oxidative stress in cultured human skin fibroblasts, J. Oleo Sci., 61, 457e464 (2012). 17. Yamamoto, S., Morita, T., Fukuoka, T., Imura, T., Yanagidani, S., Sogabe, A., Kitamoto, D., and Kitagawa, M.: The moisturizing effects of glycolipid
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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). Rau, U., Nguyen, L. A., Schulz, S., Wary, 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). Rau, U., Nguyen, L. A., Roeper, H., Koch, H., and Lang, S.: Fed-batch bioreactor production of mannosylerythritol lipids secreted by Pseudozyma aphidis, Appl. Microbiol. Biotechnol., 68, 607e613 (2005). Konishi, M., Morita, T., Fukuoka, T., Imura, T., Kakugawa, K., and Kitamoto, D.: Efficient production of mannosylerythritol lipids with high hydrophilicity by Pseudozyma hubeiensis KM-59, Appl. Microbiol. Biotechnol., 78, 37e46 (2008). Konishi, M., Nagahama, T., Fukuoka, T., Morita, T., Imura, T., Kitamoto, D., and Hatada, Y.: Yeast extract stimulates production of glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma hubeiensis SY62, J. Biosci. Bioeng., 111, 702e705 (2011). 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). Hewald, S., Linne, U., Scherer, M., Marahiel, M. A., Kämper, J., and Bölker, M.: Identification of a gene cluster of biosynthesis of mannosylerythritol lipids in the basidiomycetous fungus Ustilago maydis, Appl. Environ. Microbiol., 72, 5469e5477 (2006). Konishi, M. and Makino, M.: Selective production of deacetylated mannosylerythritol lipid, MEL-D, by acetyltransferase disruption mutant of Pseudozyma hubeiensis, J. Biosci. 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Saika, A., Koike, H., Yamamoto, S., Kishimoto, T., and Morita, T.: Enhanced production of a diastereomer type of mannosylerythritol lipid-B by the basidiomycetous yeast Pseudozyma tsukubaensis expressing lipase genes from Pseudozyma antarctica, Appl. Microbiol. Biotechnol., 101, 8345e8352 (2017). Morita, T., Habe, H., Fukuoka, T., Imura, T., and Kitamoto, D.: Convenient transformation of anamorphic basidiomycetous yeasts belonging to genus Pseudozyma induced by electroporation, J. Biosci. Bioeng., 104, 517e520 (2007). Worakitkanchanakul, W., Imura, T., Fukuoka, T., Morita, T., Sakai, H., Abe, M., Rujiravanit, R., Chavadej, S., Minamikawa, H., and Kitamoto, D.: Aqueous-phase behavior and vesicle formation of natural glycolipid biosurfactant, mannosylerythritol lipid-B, Colloids. Surf. B Biointerfaces, 65, 106e112 (2008). 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Please cite this article in press as: Saika, A., et al., Identification of the gene PtMAT1 encoding acetyltransferase from the diastereomer type of mannosylerythritol lipid-B producer Pseudozyma tsukubaensis, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.05.025
Identification of the gene PtMAT1 encoding acetyltransferase from the diastereomer type of mannosylerythritol lipid-B producer Pseudozyma tsukubaensis Azusa Saika,1 Yu Utashima,2 Hideaki Koike,3 Shuhei Yamamoto,2 Takahide Kishimoto,2 Tokuma Fukuoka,1 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 Toyobo Co., Ltd., Tsuruga Institute of Biotechnology, 10-24 Toyo-cho, Tsuruga, Fukui 914-8550, Japan,2 and Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6-9, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan3 Received 16 January 2018; accepted 29 May 2018 Available online xxx
