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.

Three-dimensional Kendrick mass plots as a tool for graphical lipid identification.

RATIONALE: The rising field of lipidomics strongly relies on the identification of lipids in complex matrices. Recent technical advances regarding liquid chromatography (LC) and high-resolution mass spectrometry (HRMS) enable the mapping of the lipidome of an organism with short data acquisition times. However, interpretation and evaluation of resulting multidimensional datasets are challenging and this is still the bottleneck regarding overall analysis times. METHODS: A novel adaption of Kendrick mass plot analysis is presented for a rapid and accurate analysis of lipids in complex matrices. Separation of lipids by their respective head group was achieved via hydrophilic interaction liquid chromatography (HILIC) coupled to HRMS. The resulting LC/HRMS datasets are processed to a list of chromatographically separated features by applying an optimized MZmine 2 workflow. All features are plotted in a three-dimensional Kendrick mass plot, which allows a fast identification of present lipid classes, based on equidistant features with fitting retention times and the same Kendrick mass defect. Suspected lipid classes are used for exact mass database matching to annotate features. A second three-dimensional Kendrick mass plot of annotated features of a single lipid class helps to reveal potential database mismatches, resulting in a curated list of identified lipid species. RESULTS: The use of the novel adaption of the Kendrick mass plot has accelerated the identification of the relevant lipid species in the green alga Chlamydomonas reinhardtii. A total of 106 species were identified within the lipid classes: phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol. CONCLUSIONS: This work shows how the addition of chromatographic information, i.e. the retention time, to a classical two-dimensional Kendrick mass plot enables rapid and accurate analysis of LC/HRMS datasets, exemplified on a green alga (C. reinhardtii) sample. Three-dimensional Kendrick mass plots have improved lipid class identification and fast spotting of falsely annotated lipid species.

Complementary approach for analysis of phospholipids by liquid chromatography hyphenated to elemental and molecular mass spectrometry.

Phospholipids are one of the most important lipid categories with multiple functions in biological systems. Their analysis can contribute to a better understanding of metabolomic and kinetic processes in living cells. Comprehensive methods based on liquid chromatography coupled to mass spectrometry are available for phospholipid identification and quantification. However, quantification of phospholipids using electrospray ionization-mass spectrometry with internal standards is still challenging due to several reasons. In particular, the detector response of phospholipid species differs with variation of the head group as well as the fatty acid chain length and double bond number. Inductively coupled plasma-tandem mass spectrometry (ICP-MS/MS) provides an alternative approach for their absolute quantification with universal detector response for phosphorus independent of its chemical form and proportional to its quantity. Therefore, a quantification method based on compound-independent calibration using hydrophilic interaction liquid chromatography (HILIC) coupled to ICP-MS/MS was developed. An inverse gradient system was implemented for constant mobile phase composition after HILIC separation, which provides steady plasma ionization conditions. Isobaric phosphorus interferences were decreased by using the oxygen reaction mode of the triple quadrupole based ICP-MS/MS instrument. Complementary molecular information was obtained by ESI-high-resolution MS and MS/MS. The applicability of this approach was demonstrated in a proof of concept by complementary analysis of a total lipid extract of baker's yeast.

Separation and identification of phospholipids by hydrophilic interaction liquid chromatography coupled to tandem high resolution mass spectrometry with focus on isomeric phosphatidylglycerol and bis(monoacylglycero)phosphate.

Changes in lipid composition of cells or tissue are often linked to various diseases. Studies indicate alterations of bis(monoacylglycero)phosphate (BMP) species in diseases such as cancer. Therefore, an extended phospholipid profiling method based on hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution mass spectrometry (MS) and data-dependent MS/MS acquisition was developed to separate and unambiguously identify BMP species. Lipid species identification was based on retention time, accurate mass and specific MS/MS fragments. The developed method was applied in a proof of concept study to lipid extracts of a cell culture model of conditional oncogene overexpression in MCF-7/NeuT breast cancer cells. Comparison of control and oncogene-induced MCF-7/NeuT breast cancer cells showed changes in BMP species distribution. Thereby, a shift from long-chain to shorter-chain fatty acid composition in BMP species was detected.