Towards bio-upcycling of polyethylene terephthalate

■ CONTENTS Established Concepts of Quantification in Metabolomics/Lipidomics 521 Recommended Absolute Quantification Approaches 521 Stable Isotope Labeling 522 Enrichment Degree and Isotopologue Distribution 522 Suite of In Vivo Synthesized Isotopically Labeled Materials 523 Applications of Stable Isotope Labeled Biomass 523 Credentialing: Isotopically Labeled Biomass for Identification 523 Isotopically Labeled Biomass for Validated Isotopologue Distribution Elucidations 524 Isotopically Labeled Biomass for Quantification 524 Harmonization and Reference Materials 526 Reference Materials and Interlaboratory Comparisons 526 Community-Based Guidelines in Metabolomics 526 Quality Control and Benchmarking 527 Nontargeted Data AnalysisIncreasing Quality by Multiple Lines of Evidence 527 Data Preprocessing 527 Elimination of Redundancies and Noise from Feature Tables 528 The Annotation Task 528 Annotation in the Field of Lipidomics 529 Retention Time and Cross Section as Orthogonal Parameter in Nontargeted Analysis 529 Retention Time for Annotation 529 Collision Cross Section Value for Compound Annotation 530 Navigating the Conflicting Goals of Metabolome Coverage and Throughput 530 Sample Preparation 530 Direct Analysis in Metabolomics and Lipidomics 531 Flow Injection-MS 531 Ultimate Throughput−Duty Cycles of Seconds Per Sample 531 ChromatographyKey Steps Toward Coverage and Throughput 532 Miniaturization of Liquid Chromatography 532 Multidimensional Chromatography 533 Dual/Parallel Chromatography 534 MS Platforms and Data Acquisition Strategies Improving Coverage, Selectivity, and Reliability 534 Toward MS-Based Multi-omics 534 Multi-omics Sample Preparation Strategies 534 Multiplatform Analysis Strategies for Metabolomics and Lipidomics 535 Merging Metabolomics and Lipidomics 536 Network Analysis and Visualization of Multiomics Workflows 536 Conclusions 538 Author Information 538 Corresponding Author 538 Authors 538 Notes 538 Biographies 538 Acknowledgments 539 References 539

Recurrent Topics in Mass Spectrometry-Based Metabolomics and Lipidomics-Standardization, Coverage, and Throughput.

Rhamnolipids are biosurfactants produced by microorganisms with the potential to replace synthetic compounds with petrochemical origin. Possible industrial applications range from pharmaceuticals to bioremediation agents. As the challenges to intensify recombinant rhamnolipid production from sugars are manifold, multidisciplinary approaches are required. Previously, P. putida KT2440 was developed as heterologous platform strain for rhamnolipid synthesis. Based on this, a multidisciplinary approach towards developing a sustainable rhamnolipid production process is presented. This includes strain development and physiological characterization, improvement of cultivation conditions, and downstream processing as well as life cycle assessment (LCA). Newly developed expression cassettes for stable integration of the rhamnolipid biosynthesis genes into the genome outperformed plasmid-based expression systems. Furthermore, genetic stability of the production strain was improved by using an inducible promoter. To enhance rhamnolipid synthesis, energy and/or carbon consuming traits were removed: Mutants negative for the synthesis of the flagellar machinery or the storage polymer PHA showed increased production by 50 %. A scale-up from shake-flasks was carried out using a 1 L bioreactor. By recycling of the foam, biomass loss could be minimized and a rhamnolipid titer of up to 1.5 g/L was achieved without using mechanical foam destroyers or anti foaming agents. The usage of a suitable minimal medium reduced undesired interphase formation in the liquid-liquid extraction step. To assess the relevant system variants by their environmental impacts, a technical scale production process was designed (150 L fermentation volume) and evaluated performing LCA. The process chains and their specific environmental impact were examined. It was found that next to biomass supply, the fermentation had the biggest environmental impact. The results are discussed in the context of the challenges of microbial biosurfactant production using hydrophilic substrates on an industrial scale, and how a multidisciplinary approach can be guided by early process evaluations.

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Integration of Genetic and Process Engineering for Optimized Rhamnolipid Production Using Pseudomonas putida.