Mannosylerythritol lipids (MELs) are biosurfactants produced from feedstocks by basidiomycetous yeasts. MELs exhibit different properties depending on their structures, such as the degree of acetylation or acylation and the chirality of the mannosylerythritol moiety. Pseudozyma tsukubaensis produces a diastereomer type of MEL-B (monoacetylated MEL); therefore, deletion of an acetyltransferase could yield a diastereomer type of MEL-D (deacetylated MEL), which has only been produced in in vitro reactions of lipase using MEL-B as a substrate. Here, we deleted the gene PtMAT1 in P. tsukubaensis 1E5 encoding an acetyltransferase related to MEL biosynthesis via targeted gene deletion and generated a producer of the diastereomer type of MEL-D. The uracil auxotrophic mutant of P. tsukubaensis 1E5 (PtURA5mutant) was used as a host strain for gene deletion. The gene PtMAT1 was replaced with a PtURA5 cassette by homologous recombination using uracil auxotrophy as a selectable marker. According to thin-layer chromatography and nuclear magnetic resonation spectroscopy, we identified the strain DPtMAT1 as a producer of the diastereomer type of MEL-D instead of MEL-B. Ó 2018, The Society for Biotechnology, Japan. All rights reserved. [Keywords: Acetyltransferase; Basidiomycetous yeast; Diastereomer type of mannosylerythritol lipid; Gene disruption; Pseudozyma tsukubaensis]
Biosurfactants (BSs) are a type of surfactant produced from renewable feedstock by various fungi and bacteria. BSs have attracted considerable interest because of their excellent interfacial activity, biodegradability, biocompatibility and mild production condition. Recently, some BSs are commercialized in several countries for cosmetics, laundry detergent and personal care. Many types of BSs have been reported, such as sophorolipids, mannosylerythritol lipids (MELs), and rhamnolipids (1e5), among which MELs have received much attention because of their high structural variation. The basic structure of MELs includes mannose and erythritol as a hydrophilic moiety and fatty acid chain and acetyl group as a hydrophobic moiety. MELs and their derivatives can be categorized based on four structural components: the degree of acetylation (MEL-A, MEL-B, MEL-C, and MEL-D) (6); the degree of acylation (mono-, di-, and tri-acylated MEL) (7,8); the chirality of the erythritol bond (conventional type and diastereomer type) (9); and the sugar alcohol type (mannosylmannitol lipid, mannosylribitol lipid, and mannosylarabitol lipid) (10,11). These MELs show various and unique properties, such as selfassembling, gene deliver, anti-tumor, anti-bacteria, anti-oxidation and damaged human skin and hair repair, depend on their structures (12e18). Thus, MELs are promising for cosmetics,
* Corresponding author. Tel.: þ81 29 861 4426; fax: þ81 29 861 4457. E-mail address: [email protected] (T. Morita).
pharmaceutical, agriculture, food and environmental application, and manipulation of the structure of MEL is important for expanding its industrial applications. Most yeast species in the genus Pseudozyma (e.g., P. antarctica [currently designated as Moesziomyces antarcticus], P. parantarctica [currently designated as M. parantarcticus], P. aphidis [currently designated as M. aphidis], and P. rugulosa [currently designated as M. rugulosus]), produce a MEL mixture consisting of MEL-A (>70% of all produced MELs; di-acetylated), MEL-B (mono-acetylated), and MEL-C (mono-acetylated) (3,19e23). However, P. tsukubaensis, P. graminicola, P. hubeiensis, P. siamensis, and P. shanxiensis predominantly produce MEL-B or MEL-C (24e26). Meanwhile, there are no reports of natural MEL producers that dominantly or selectively produce MEL-D (deacetylated). Acetyltransferase deletion strains of Ustilago maydis and P. hubeiensis, conventional types of MEL producers, have been shown to produce the conventional type of MEL-D (27,28). Fukuoka et al. (29) reported that MEL-D was produced via deacetylation reaction of lipases from MEL-B as a substrate in vitro. MEL-D has advantageous properties, such as higher critical aggregation concentration, higher hydration ability, and self-assembly into a lamellar structure at a remarkably wide range of concentrations. In addition, compared with the conventional type of MEL-D, the diastereomer type of MEL-D shows higher hydrophilicity and hydration ability (6). Therefore, the diastereomer type of MEL-D is
