Biosurfactants produced by <i>R</i>. <i>taiwanensis</i> MD1149 are polyol esters of fatty acids (PEFA).

<p>Biosurfactant compounds were purified using solid phase extraction (reversed phase), and eluted from the column using 100% methanol as detected by LC-MS analysis (A). The organic solvent was subsequently evaporated, and the dried material was digested with methanolic HCl, derivatized (silylated), and analyzed by gas chromatography-mass spectrometry (B). GC-MS analysis revealed that the biosurfactant mixture was composed of glycolipids containing the sugar alcohols mannitol and arabitol (TMS derivatives), as well six main fatty acid constituents.</p

Biosurfactant production of <i>R</i>. <i>bogoriensis</i> (control) compared to <i>R</i>. <i>taiwanensis</i> MD1149.

<p><i>R</i>. <i>bogoriensis</i> produced known sophorolipids that markedly reduced the surface tension of the culture medium (A). The presence of these sophorolipid biosurfactants was readily detected in the LC-MS total ion chromatograms (B, green triangle), and the accurate mass of the four main sophorolipid species was measured and confirmed (C). <i>R</i>. <i>taiwanensis</i> MD1149 produced biosurfactants that transiently lowered the surface tension of the culture medium (D), which corresponded with their appearance, and subsequent disappearance, in the LC-MS total ion chromatograms (E, blue triangle).</p

Biosurfactants produced by <i>R</i>. <i>taiwanensis</i> MD1149 are transiently present in the culture medium.

<p>MD1149 was cultured for four days (d4) then split; half of the culture was allowed to continue shaking with cells (left chromatograms), while in the other half, cells were removed via centrifugation, and allowed to continue shaking (right chromatograms). The two flasks were monitored for an additional three days (d5, d6, d7) by LC-MS.</p

<i>Rhodotorula taiwanensis</i> MD1149 produces hypoacetylated PEFA compounds with increased surface activity compared to <i>Rhodotorula babjevae</i> MD1169

<div><p>Biosurfactants have several desirable characteristics in the industrial sector: detergency, antimicrobial effects, skin hydration, and emulsibility. Several yeast glycolipids are currently being utilized in these capacities: sophorolipids, ustilagic acid, and mannosylerythritol lipids (MELs). An emerging class of glycolipids, termed polyol esters of fatty acids (PEFA), have recently been reported for <i>Rhodotorula babjevae</i>, a basidiomycetous yeast species that secretes hyperacetylated congeners of PEFA (typically with 3–6 acetylation modifications). While screening Rhodotorula species for surfactant production, we identified a new environmental isolate identified as <i>Rhodotorula taiwanensis</i> MD1149 that dropped the surface tension of the liquid medium, indicating that it produced a potent biosurfactant. Acid depolymerization of the purified biosurfactants, followed by gas chromatography-mass spectrometry (GC-MS) analysis revealed that the biosurfactants were composed of PEFA compounds composed mainly of mannitol and arabitol esters of 3-hydroxy fatty acid, 3-methoxy fatty acid, and fatty acids with a single double bond; chain lengths were mainly C16 and C18. Liquid chromatography-mass spectrometry (LC-MS) confirmed the predicted accurate mass of these compounds. Interestingly, PEFA compounds produced by <i>Rhodotorula taiwanensis</i> MD1149 were more surface active due to their hypoacetylation profile (0–4 acetylation modifications) compared to <i>Rhodotorula babjevae</i> MD1169. These disparate surface active properties, based on acetylation, change the hydrophilic-lipophilic balance (HLB) of these compounds, and their potential utility within industrial applications.</p></div

Biosurfactants produced by <i>R</i>. <i>taiwanensis</i> MD1149 are polyol esters of fatty acids (PEFA).

<p>Biosurfactant compounds were purified using solid phase extraction (reversed phase), and eluted from the column using 100% methanol as detected by LC-MS analysis (A). The organic solvent was subsequently evaporated, and the dried material was digested with methanolic HCl, derivatized (silylated), and analyzed by gas chromatography-mass spectrometry (B). GC-MS analysis revealed that the biosurfactant mixture was composed of glycolipids containing the sugar alcohols mannitol and arabitol (TMS derivatives), as well six main fatty acid constituents.</p

Biosurfactants produced by <i>R</i>. <i>babjevae</i> MD1169 are polyol fatty acid esters with a similar composition profile to <i>R</i>. <i>taiwanensis</i> MD1149.

