Hyphenation of supercritical fluid chromatography with different detection methods for identification and quantification of liamocin biosurfactants.

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-ß-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.

Comprehensive liamocin biosurfactants analysis by reversed phase liquid chromatography coupled to mass spectrometric and charged-aerosol detection.

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.

Exploiting the Natural Diversity of RhlA Acyltransferases for the Synthesis of the Rhamnolipid Precursor 3-(3-Hydroxyalkanoyloxy)Alkanoic Acid.

While rhamnolipids of the Pseudomonas aeruginosa type are commercially available, the natural diversity of rhamnolipids and their origin have barely been investigated. Here, we collected known and identified new rhlA genes encoding the acyltransferase responsible for the synthesis of the lipophilic rhamnolipid precursor 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA). Generally, all homologs were found in Betaproteobacteria and Gammaproteobacteria A likely horizontal gene transfer event into Actinobacteria is the only identified exception. The phylogeny of the RhlA homologs from Pseudomonas and Burkholderia species is consistent with the organism phylogeny, and genes involved in rhamnolipid synthesis are located in operons. In contrast, RhlA homologs from the Enterobacterales do not follow the organisms' phylogeny but form their own branch. Furthermore, in many Enterobacterales and Halomonas from the Oceanospirillales, an isolated rhlA homolog can be found in the genome. The RhlAs from Pseudomonas aeruginosa PA01, Pseudomonas fluorescens LMG 05825, Pantoea ananatis LMG 20103, Burkholderia plantarii PG1, Burkholderia ambifaria LMG 19182, Halomonas sp. strain R57-5, Dickeya dadantii Ech586, and Serratia plymuthica PRI-2C were expressed in Escherichia coli and tested for HAA production. Indeed, except for the Serratia RhlA, HAAs were produced with the engineered strains. A detailed analysis of the produced HAA congeners by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) highlights the congener specificity of the RhlA proteins. The congener length varies from 4 to 18 carbon atoms, with the main congeners consisting of different combinations of saturated or monounsaturated C10, C12, and C14 fatty acids. The results are discussed in the context of the phylogeny of this unusual enzymatic activity.IMPORTANCE The RhlA specificity explains the observed differences in 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) congeners. Whole-cell catalysts can now be designed for the synthesis of different congener mixtures of HAAs and rhamnolipids, thereby contributing to the envisaged synthesis of designer HAAs.

A pH shift induces high-titer liamocin production in Aureobasidium pullulans.

Liamocins are biosurfactants produced by the fungus Aureobasidium pullulans. A. pullulans belongs to the black yeasts and is known for its ability to produce pullulan and melanin. However, the production of liamocins has not been investigated intensively. Initially, HPLC methods for the quantification of liamocin and the identification of liamocin congeners were established. Eleven congeners could be detected, differing in the polyol head groups arabitol or mannitol. In addition, headless molecules, so-called exophilins, were also identified. The HPLC method reported here allows quick and reliable quantification of all identified congeners, an often-overlooked prerequisite for the investigation of valuable product formation. Liamocin synthesis was optimized during cultivation in lab-scale fermenters. While the pH can be kept constant, the best strategy for liamocin synthesis consists of a growth phase at neutral pH and a subsequent production phase induced by a manual pH shift to pH 3.5. Finally, combining increased nitrogen availability with a pulsed fed-batch fermentation, cell growth, and liamocin titers could be enhanced. Here, the maximal titers of above 10 g/L that were reached are the highest reported to date for liamocin synthesis using A. pullulans in lab-scale fermenters.

Analysis of steroids in urine by gas chromatography-capillary photoionization-tandem mass spectrometry.