1389-1723/$ e see front matter Ó 2018, The Society for Biotechnology, Japan. All rights reserved. https://doi.org/10.1016/j.jbiosc.2018.05.025
Please cite this article in press as: Saika, A., et al., Identification of the gene PtMAT1 encoding acetyltransferase from the diastereomer type of mannosylerythritol lipid-B producer Pseudozyma tsukubaensis, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.05.025
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expected to be useful in aqueous applications that would expand the utility of MELs in industrial applications. However, no diastereomer type of MEL-D producer has been reported. P. tsukubaensis is the only effective producer of a diastereomer type of MEL. The P. tsukubaensis strain 1E5 produces over 70 g L1 of MEL-B selectively from olive oil as a carbon source (26). Fig. 1 shows the biosynthesis pathway of MELs in P. tsukubaensis. Plant oil is hydrolyzed into glycerol and fatty acids by lipase catalysis, which are introduced into the cell and converted into guanosine diphosphate (GDP)-mannose and erythritol as MEL precursors. Subsequently, GDP-mannose is transferred to erythritol via PtEMT1p-encoding glycosyltransferase, yielding mannosylerythritol. PtMAC1p- and PtMAC2p-encoding acyltransferases catalyze the transition of medium-chain acyl-CoA into mannose. Finally, acetyl-CoA is transferred to mannose at the C-6 0 position by PtMAT1p-encoding acetyltransferase. Recently, whole-genome sequencing of strain 1E5 was performed, and the gene cluster related to MEL biosynthesis was identified (30). We performed the functional characterization of PtMAC2p in P. tsukubaensis 1E5, and found that PtMAC2p catalyzed acylation at the C-3 0 position of mannose in MEL (30). In addition, the putative acetyltransferase gene (PtMAT1) of P. tsukubaensis 1E5 was obtained by whole-genome sequencing. PtMAT1 is likely related to MEL acetylation; therefore, we hypothesized that a PtMAT1-deleted strain would produce the diastereomer type of MEL-D. Here, we identified the function of PtMAT1p in P. tsukubaensis 1E5 via gene deletion. Targeted gene deletion was performed by homologous recombination with uracil auxotrophy as a selective marker. Strain DPtMAT1 produced MEL-D selectively, and its complemented strain DPtMAT1 harboring pUC_UARSneo-PtMAT1 restored MEL acetylation. Therefore, we concluded that PtMAT1p catalyzes the transfer reaction of acetyl-CoA to the mannose moiety. MATERIALS AND METHODS Strains and plasmids P. tsukubaensis 1E5 (JCM16987) and its uracilauxotrophic mutant strain PtURA5-mutant were used as the host strains for MEL production (26,30). The plasmids pUC-PtMAT1-URA5 and pUC_UARSneo-PtMAT1 used for PtMAT1 deletion and complementation were constructed as described below. These plasmids were introduced into the strains via electroporation as
