Tandem-Mass-Spectrometry-Driven Investigation of the Anaplerotic Reactions in Corynebacterium glutamicum

The anaplerotic reactions form the basis of a circular reaction network around the metabolites phosphoenolpyruvate, pyruvate, oxaloacetate, and malate, comprising specific carboxylation and decarboxylation reactions which link glycolysis to the tricarboxylic acid cycle. The net carboxylation activity of anaplerotic reactions is essential for the replenishment of tricarboxylic acid cycle intermediates and therefore directly limits the biosynthetic flux towards amino acids derived from it, most notable L-lysine and L-glutamate. The microbial production of these amino acids is primarily accounted for by the biotechnological usage of Corynebacterium glutamicum, a major workhorse of industrial biotechnology and model organism of systems biology alike. The pivotal role of anaplerotic reactions in the biotechnological usage of C. glutamicum motivates the present thesis which is dedicated to a thorough study of these reactions in this organism. First, a theoretical analysis establishes the reason for the unsuccessfully determined anaplerotic reaction activities by 13C-Metabolic Flux Analysis (13C-MFA) in previous studies. Using a focused isotopomer network of the anaplerotic node, it is shown in full generality that for C. glutamicum only certain anaplerotic deletion mutants allow to uniquely determine the anaplerotic fluxes from stationary 13C-isotopomer data. This result prompted the investigation of a set of single and double deletion mutants whose metabolome was investigated by a protocol employing a liquid chromatography electrospray ionization triple quadrupole mass spectrometry (LC-ESI-QqQ MS) platform. By means of quadrupole-time-of-flight technology (QqTOF) a proteome data set for the investigated deletion mutants was generated and evaluated with a novel regression approach, yielding precise fold changes for almost a third of all open reading frames of C. glutamicum. A major biological finding arising from the total data set consists in the proof that the glyoxylate shunt can be active as anaplerotic reaction in C. glutamicum under solely glycolytic conditions, which is brought about by a metabolite-regulator interaction. The finding of inconsistently estimated fluxes in a 13C-Metabolic Flux Analysis of the investigated strains prompted the development of a novel LC-ESI-QqTOF MS-based 13C-profiling method, which harvests the collision induced dissociation (CID) of product anions of central metabolism as well as product cations of amino acids for the positional resolution of 13C-label enrichment. For this purpose, accurate mass spectrometry, selectively-labeled standards, and published fragmentation pathways are used to structurally annotate all dominant mass peaks of a large collection of metabolites, resulting in the most detailed map of the carbon atom fate of LC-ESI-MS/MS collisional fragments yet. Apart from providing positional resolution of 13C-enrichment, it is shown that the CID of amino acids can be harvested to separate 13C- and 15N-isotopic species of these metabolites without resolution in the m/z domain

Tailoring Corynebacterium glutamicum towards increased malonyl-CoA availability for efficient synthesis of the plant pentaketide noreugenin

Background:In the last years, different biotechnologically relevant microorganisms have been engineered for the synthesis of plant polyphenols such as flavonoids and stilbenes. However, low intracellular availability of malonyl-CoA as essential precursor for most plant polyphenols of interest is regarded as the decisive bottleneck preventing high product titers.Results:In this study, Corynebacterium glutamicum, which emerged as promising cell factory for plant polyphenol production, was tailored by rational metabolic engineering towards providing significantly more malonyl-CoA for product synthesis. This was achieved by improving carbon source uptake, transcriptional deregulation of accBC and accD1 encoding the two subunits of the acetyl-CoA carboxylase (ACC), reduced flux into the tricarboxylic acid (TCA) cycle, and elimination of anaplerotic carboxylation of pyruvate. The constructed strains were used for the synthesis of the pharmacologically interesting plant pentaketide noreugenin, which is produced by plants such as Aloe arborescens from five molecules of malonyl-CoA. In this context, accumulation of the C1/C6 cyclized intermediate 1-(2,4,6-trihydroxyphenyl)butane-1,3-dione (TPBD) was observed, which could be fully cyclized to the bicyclic product noreugenin by acidification.Conclusion:The best strain C. glutamicum Nor2 C5 mufasOBCD1 PO6-iolT1 ∆pyc allowed for synthesis of 53.32 mg/L (0.278 mM) noreugenin in CGXII medium supplemented with casamino acids within 24 h

Comprehensive and accurate tracking of carbon origin of LC-tandem mass spectrometry collisional fragments for 13C-MFA