<p><i>R</i>. <i>babjevae</i> biosurfactants were purified using solid phase extraction (reversed phase), and eluted from the column using 100% methanol as detected by LC-MS analysis (A). <i>R</i>. <i>babjevae</i> MD1169 compounds are illustrated in the green LC-MS total ion chromatogram, and are overlayed with the LC-MS total ion chromatogram from <i>R</i>. <i>taiwanensis</i> MD1149 (black trace). Interestingly, GC-MS analysis of <i>R</i>. <i>babjevae</i> MD1169 revealed that the biosurfactant mixture was composed of the same sugar alcohol and fatty acid constituents as <i>R</i>. <i>taiwanensis</i>, but at different ratios (B). The GC-MS total ion chromatograms (between the two samples) was normalized for mannitol concentration, and the ratios of the other constituents were relative to it.</p

Biosurfactants produced by <i>R</i>. <i>taiwanensis</i> MD1149 and <i>R</i>. <i>babjevae</i> MD1169 have distinct acetylation profiles that impact their surface-active properties.

<p>Spent liquid medium was harvested during peak production of biosurfactants in three replicate experiments (for each organism), and analyzed by LC-MS. The individual areas for each acetyl group species were then added together to create an acetylation profile of all of the detectable compounds produced by <i>R</i>. <i>taiwanensis</i> (blue) versus <i>R</i>. <i>babjevae</i> (green) (A). The surface tension of the spent liquid medium for each of the three biological replicates was also measured, averaged together, and compared between <i>R</i>. <i>taiwanensis</i> (blue) versus <i>R</i>. <i>babjevae</i> (green) (B). A p-value less than 0.001 was indicated by the three asterisks.</p

Arabitol 3-hydroxy C18 exists as an acetylation series.

<p>High-resolution mass spectrometry confirmed non-acetylated and acetylated arabitol 3-hydroxy C18 congeners in the spent liquid medium (A). The 3-methoxy and unsaturated fatty acid versions of these compounds were also detected. The calculated formulae also match the double bond equivalents (DBE) for the proposed structures (B and C). The potential acetylation sites (“R”) are highlighted on the different arabitol C18 congeners (C), as well as the potential number of structural combinations that exist for 3 acetyl groups—the most abundant type of arabitol 3-hydroxy C18.</p

Evolutionary Divergence in the Catalytic Activity of the CAM-1, ROR1 and ROR2 Kinase Domains

<div><p>Receptor tyrosine kinase-like orphan receptors (ROR) 1 and 2 are atypical members of the receptor tyrosine kinase (RTK) family and have been associated with several human diseases. The vertebrate RORs contain an ATP binding domain that deviates from the consensus amino acid sequence, although the impact of this deviation on catalytic activity is not known and the kinase function of these receptors remains controversial. Recently, ROR2 was shown to signal through a Wnt responsive, β-catenin independent pathway and suppress a canonical Wnt/β-catenin signal. In this work we demonstrate that both ROR1 and ROR2 kinase domains are catalytically deficient while CAM-1, the <i>C</i>. <i>elegans</i> homolog of ROR, has an active tyrosine kinase domain, suggesting a divergence in the signaling processes of the ROR family during evolution. In addition, we show that substitution of the non-consensus residues from ROR1 or ROR2 into CAM-1 and MuSK markedly reduce kinase activity, while restoration of the consensus residues in ROR does not restore robust kinase function. We further demonstrate that the membrane-bound extracellular domain alone of either ROR1 or ROR2 is sufficient for suppression of canonical Wnt3a signaling, and that this domain can also enhance Wnt5a suppression of Wnt3a signaling. Based on these data, we conclude that human ROR1 and ROR2 are RTK-like pseudokinases.</p></div

Comparison of kinetic parameters for MuSK, CAM-1, ROR1 and ROR2 tyrosine phosphorylation activity.

<p>CAM-1 and ROR data were obtained from a [γ−³²P]ATP-based phosphocellulose binding assay. Data are from three technical replicates, ± standard error. ND, Not determined. ^aMuSK data are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102695#pone.0102695-Till1" target="_blank">[24]</a>.</p