described by Saika et al. (30). The DNA Data Bank of Japan (DDBJ) accession numbers of PtMAT1p and PtURA5p from P. tsukubaensis 1E5 are LC274623 and LC274622, respectively. Sequence analysis The draft genome sequence of P. tsukubaensis 1E5 was analyzed using the framework of the Adaptable and Seamless Technology Transfer Program through Target-driven R&D (30). The BLAST program was used to determine sequence similarity by searching the database available on the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignments were displayed using ClustalW. Phylogenetic analysis was performed using the neighbor-joining method (31) with the program MEGA7 (32) and bootstrap analysis based on 1000 replicates (33). Plasmid construction The pUC-PtMAT1-URA5 plasmid (Fig. 2A) was constructed as follows. A PtMAT1 fragment (upstream of PtMAT1 [2 kb]-PtMAT1downstream of PtMAT1 [2 kb]; 5.7 kb) was amplified by PCR using the genomic DNA of P. tsukubaensis 1E5 as a template and the following set of oligonucleotide primers: 50 -CTCTAGAGGATCCCCTGTCTACTCGCTCGACTTT-30 (forward) and 50 TCGAGCTCGGTACCCCAAGCACTCCTGCAAGCA-30 (reverse). The 5.7-kb PtMAC2 fragment was inserted into SmaI-digested pUC18 using the In-Fusion Cloning Kit (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions, yielding pUC_PtMAT1_2K. The PtURA5 fragment (promoter [1 kb]-PtURA5-terminator [0.5 kb]; 2.3 kb) was amplified by PCR using P. tsukubaensis 1E5 genomic DNA as a template and the following set of oligonucleotide primers: 50 -TTTCAAGTG(forward) and 50 -GTAGCTAAAGTAATAAGTATTCCGAAGGTCATGGTGTTCC-30 ACAAGCCAGATCAAGTTCGTC-30 (reverse). The linearized vector was prepared by inverse PCR using pUC_PtMAT1_2K as a template and the following set of oligonucleotide primers: 50 -TATTACTTTAGCTACCCATCTTCTTG-30 (forward) and 50 AATACTCACTTGAAAGTGCACGCATC-30 (reverse). Then, it was ligated with the PtURA5 fragment using the In-Fusion Cloning Kit according to the manufacturer’s instructions, yielding pUC-PtMAT1-URA5. The plasmid pUC_UARSneo-PtMAT1 (Fig. 2B) was constructed as follows. A PtMAT1 fragment containing promoter and terminator sequences was amplified by PCR using the genomic DNA of P. tsukubaensis 1E5 as the template and the following set of oligonucleotide primers: 50 -CTCTAGAGGATCCCCTCAGGCGCTCCACACTTT-30 (forward) and 50 -TCGAGCTCGGTACCCTCAAGAAGGTCTATGCCGA-30 (reverse). The 3.2-kb PtMAT1 fragment was inserted into linearized SmaI-digested pUC_UARSneo (34) using the In-Fusion Cloning Kit according to the manufacturer’s instructions, yielding the plasmid pUC_UARSneo-PtMAT1. PtMAT1 gene deletion To obtain a diastereomer type of MEL-D-producing strain, the PtMAT1 gene was deleted by homologous recombination in the uracilauxotrophic strain of the PtURA5-mutant as described by Saika et al. (30). The DNA fragment for PtMAT1 deletion, termed PtMAT1D::URA5, was prepared by KpnI and XbaI digestion of pUC-PtMAT1-URA5. PtMAT1 deletion was confirmed with the colony PCR method using two sets of oligonucleotide primers: 50 -CTCCTCGTCGACTTGTCCTC-30 (forward) and 50 -TTGAGCGACTCCTTGATGTG-30 (reverse) (ColonyPCR_B); and 50 -TTGCGTCGGGAGGACTGGCTGG-30 (forward) and 50 TGCATGCCTGAACCTGGTCG-30 (reverse) (ColonyPCR_C). The resulting colonies were selected on a yeast nitrogen base without amino acid (YNB) plate (YNB-ura) (1.7 g L1 YNB, 5 g L1 (NH4)2SO4, 20 g L1 glucose, and 20 g L1 agar) to estimate the ability to recover uracil synthesis.
FIG. 1. Biosynthetic pathway of the diastereomer type of mono-acetylated mannosylerythritol lipid (MEL-B) produced in Pseudozyma tsukubaensis from plant oil. PtEMT1p, erythritol/mannose transferase; PtMAC1p and PtMAC2p, acyltransferases; PtMAT1p, acetyltransferase; and PtMMF1p, putative transporter.
Please cite this article in press as: Saika, A., et al., Identification of the gene PtMAT1 encoding acetyltransferase from the diastereomer type of mannosylerythritol lipid-B producer Pseudozyma tsukubaensis, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.05.025
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FIG. 2. The plasmids used in this study. (A) pUC_PtMAT1-URA5; (B) pUC_UARSneo-PtMAT1. PtMAT1 and PtURA5 are derived from P. tsukubaensis 1E5. URA5-P, promoter region of PtURA5; and URA5-T, terminator region of PtURA5.
