Liamocins, a group of high-density glycolipids, are only produced by certain strains of the yeast-like fungi in the genus Aureobasidium. Until now, few studies have focused on the surfactant properties of liamocins produced from the highly diverse tropical strains of Aureobasidium. Therefore, the aims of this research were to screen the liamocin production from tropical strains of Aureobasidium spp. and to characterize their surfactant properties. A total of 41 strains of Thai Aureobasidium spp. were screened for their ability to produce liamocins, and the products were detected using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and thin-layer chromatography. Of those strains, 30 strains of Aureobasidium spp. tested were found to produce liamocins with yields ranging from 0.53 to 10.60 g/l. The nature of all crude liamocins was heterogeneous, with different compositions and ratios depending on the yeast strain. These liamocins exhibited relatively high emulsifying activity against vegetable oils tested, with an emulsification index of around 40-50%; the emulsion stability of some liamocins was up to 30 days. The obtained critical micelle concentration values were varied, with those ​​of liamocins produced from A. pullulans, A. melanogenum and A. thailandense falling in ranges from 7.70 to 119.78, 10.73 to > 1,000, and 68.56 to > 1,000 mg/l, respectively. The emulsification activity of liamocins was higher than that of the analytical grade rhamnolipids. These compounds showed strong surface tension reduction in a sodium chloride concentration range of 2-12% (w/v), pH values between 3 and 7, and temperatures between 4 and 121 °C. This is the first report of liamocins produced by A. thailandense.

Aureobasidium melanogenum was found to be grown the best at the constant pH 7.0 and to produce the highest amount of liamocins at the constant pH 3.0. Therefore, the wild type strain A. melanogenum 9-1 and the engineered strain V33 constructed in the laboratory were grown at the constant pH 7.0 for 48 h, then, they were continued to be cultivated at the constant pH 3.0. Under such conditions, A. melanogenum 9-1 produced 36.51 ± 0.55 g L-1 of liamocin and its cell mass was 27.43 ± 0.63 and 6.00 ± 0.11 g L-1 of glucose was left in the finished medium within 168 h while the engineered strain V33 secreted 70.86 ± 2.04 g L-1 of liamocin, its cell mass was 31.63 ± 0.74 g L-1 , 0.16 ± 0.01 g L-1 of glucose was maintained in the finished medium. Then, Massoia lactone was released from the produced liamocins. The released Massoia lactone loaded in the nanoemulsions could be used to actively damage cell wall and cell membrane of both spores and mycelia of Aspergillus flavus, leading to its cell necrosis. Massoia lactone loaded in the nanoemulsions also actively inhibited cell growth of A. flavus, its conidia production and aflatoxin biosynthesis on peanuts, indicating that Massoia lactone loaded in the nanoemulsions had highly potential application in controlling cell growth of A. flavus and aflatoxin biosynthesis in foods and feedstuffs.

Liamocins and Massoia lactone have many applications. In this study, the glucose-derepressed mutant Δcrea5 in which the CREA gene was removed could produce 36.5 g/L of liamocins. Furthermore, overexpression of the MSN2 gene in the mutant Δcrea5 made the transformant M60 produce 41.4 g/L of liamocins and further overexpression of the GAL1 gene in the transformant M60 rendered the transformant G40 to produce 49.5 ± 0.4 g/L of liamocins during the 10-L fermentation while their wild type strain 9-1 made only 26.3 g/L of liamocins. The expressed transcription activators Msn2 and Gal1 were localized in the nuclei, promoting expression of the genes responsible for liamocins biosynthesis and sugar transport. Massoia lactone prepared from the produced liamocins could actively kill the spores of the pathogenic fungi from the diseased human skin by inhibiting spore germination and causing cellular necrosis of the fungal spores.