Lipids represent a very diverse group of compounds with a high variety of physicochemical properties determining their functional activities. For omics-wide identification of lipid species from complex biological samples, several crucial analytical steps including extraction, chromatographic separation and mass spectrometry analysis need to be carefully considered and validated. Here we review applications of three main chromatography techniques—reversed phase, normal phase and hydrophilic interaction liquid chromatography—for analysis of complex natural lipidomes aiming to uncover the diversity of lipid species. To correlate lipid separation with their physicochemical properties, lipid chemical space was reconstructed and used to explain principles underlying different chromatographic techniques. Furthermore, examples of available methods for analysis of complex natural lipidomes characterized by high diversity and dynamic range of lipid concentrations are illustrated for adipose tissue lipidomics.

Liquid Chromatography Techniques in Lipidomics Research

Glycerophospholipids with two, non-equivalent fatty acyl chains can adopt one of two isomeric forms depending on the relative position of substitutions on the glycerol backbone. These so-called sn-positional isomers can have distinct biophysical and biochemical behaviors making it desirable to uniquely assign their regiochemistries. Unambiguous assignment of such similar molecular structures in complex biological extracts is a significant challenge to current analytical technologies. We have recently reported a novel mass spectrometric method that combines collision- and ozone-induced dissociation in series (CID/OzID) to yield product ions characteristic of acyl chain substitution patterns in glycerophospholipids. Here phosphatidylcholines are examined using the CID/OzID protocol combined with desorption electrospray ionization (DESI) to facilitate the rapid exploration of sample arrays comprised of a wide variety of synthetic and biological sources. Comparison of the spectra acquired from different extracts reveals that the sn-positional isomers PC 16:0/18:1 and PC 18:1/16:0 (where the 18:1 chain is present at the sn-2 and sn-1 position of the glycerol backbone, respectively) are most often found together in lipids of either natural or synthetic origin. Moreover, the proportions of the two isomers vary significantly between extracts from different organisms or even between adjacent tissues from the same organism.

A rapid ambient ionization-mass spectrometry approach to monitoring the relative abundance of isomeric glycerophospholipids

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.

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

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

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.

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

Rationale Lipids are a group of biomolecules known for their importance in a broad variety of biological tasks, which is attributed to the diversity of their molecular structures. The exact position of the double bond in a lipid affects its biological properties. Therefore, the determination of its position is highly important. Nevertheless, the unambiguous assignment is still a challenging task and requires the development of new approaches. Methods A novel method for pinpointing double-bond positions in lipids is presented based on the photochemical Paternὸ-Buchi reaction. Therefore, the reaction was performed using a post-column derivatization by ultraviolet (UV) light irradiation (254 nm) and an additional constant flow of acetone. The acetone adduct was formed due to the reaction of acetone with the double bond resulting in an oxetane. Subsequent fragmentation experiments generated diagnostic fragments allowing the localization of double-bond positions. Results Intending online hyphenation with high-performance liquid chromatography (HPLC)/tandem mass spectrometry (MS/MS), the reaction was optimized regarding parameters such as reaction time and the influence of trace oxygen as well as solvents. An additional separation of the reaction products by means of HPLC enabled further characterization of the chemical structures formed by UV irradiation. To demonstrate the applicability of the developed method, a mixture of the constitutional phospholipid isomers bis(monooleoylglycero) phosphate and dioleoyl-phosphatidylglycerol was analyzed regarding their double-bond positions, and lipids from an extract of the green alga Chlamydomonas reinhardtii were investigated. Conclusions The successful hyphenation of HPLC/MS/MS with the online reaction was demonstrated for localization of double bonds in single lipids, lipid mixtures, and complex biological samples. Matrix effects can be reduced and further structural information, for example in case of constitutional isomers, can be obtained. This opens up new avenues for pinpointing lipid double-bond positions even in complex samples.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/rcm.8262

Localization of double-bond positions in lipids by tandem mass spectrometry succeeding high-performance liquid chromatography with post-column derivatization

https://chemistry-europe.onlinelibrary.wiley.com/doi/pdf/10.1002/ansa.20190009

Christian Vosse

Papers

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

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

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

Localization of double-bond positions in lipids by tandem mass spectrometry succeeding high-performance liquid chromatography with post-column derivatization

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