Transformation of plasmid pUC_UARSneo-PtMAT1 The expression plasmid pUC_UARSneo-PtMAT1 was introduced into the chromosome of strain DPtMAT1 by non-homologous recombination using electroporation according to Saika et al. (34). Following electroporation, the resulting colonies were grown at 25 C for 3 days on YM plates (3 g L1 yeast extract, 3 g L1 malt extract, 5 g L1 peptone, and 10 g L1 glucose) containing 500 mg mL1 G418. Three different clones for each recombinant strain were selected and used in subsequent experiments. MEL production The gene-deleted and gene-complemented strains of P. tsukubaensis 1E5 were cultivated in YM medium containing 50 g L1 glycerol at 25 C for 2 days with shaking at 250 rpm. An aliquot of the seed culture (1 mL) was inoculated in 20 mL of MEL production medium (5 g L1 yeast extract, 3 g L1 NaNO3, 0.3 g L1 KH2PO4, and 0.3 g L1 MgSO4$7H2O) containing 3% (w/v) olive oil and 2% (w/v) glycerol, and cultivated at 25 C for 4 days with shaking at 250 rpm. MELs produced in the culture were extracted with an equal volume of ethyl acetate. Thin-layer chromatography analysis MELs were detected using thin-layer chromatography (TLC) (35). The extracted MELs were analyzed by TLC using chloroform, methanol, and 12% NH4OH in a 65:15:2 (v:v:v) ratio as an eluent. MELs were detected on the TLC plate by spraying the plate with 2% anthronesulfate reagent and heating at 150 C for 5 min. Purified MEL mixture produced by P. antarctica was used as a reference. Structural analysis The structure of purified MEL was characterized by H nuclear magnetic resonance spectroscopy (NMR) with a Bruker AVANCE 400 (400 MHz) at 30 C in a CDCl3 solution (diastereomer type of MEL-B) or CDCl3 containing 10 % (v/v) methanol-d4 (diastereomer type of MEL-D). Tetramethylsilane was used as an internal chemical shift standard. 1
RESULTS Amino acid sequence analysis of PtMAT1p from P. tsukubaensis 1E5 Based on BLASTP analyses, the putative acetyltransferase gene (PtMAT1) was found in the draft genome of P. tsukubaensis 1E5. An amino acid sequence analysis showed that PtMAT1p shared approximately 40e60% of its identity with 13 homologous proteins from 10 strains belonging to the genera Ustilago, Melanopsichium, Pseudozyma, and Sporisorium (Table S1). There is no putatively conserved domain found. Among the 10 strains, U. maydis, P. antarctica, P. aphidis, P. hubeiensis, and S. scitamineum are well-known MEL producers. We aligned the deduced amino acid sequences of 15 proteins (13 homologous proteins and PtMAT1p from P. tsukubaensis NBRC1940 and 1E5) using ClustalW, and created a phylogenetic tree (Figs. 3 and S1). The phylogenetic analysis placed PtMAT1p from P. tsukubaensis in a separate clade from those of the other MEL producers. PtMAT1 deletion in the PtURA5-mutant An acetyltransferase (PtMAT1)-deleted stain of the PtURA5-mutant (uracil auxotrophic mutant of P. tsukubaensis 1E5) was generated by homologous recombination. In total, 84 candidate strains were obtained by plate selection, and deletion of PtMAT1 was evaluated by colony
PCR (Fig. 4). Primer set B (Fig. 4B) amplified the 1.0-kb inner region of PtMAT1. There was no corresponding band in strain DPtMAT1 (Fig. 4B, lanes 2e6), but a 1.0-kb band was apparent in the PtURA5-mutant (Fig. 4B, lane 1). In addition, 2.1 kb in length corresponding to the fragment PtMAT1D::URA5 was detected in strain DPtMAT1 (Fig. 4C, lanes 2e6). From the colony PCR results, five strains were selected as PtMAT1 deletion strains (DPtMAT1 No. 1e5). Uracil biosynthesis was restored in the PtMAT1 deletion strains, as they could grow without uracil supplementation in the medium (Fig. S2). Based on these observations, PtMAT1 deletion was successful in the PtURA5mutant of P. tsukubaensis. MEL biosynthesis of strain DPtMAT1 Five strains of DPtMAT1 were cultivated in MEL production medium containing 3%
(w/v) olive oil for 4 days at 25 C, and the MEL structure was evaluated by TLC analysis (Fig. 5A). The strain 1E5 (parent strain of PtURA5-mutant) was used as a control strain. The produced MELs were extracted with ethyl acetate and detected with TLC using anthrone-sulfate reagent. In the TLC analysis, only two strains produced MEL (DPtMAT1 No. 2 and No. 4). MEL-B produced by strain 1E5 was detected in the upper position on the TLC plate, whereas the spot from the extract of strain DPtMAT1 was detected in a lower position. Because MEL-D has no acetyl groups, it shows higher hydrophilicity than MEL-B. The TLC analysis strongly suggested that strain DPtMAT1 produced MEL-D due to PtMAT1 deletion.
FIG. 3. Phylogenetic relationships among P. tsukubaensis 1E5 and homologous strains based on the MAT1p amino acid sequence.