Massoia lactone could be released from liamocins produced by Aureobasidium melanogenum M39. The obtained Massoia lactone was very stable and highly active against many fungal crop pathogens which cause many plant diseases and food unsafety. Massoia lactone treatment not only could effectively inhibit their hyphal growth and spore germination, but also caused pore formation in cell membrane, reduction of ergosterol content, rise in intracellular ROS levels, and leakage of intracellular components, consequently leading to cellular necrosis and cell death. The direct contact of Massoia lactone with Fusarium graminearum spores could stop the development of Fusarium head blight symptom in the diseased wheats. Therefore, Massoia lactone could be a promising candidate for development as an effective and green bio-fungicide because of its high anti-fungal activity and the multiplicity of mode of its action.

Liamocins synthesized by Aureobasidium spp. are glycolipids composed of a single mannitol or arabitol headgroup linked to either three, four or even six 3,5-dihydroxydecanoic ester tail-groups. The highest titer of liamocin achieved was over 40.0 g/L. The substrates for liamocins synthesis include glucose, sucrose, xylose, mannitol, and others. The Pks1 is responsible for the biosynthesis of the tail-group 3,5-dihydroxydecanoic acid, both mannitol dehydrogenase (MDH) and mannitol 1-phosphate 5-dehydrogenase (MPDH) catalyze the mannitol biosynthesis and the arabitol biosynthesis is controlled by arabitol dehydrogenase (ArDH). The ester bond formation between 3,5-dihydroxydecanoic acid and mannitol or arabitol is catalyzed by the esterase (Est1). Liamocin biosynthesis is regulated by the specific transcriptional activator (Gal1), global transcriptional activator (Msn2), various signaling pathways, acetyl-CoA flux while Pks1 activity is controlled by PPTase activity. The synthesized liamocins have high bioactivity against the pathogenic bacteria Streptococcus spp. and some kinds of cancer cells while Massoia lactone released liamocins which exhibited obvious antifungal and anticancer activities. Therefore, liamocins and Massoia lactone have many applications in various sectors of biotechnology.

The coproduction of polymalic acid (PMA) and liamocins, two important metabolites secreted by Aureobasidium pullulans, from two waste by-products from the xylitol and gluconate industries was investigated in shake flasks and fermentors, confirming that waste xylose mother liquor (WXML) could be utilized as an economical feedstock without any pretreatment. Gluconate could strengthen carbon flux and NADPH supply for the synergetic biosynthesis of PMA and liamocins. High PMA and liamocin titers of 82.9 ± 2.1 and 28.3 ± 2.7 g/L, respectively, were obtained from the coupled WXML and waste gluconate mother liquor (WGML) in batch fermentation, with yields of 0.84 and 0.25 g/g, respectively. These results are comparable to those obtained from renewable feedstocks. Economic assessment of the process revealed that PMA and liamocins could be coproduced from two by-products at costs of $1.48/kg or $0.67/kg (with liamocins credit), offering an economic and sustainable process for the application of waste by-products.

Liamocins, as the secondary metabolites synthesized and secreted by Aureobasidium spp., consist of a single mannitol or a single arabitol head group partially O-acylated with three 3,5-dihydroxydecanoic ester groups or directly esterified with three or four 3,5-dihydroxydecanoic ester tails. Very recently, the whole synthetic pathway of liamocins in A. melanogenum 6-1-2 has been elucidated. It was found that the promoter sequences of all the genes related to liamocin synthesis in A. melanogenum 6-1-2 had stress regulatory elements with core sequences of AGGGG or CCCCT. Therefore, expression of all the genes would be regulated by the Msn2. In this study, it was found that removal of the single one MSN2 gene in A. melanogenum 6-1-2 made the mutant decrease yield of extracellular liamocin by 92.28 %, while complementation of the MSN2 gene in the mutant rendered liamocin synthesis to be restored. When A. melanogenum 6-1-2 was cultured in the liamocin fermentation medium with high glucose and low nitrogen, the Msn2 was localized in the nucleus and positively regulated the expression of the genes related to liamocin biosynthesis. Furthermore, when the key BCY1 gene encoding regulatory subunit of the cAMP-PKA signaling pathway in A. melanogenum 6-1-2 was knocked out, the amount of extracellular liamocins synthesized by the mutant was decreased by 96.73 % and the Msn2 was localized in the cytoplasm. Similarly, when the key HOG1 gene in the HOG1 signaling pathway was deleted, liamocin biosynthesis in the knockout strain was decreased by 98.09 %. However, it was found that the Hog1 may be one part of the general transcription complex to regulate the transcription of the MSN2 gene, leading to the reduced Msn2 and liamocin synthesis in the mutant. In addition, the key TOR1 gene and SNF1 gene in the TOR1 signaling pathway and the SNF1 signaling pathway were not involved in the regulation of the Msn2 activity and liamocin synthesis. It was concluded that the transcriptional activator Msn2, the HOG1 signaling pathway and the cAMP-PKA signaling pathway were involved in the regulation of liamocin biosynthesis and production.