A new heated capillary photoionization (CPI) ion source design was developed to photoionize analytes inside a transfer capillary between a gas chromatograph (GC) and a mass spectrometer (MS). The CPI setup included a wide, oval-shaped vacuum-ultraviolet (VUV) transparent magnesium fluoride (MgF2) window to maximize photoionization efficiency and thus sensitivity. The source contained a nitrogen housing around the ionization chamber inlet to avoid undesirable hydrolysis and oxidation reactions with ambient air and to maximize the proportion of formed molecular radical cations of analytes. The feasibility of the ion source was studied by analyzing 18 endogenous steroids in urine as their trimethylsilyl (TMS) derivatives with gas chromatography-tandem mass spectrometry (GC-MS/MS). The method was validated and applied to human urine samples. To our best knowledge, this is the first time that a capillary photoionization ion source has been applied for quantitative analysis of biological samples. The GC-CPI-MS/MS method showed good chromatographic resolution (peak half-widths between 3.1 to 5.3 s), acceptable linearity (coefficient of determination between 0.981 to 0.996), and repeatability (relative standard deviation (RSD%) between 5 to 18%). Limits of detection (LOD) were between 2 to 100 pg mL-1 and limits of quantitation (LOQ) were between 0.05 to 2 ng mL-1. In total, 15 steroids were quantified either as a free steroid or glucuronide conjugate from the urine of volunteers. The new CPI source design showed excellent sensitivity for analysis of steroids in complex biological samples.

Mass spectrometric characterization of siderophores produced by Pseudomonas taiwanensis VLB120 assisted by stable isotope labeling of nitrogen source.

The structures of three previously unknown siderophores produced by the fluorescent, biotechnologically relevant Pseudomonas taiwanensis (P. taiwanensis) VLB120 bacteria were elucidated by means of hydrophilic interaction liquid chromatography (HILIC) hyphenated to high-resolution tandem mass spectrometry (HRMS/MS). They could be verified as iron(III)- and aluminum(III) complexes as well as the protonated molecules of the siderophores formed by in-source fragmentation. The siderophores were separated according to their different acyl side chains and additionally according their central ions. One of the siderophores was identified as pyoverdine with a malic acid (hydroxy succinic acid) amide side chain and a peptide moiety consisting of Orn-Asp-OHAsn-Thr-AcOHOrn-Ser-cOHOrn. The other analytes were assigned to an azotobactin with the identical peptide chain linked to the characteristic chromophoric unit and a pyoverdine with a variation in the amino acid sequence. Proline is directly linked to the pyoverdine chromophore instead of ornithine. Acidic and enzymatic hydrolyses were carried out to analyze the individual amino acids. Beside OHAsn, each amino acid of the peptide part was identified by HILIC-HRMS and comparison to authentic standards. Additionally, 15N-labeled cellular supernatants were analyzed by means of HRMS/MS. The results of the MS/MS experiments complemented by accurate mass data facilitated elucidation of the structures studied in this work and provided further confirmation of the three recently described pyoverdines of P. taiwanensis VLB120 (Baune et al. in Biometals 30:589-597, 2017. https://doi.org/10.1007/s10534-017-0029-7 ).

Structural characterization of pyoverdines produced by Pseudomonas putida KT2440 and Pseudomonas taiwanensis VLB120.

The previously unknown sequences of several pyoverdines (PVD) produced by a biotechnologically-relevant bacterium, namely, Pseudomonas taiwanensis VLB120, were characterized by high performance liquid chromatography (HPLC)-high resolution mass spectrometry (HRMS). The same structural characterization scheme was checked before by analysis of Pseudomonas sp. putida KT2440 samples with known PVDs. A new sample preparation strategy based on solid-phase extraction was developed, requiring significantly reduced sample material as compared to existing methods. Chromatographic separation was performed using hydrophilic interaction liquid chromatography with gradient elution. Interestingly, no signals for apoPVDs were detected in these analyses, only the corresponding aluminum(III) and iron(III) complexes were seen. The chromatographic separation readily enabled separation of PVD complexes according to their individual structures. HPLC-HRMS and complementary fragmentation data from collision-induced dissociation and electron capture dissociation enabled the structural characterization of the investigated pyoverdines. In Pseudomonas sp. putida KT2240 samples, the known pyoverdines G4R and G4R A were readily confirmed. No PVDs have been previously described for Pseudomonas sp. taiwanensis VLB120. In our study, we identified three new PVDs, which only differed in their acyl side chains (succinic acid, succinic amide and malic acid). Peptide sequencing by MS/MS provided the sequence Orn-Asp-OHAsn-Thr-AcOHOrn-Ser-cOHOrn. Of particular interest is the presence of OHAsn, which has not been reported as PVD constituent before.