Please cite this article in press as: Saika, A., et al., Identification of the gene PtMAT1 encoding acetyltransferase from the diastereomer type of mannosylerythritol lipid-B producer Pseudozyma tsukubaensis, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.05.025
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J. BIOSCI. BIOENG., harboring pUC_UARSneo-PtMAT1 was in the same position as that of the MEL-B standard. In contrast, the mobility of MEL produced by strain DPtMAT1 harboring the empty vector was unchanged. Based on the change in mobility, PtMAT1 expression restores MEL acetylation. Structural analysis of MELs The structure of MEL produced by strain DPtMAT1 was identified by NMR analysis, and the signal pattern was compared with the diastereomer type of MEL-B produced by the strain 1E5 (Fig. 6). Each signal was assigned as described previously (29). The 1H NMR spectrum of MEL produced by strain 1E5 and DPtMAT1 were similar; however, the signal assigned to the acetyl group (2.13 ppm) in MEL-B produced by strain 1E5 was not apparent for the MEL produced by strain DPtMAT1. In addition, the signal assigned to H-60 was shifted to a higher magnetic field in strain DPtMAT1. The signal assigned to H4a and H-4b showed that the MEL extracted from strain DPtMAT1 was a diastereomer; therefore, PtMAT1 deletion did not affect the chirality of the mannosylerythritol moiety. According to the NMR analysis, strain DPtMAT1 produced the diastereomer type of MEL-D by PtMAT1 deletion. DISCUSSION
FIG. 4. PCR analysis of parental strain 1E5 and strain DPtMAT1. (A) Primer sets were used in the PCR amplification to assess PtMAT1 deletion. Agarose gel electrophoresis of amplified DNA fragments was performed to confirm gene disruption. Gene fragments were amplified using primer set ColonyPCR_B (B) and primer set ColonyPCR_C (C). Lane M, DNA marker; lane 1, PtURA5-mutant5; lanes 2e6, strains DPtMAT1 No. 1, No. 2, No. 3, No. 4, and No. 5.
Gene complementation of strain DPtMAT1 using pUC_URASneo-PtMAT1 To obtain further evidence of the function of PtMAT1p, PtMAT1 underwent complementation using the expression plasmid pUC_UARSneo-PtMAT1. The empty vector pUC_UARSneo was used as a negative control. A plasmid harboring PtMAT1 from P. tsukubaensis 1E5 was introduced into strain DPtMAT1 No. 2, which was cultivated in MEL production medium containing 3% (w/v) olive oil for 4 days at 25 C. Fig. 5B shows the TLC pattern of the complementary strain of DPtMAT1. The corresponding spot of MEL produced by strain DPtMAT1
In our previous studies, the diastereomer type of MEL-B produced by P. tsukubaensis showed higher water-holding capacity and greater self-assembling properties across a wide range of concentrations and temperatures compared to the conventional type of MELs (6,36). Therefore, the diastereomer type of MEL has a greater potential for use in aqueous solutions. We recently demonstrated the expansion of the diastereomer type of MEL structures using gene engineering methods. We focused on PtEMT1p, which is crucial in determining the sugar conformation of MEL, and succeeded in producing the diastereomer type of MEL-A using the recombinant strain of P. antarctica expressing PtEMT1 from P. tsukubaensis (37). In addition, the mono-acylated diastereomer type of MEL-D producer was generated by targeted gene-disruption of PtMAC2 from P. tsukubaensis as a host strain (30). As demonstrated by these results, modification of the MEL biosynthesis pathway by gene engineering techniques enables the tailor-made production of the diastereomer type of MELs.
FIG. 5. Effect of PtMAT1 deletion on MEL production. The MEL production of the strain 1E5 (parent strain of PtURA5-mutant5), strain DPtMAT1, and PtMAT1-complemented strain was determined using MEL production medium containing and 3% olive oil at 25 C for 4 days. The produced MEL was extracted with ethyl acetate and visualized using anthrone reagent. (A) Lanes 1e5, strains DPtMAT1 No. 1, No. 2, No. 3, No. 4, and No. 5; lane 6, strain 1E5; (B) lane 1, strain DPtMAT1 No. 2 harboring pUC_UARSneo; lane 2, strain DPtMAT1 harboring pUC_UARSneo-PtMAT1; STD, MEL mixture of MEL-A, MEL-B, and MEL-C.