Liamocins are a class of biosurfactants with growing interest. However, methods for identification and quantification of liamocins on the molecular level are lagging behind. Therefore, we developed a chromatographic separation based on supercritical fluid chromatography (SFC) for liamocins and structurally related exophilins. The different congeners could be separated on a charge modulated hydroxyethyl amide functionalized silica-based column. Coupling to high-resolution mass spectrometry (MS) revealed four exophilin species and four liamocin species with mannitol and arabitol as polyol head group in a sample of the yeast-like fungus Aureobasidium pullulans (A. pullulans). In contrast to a recently published reversed phase high-performance liquid chromatography (HPLC) method, the different subclasses (exophilins, mannitol liamocins and arabitol liamocins) were additionally separated by means of SFC. The structures were confirmed by their accurate masses and tandem mass spectrometry (MS/MS). A complementary quantification method was developed using SFC coupled to charged-aerosol detection (CAD) to overcome the disadvantages of quantification by means of MS without authentic standards. A flow compensation by varying the make-up flow was used to obtain a constant composition of the mobile phase during detection and to ensure a stable detector response. The concentrations of the individual liamocin species were determined using an external calibration with n-octyl-β-d-glycopyranoside. The total amount of these concentrations agrees with the dry weight of an aliquot of the heavy oil. The developed SFC-based method has the advantage of shorter analysis time and superior selectivity compared to the previously published LC separation. In brief, the here presented SFC hyphenations enable comprehensive analysis of liamocin biosurfactants providing identification and absolute quantification of individual congeners.

Liamocin biosurfactants and structurally related exophilins secreted by the Aureobasidium pullulans (A. pullulans) strain NRRL62031 were firstly analyzed by hyphenation of high-performance liquid chromatography (HPLC) with high-resolution mass spectrometry (HRMS). Ten different analytes were detected and identified by their accurate masses and divided into subclasses according to their different head groups: three liamocins with arabitol as head group, three mannitol liamocins, and four exophilins. A baseline separation of congeners within the subclasses was achieved by reversed phase HPLC on a C18 stationary phase, whereas an overlap of subclasses occurred. The structures were simultaneously confirmed by online tandem mass spectrometry (MS/MS) experiments in positive and negative ionization mode. The assigned polyol head groups and thus the feasibility of this method were confirmed by gas chromatography (GC)-MS data obtained after hydrolysis and derivatization of the liamocins. Based on the varying structural characteristics of liamocins, e.g. the polyol head group (or even none for exophilins) and the degree of acetylation, different detector response in LC-MS was expected, impairing relative quantification of congeners. Therefore, a complementary quantification method was developed using HPLC coupled to charged-aerosol detection (CAD), which allows the determination of the amount of the individual liamocin species without authentic liamocin standards. Hence, the here presented hyphenated techniques facilitate comprehensive analysis of liamocin biosurfactants.

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