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.

The aim of this study was to determine if the novel anti-streptococcal inhibitors, liamocins, also inhibit biofilm formation by S. mutans and S. sobrinus. S. mutans strain ATCC 25175 and S. sobrinus strain ATCC 33478 were tested for biofilm formation in a rapid microtiter plate (MTP) assay and the effects of added liamocins were determined. This assay measures relative biofilm growth on pin lids. Results were verified in a biofilm flow cell assay, using hydroxyapatite-coated coupons to simulate dental material. Planktonic cultures of S. mutans and S. sobrinus were inhibited by 0.1 mg liamocins/ml. When liamocins were added after the adhesion phase in a rapid microtiter plate assay, S. mutans was inhibited 53% by 5 mg liamocins/ml, while S. sobrinus was more sensitive, showing 100% inhibition at 0.5 mg liamocins/ml. When liamocins were added during the adhesion phase, biofilms of S. mutans showed 78% inhibition at 3.0 mg liamocins/ml. In a biofilm flow cell assay, liamocins added after the adhesion phase at 0.5 mg liamocins/ml inhibited biofilms of S. sobrinus, and appeared to remove biofilms over time. Liamocins were shown for the first time to inhibit biofilm formation by S. mutans and S. sobrinus. Since liamocins are specific for Streptococcus spp., they are potential new inhibitors of oral streptococcal biofilms that should not affect normal oral microflora.

Liamocins are unique heavier-than-water \"oils\" produced by certain strains of the fungus Aureobasidium pullulans. Liamocins have antibacterial activity with specificity for Streptococcus sp. Previous studies reported that liamocin yields were highest from strains of A. pullulans belonging to phylogenetic clades 8, 9, and 11, cultured on medium containing sucrose. In this study, 27 strains from these clades were examined for the first time for production of liamocins from agricultural biomass substrates. Liamocin yields were highest from strains in phylogenetic clade 11, and yields were higher from cultures grown on sucrose than from those grown on pretreated wheat straw. However, when supplementary enzymes (cellulase, β-glucosidase, and xylanase) were added, liamocin production on pretreated wheat straw was equivalent to that on sucrose. Liamocins produced from wheat straw were free of the melanin contamination common in sucrose-grown cultures. Furthermore, MALDI-TOF MS analysis showed that liamocins produced from wheat straw were under-acetylated, resulting in higher proportions of the mannitol A1 and B1 species of liamocin, the latter of which has the highest biological activity against Streptococcus sp.