Please cite this article in press as: Saika, A., et al., Identification of the gene PtMAT1 encoding acetyltransferase from the diastereomer type of mannosylerythritol lipid-B producer Pseudozyma tsukubaensis, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.05.025
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FIG. 6. Partial 1H NMR spectra. (A) Diastereomer type of MEL-B synthesized by strain 1E5, and (B) diastereomer type of MEL-D synthesized by strain DPtMAT1.
In the phylogenetic analysis (Fig. 3), PtMAT1p from P. tsukubaensis NBRC1940 and 1E5 were considered as a separate clade from the other MEL producers. U. maydis, S. scitamineum, P. antarctica, P. aphidis, and P. hubeiensis produce MEL mixtures containing MEL-A (acetylated at the C-40 and C-60 positions of mannose), MEL-B (acetylated at the C-60 position of mannose), and MEL-C (acetylated at the C-40 position of mannose). In other words, MAT1p from these five strains shows relaxed regioselectivity. Conversely, PtMAT1p from P. tsukubaensis only catalyzes the transfer of acetyl-CoA to the C-60 position of the mannosyl moiety, because P. tsukubaensis selectively produces MEL-B. From these results, the protein structure and substrate-binding mechanism of PtMAT1p may differ from other MEL producers. Although 84 strains lacking uracil auxotrophy were obtained on the YNB-ura plate, only five were PtMAT1-deleted strains (Fig. 4), suggesting that non-homologous recombination occurred at a high frequency. In addition, according to the TLC analysis, strains DPtMAT1 No. 1, No. 3, and No. 5 lacked MEL productivity (Fig. 5A). Non-homologous recombination occurred simultaneously during PtMAT1 deletion; therefore, these three strains may be affected by undesired gene deletion via nonhomologous recombination. A previous study demonstrated that Ku70 deletion led to improved targeted gene deletion efficiency in Saccharomyces macrospora (38). Therefore, further modification of the gene deletion method, such as Ku70 deletion, could improve the gene deletion technique in yeast strains of the genus Pseudozyma.
In the TLC analysis (Fig. 5A), the MEL productivity of the strains
DPtMAT1 No. 2 and No. 4 decreased compared with the parent strain. Non-homologous recombination with the fragment PtMAT1D::URA5 was considered to be the cause of the decrease in MEL production. In another study, the MAT1-deleted strain of P. hubeiensis maintained about 70% of MEL productivity (91.6/ 129 g L1 ¼ MAT1-deleted strain/parent strain) (28). P. tsukubaensis can produce over 70 g L1 of the diastereomer type of MEL-B; therefore, strain DPtMAT1 has the potential to produce abundant levels of the diastereomer type of MEL-D. In addition, because antibiotics selection is not required during cultivation, strain DPtMAT1 may be suitable for industrial-scale production. We demonstrated that lipase expression in P. tsukubaensis strain 1E5 led to an increase in diastereomer type of MEL-B production using olive oil as a carbon source (34). The overexpression of the lipase gene in strain DPtMAT1 may efficiently increase productivity of the diastereomer type of MEL-D. In conclusion, we studied PtMAT1 in P. tsukubaensis and successfully generated the PtMAT1 deletion strain via homologous recombination. Based on TLC and NMR analyses, PtMAT1p catalyzes the transfer reaction of the acetyl group to the mannose moiety of MEL, and strain DPtMAT1 produces the diastereomer type of MEL-D (i.e., non-acetylated MEL). These results can be used to promote the development of tailor-made MEL production, and support the expansion of industrial applications of MELs. Supplementary data related to this article can be found at https://doi.org/10.1016/j.jbiosc.2018.05.025.