In recent years the benefit of measuring positionally resolved 13C-labeling enrichment from tandem mass spectrometry (MS/MS) collisional fragments for improved precision of 13C-Metabolic Flux Analysis (13C-MFA) has become evident. However, the usage of positional labeling information for 13C-MFA faces two challenges: (1) The mass spectrometric acquisition of a large number of potentially interfering mass transitions may hamper accuracy and sensitivity. (2) The positional identity of carbon atoms of product ions needs to be known. The present contribution addresses the latter challenge by deducing the maximal positional labeling information contained in LC-ESI-MS/MS spectra of product anions of central metabolism as well as product cations of amino acids. For this purpose, we draw on accurate mass spectrometry, selectively labeled standards, and published fragmentation pathways to structurally annotate all dominant mass peaks of a large collection of metabolites, some of which with a complete fragmentation pathway. Compiling all available information, we arrive at the most detailed map of carbon atom fate of LC-ESI-MS/MS collisional fragments yet, comprising 170 intense and structurally annotated product ions with unique carbon origin from 76 precursor ions of 72 metabolites. Our 13C-data proof that heuristic fragmentation rules often fail to yield correct fragment structures and we expose common pitfalls in the structural annotation of product ions. We show that the positionally resolved 13C-label information contained in the product ions that we structurally annotated allows to infer the entire isotopomer distribution of several central metabolism intermediates, which is experimentally demonstrated for malate using quadrupole-time-of-flight MS technology. Finally, the inclusion of the label information from a subset of these fragments improves flux precision in a Corynebacterium glutamicum model of the central carbon metabolism

Hot isopropanol quenching procedure for automated microtiter plate scale 13C-labeling experiments

BACKGROUND: Currently, the generation of genetic diversity for microbial cell factories outpaces the screening of strain variants with omics-based phenotyping methods. Especially isotopic labeling experiments, which constitute techniques aimed at elucidating cellular phenotypes and supporting rational strain design by growing microorganisms on substrates enriched with heavy isotopes, suffer from comparably low throughput and the high cost of labeled substrates. RESULTS: We present a miniaturized, parallelized, and automated approach to (13)C-isotopic labeling experiments by establishing and validating a hot isopropanol quenching method on a robotic platform coupled with a microbioreactor cultivation system. This allows for the first time to conduct automated labeling experiments at a microtiter plate scale in up to 48 parallel batches. A further innovation enabled by the automated quenching method is the analysis of free amino acids instead of proteinogenic ones on said microliter scale. Capitalizing on the latter point and as a proof of concept, we present an isotopically instationary labeling experiment in Corynebacterium glutamicum ATCC 13032, generating dynamic labeling data of free amino acids in the process. CONCLUSIONS: Our results show that a robotic liquid handler is sufficiently fast to generate informative isotopically transient labeling data. Furthermore, the amount of biomass obtained from a sub-milliliter cultivation in a microbioreactor is adequate for the detection of labeling patterns of free amino acids. Combining the innovations presented in this study, isotopically stationary and instationary automated labeling experiments can be conducted, thus fulfilling the prerequisites for (13)C-metabolic flux analyses in high-throughput. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-022-01806-4

Identification of the phd gene cluster responsible for phenylpropanoid utilization in Corynebacterium glutamicum

Phenylpropanoids as abundant, lignin-derived compounds represent sustainable feedstocks for biotechnological production processes. We found that the biotechnologically important soil bacterium Corynebacterium glutamicum is able to grow on phenylpropanoids such as p-coumaric acid, ferulic acid, caffeic acid, and 3-(4-hydroxyphenyl)propionic acid as sole carbon and energy sources. Global gene expression analyses identified a gene cluster (cg0340-cg0341 and cg0344-cg0347), which showed increased transcription levels in response to phenylpropanoids. The gene cg0340 (designated phdT) encodes for a putative transporter protein, whereas cg0341 and cg0344-cg0347 (phdA-E) encode enzymes involved in the β-oxidation of phenylpropanoids. The phd gene cluster is transcriptionally controlled by a MarR-type repressor encoded by cg0343 (phdR). Cultivation experiments conducted with C. glutamicum strains carrying single-gene deletions showed that loss of phdA, phdB, phdC, or phdE abolished growth of C. glutamicum with all phenylpropanoid substrates tested. The deletion of phdD (encoding for putative acyl-CoA dehydrogenase) additionally abolished growth with the α,β-saturated phenylpropanoid 3-(4-hydroxyphenyl)propionic acid. However, the observed growth defect of all constructed single-gene deletion strains could be abolished through plasmid-borne expression of the respective genes. These results and the intracellular accumulation of pathway intermediates determined via LC-ESI-MS/MS in single-gene deletion mutants showed that the phd gene cluster encodes for a CoA-dependent, β-oxidative deacetylation pathway, which is essential for the utilization of phenylpropanoids in C. glutamicu

Parallelized disruption of prokaryotic and eukaryotic cells via miniaturized and automated bead mill

The application of integrated microbioreactor systems is rapidly becoming of more interest to accelerate strain characterization and bioprocess development. However, available high‐throughput screening capabilities are often limited to target extracellular compounds only. Consequently, there is a great demand for automated technologies allowing for miniaturized and parallel cell disruption providing access to intracellular measurements. In this study, a fully automated bead mill workflow was developed and validated for four different industrial platform organisms: Escherichia coli , Corynebacterium glutamicum , Saccharomyces cerevisiae , and Aspergillus niger . The workflow enables up to 48 parallel cell disruptions in microtiter plates and is applicable at‐line to running lab‐scale cultivations. The resulting cell extracts form the basis for quantitative omics studies where no rapid metabolic quenching is required (e.g., genomics and proteomics)