Please cite this article in press as: Saika, A., et al., Identification of the gene PtMAT1 encoding acetyltransferase from the diastereomer type of mannosylerythritol lipid-B producer Pseudozyma tsukubaensis, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.05.025
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J. BIOSCI. BIOENG., ACKNOWLEDGMENTS
This work was supported by the Japan Science and Technology Agency under the Adaptable and Seamless Technology Transfer Program through Target-driven R&D (AS2621413N). YU, SY, and TK are employees of Toyobo Co., Ltd.; however, Toyobo Co., Ltd. did not have any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. AS, HK, TF, SY, TK, and TM are the inventors of a submitted patent related to this study (JP2017-008703). The authors have declared that no other competing interests exist. This article does not contain any studies with human participants or animals performed by any of the authors. References 1. 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). 2. Rau, U., Hammen, S., Hechmann, R., Wray, V., and Lang, S.: Sophorolipids: a source for novel compounds, Ind. Crop. Prod., 13, 85e92 (2001). 3. 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). 4. Kitamoto, D., Akiba, S., Hioki, C., and Tabuchi, T.: Extracellular accumulation of mannosylerythritol lipids by a strain Candida antarctica, Agric. Biol. Chem., 54, 31e36 (1990). 5. Maier, R. M. and Soberón-chávez, G.: Pseudomonas aeruginosa rhamnolipids: biosynthesis and potential applications, Appl. Microbiol. Biotechnol., 54, 625e633 (2000). 6. Fukuoka, T., Yanagihara, T., Imura, T., Morita, T., Sakai, H., Abe, M., and Kitamoto, D.: The diastereomers of mannosylerythritol lipids have different interfacial properties and aqueous phase behavior, reflecting the erythritol configuration, Carbohydr. Res., 351, 81e86 (2012). 7. 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). 8. Fukuoka, T., Morita, T., Konishi, M., Imura, T., and Kitamoto, D.: Characterization of new glycolipid biosurfactants, tri-acylated mannosylerythritol lipids, produced by Pseudozyma yeasts, Biotechnol. Lett., 29, 1111e1118 (2007). 9. Fukuoka, T., Morita, T., Konishi, M., Imura, T., and Kitamoto, D.: A basidiomycetous yeast, Pseudozyma tsukubaensis, efficiently produces a novel glycolipid biosurfactant. The identification of a new diastereomer of mannosylerythritol lipid-B, Carbohydr. Res., 343, 555e560 (2008). 10. Morita, T., Fukuoka, T., Imura, T., and Kitamoto, D.: Formation of the two novel glycolipid biosurfactants, mannosylribitol lipid and mannosylarabitol lipid, by Pseudozyma parantarctica JCM 11752T, Appl. Microbiol. Biotechnol., 96, 931e938 (2012). 11. Morita, T., Fukuoka, T., Konishi, M., Imura, T., Yamamoto, S., Kitagawa, M., Sogabe, A., and Kitamoto, D.: Production of a novel glycolipid biosurfactant, mannosylmannitol lipid, by Pseudozyma parantarctica and its interfacial properties, Appl. Microbiol. Biotechnol., 83, 1017e1025 (2009). 12. Kitamoto, D., Morita, T., Fukuoka, T., Konishi, M., and Imura, T.: Selfassembling properties of glycolipid biosurfactants and their potential applications, Curr. Opin. Colloid Interface Sci., 14, 315e328 (2009). 13. Kitamoto, D., Isoda, H., and Nakahara, T.: Functions and potential applications of glycolipid biosurfactants d from energy-saving materials to gene delivery carriers, J. Biosci. Bioeng., 94, 187e201 (2002). 14. Zhao, X., Murata, T., Ohno, S., Day, N., Song, J., Nomura, N., Nakahara, T., and Yokoyama, K. K.: Protein kinase C_ plays a critical role in mannosylerythritol lipid-induced differentiation of melanoma B16 cells, J. Biol. Chem., 276, 39903e39910 (2001). 15. Kitamoto, D., Yanagishita, S., Shinbo, T., Nakane, T., Kamisawa, C., and Nakahara, T.: Surface active properties and antimicrobial activities of mannosylerythritol lipids as biosurfactants produced by Candida antarctica, J. Biotechnol., 29, 91e96 (1993). 16. Takahashi, M., Morita, T., Fukuoka, T., Imura, T., and Kitamoto, D.: Glycolipid biosurfactants, mannosylerythritol lipids, show antioxidant and protective effects against H2O2-induced oxidative stress in cultured human skin fibroblasts, J. Oleo Sci., 61, 457e464 (2012). 17. Yamamoto, S., Morita, T., Fukuoka, T., Imura, T., Yanagidani, S., Sogabe, A., Kitamoto, D., and Kitagawa, M.: The moisturizing effects of glycolipid
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Please cite this article in press as: Saika, A., et al., Identification of the gene PtMAT1 encoding acetyltransferase from the diastereomer type of mannosylerythritol lipid-B producer Pseudozyma tsukubaensis, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.05.025