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1.
Microb Cell Fact ; 21(1): 78, 2022 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-35527247

RESUMO

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 13C-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 13C-metabolic flux analyses in high-throughput.


Assuntos
2-Propanol , Corynebacterium glutamicum , 2-Propanol/metabolismo , Aminoácidos/metabolismo , Isótopos de Carbono/metabolismo , Corynebacterium glutamicum/metabolismo , Marcação por Isótopo/métodos
2.
Biotechnol J ; 16(9): e2100043, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34089621

RESUMO

BACKGROUND: Lignocellulosic biomass is the most abundant raw material on earth. Its efficient use for novel bio-based materials is essential for an emerging bioeconomy. Possible building blocks for such materials are the key TCA-cycle intermediates α-ketoglutarate and succinate. These organic acids have a wide range of potential applications, particularly in use as monomers for established or novel biopolymers. Recently, Corynebacterium glutamicum was successfully engineered and evolved towards an improved utilization of d-xylose via the Weimberg pathway, yielding the strain WMB2evo . The Weimberg pathway enables a carbon-efficient C5-to-C5 conversion of d-xylose to α-ketoglutarate and a shortcut route to succinate as co-product in a one-pot process. METHODS AND RESULTS: C. glutamicum WMB2evo was grown under dynamic microaerobic conditions on d-xylose, leading to the formation of comparably high amounts of succinate and only small amounts of α-ketoglutarate. Subsequent carbon isotope labeling experiments verified the targeted production route for both products in C. glutamicum WMB2evo . Fed-batch process development was initiated and the effect of oxygen supply and feeding strategy for a growth-decoupled co-production of α-ketoglutarate and succinate were studied in detail. The finally established fed-batch production process resulted in the formation of 78.4 mmol L-1 (11.45 g L-1 ) α-ketoglutarate and 96.2 mmol L-1 (11.36 g L-1 ) succinate. CONCLUSION: The developed one-pot process represents a promising approach for the combined supply of bio-based α-ketoglutarate and succinate. Future work will focus on tailor-made down-stream processing of both organic acids from the fermentation broth to enable their application as building blocks in chemical syntheses. Alternatively, direct conversion of one or both acids via whole-cell or cell-free enzymatic approaches can be envisioned; thus, extending the network of value chains starting from cheap and renewable d-xylose.


Assuntos
Corynebacterium glutamicum , Corynebacterium glutamicum/genética , Ácidos Cetoglutáricos , Engenharia Metabólica , Succinatos , Ácido Succínico , Xilose
3.
Eng Life Sci ; 20(8): 350-356, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32774207

RESUMO

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).

4.
Front Bioeng Biotechnol ; 8: 602936, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33553115

RESUMO

Wild-type C. glutamicum ATCC 13032 is known to possess two enzymes with anaplerotic (C4-directed) carboxylation activity, namely phosphoenolpyruvate carboxylase (PEPCx) and pyruvate carboxylase (PCx). On the other hand, C3-directed decarboxylation can be catalyzed by the three enzymes phosphoenolpyruvate carboxykinase (PEPCk), oxaloacetate decarboxylase (ODx), and malic enzyme (ME). The resulting high metabolic flexibility at the anaplerotic node compromises the unambigous determination of its carbon and energy flux in C. glutamicum wild type. To circumvent this problem we performed a comprehensive analysis of selected single or double deletion mutants in the anaplerosis of wild-type C. glutamicum under defined d-glucose conditions. By applying well-controlled lab-scale bioreactor experiments in combination with untargeted proteomics, quantitative metabolomics and whole-genome sequencing hitherto unknown, and sometimes counter-intuitive, genotype-phenotype relationships in these mutants could be unraveled. In comparison to the wild type the four mutants C. glutamiucm Δpyc, C. glutamiucm Δpyc Δodx, C. glutamiucm Δppc Δpyc, and C. glutamiucm Δpck showed lowered specific growth rates and d-glucose uptake rates, underlining the importance of PCx and PEPCk activity for a balanced carbon and energy flux at the anaplerotic node. Most interestingly, the strain C. glutamiucm Δppc Δpyc could be evolved to grow on d-glucose as the only source of carbon and energy, whereas this combination was previously considered lethal. The prevented anaplerotic carboxylation activity of PEPCx and PCx was found in the evolved strain to be compensated by an up-regulation of the glyoxylate shunt, potentially in combination with the 2-methylcitrate cycle.

5.
Anal Chem ; 91(21): 13407-13417, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31577133

RESUMO

Computational and experimental advances of recent years have culminated in establishing 13C-Metabolic Flux Analysis (13C-MFA) as a routine methodology to unravel the fluxome. As the acronym suggests, 13C-MFA has relied on the relative abundance of 13C-isotopes in metabolites for flux inference, most commonly measured by mass spectrometry. In this manuscript we expand the scope of labeling measurements to the case of simultaneous 13C- and 15N-labeling of amino acids. Analytically, the separation of isotopologues of this metabolite class can only be achieved at resolving power beyond 65,000. In this manuscript we harvest an overlooked property of the collision induced dissociation of amino acid adducts to discern 13C- and 15N- isotopologues of amino acids with a primary amine without separating them in the m/z domain.

6.
Microb Cell Fact ; 18(1): 71, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975146

RESUMO

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.


Assuntos
Corynebacterium glutamicum/metabolismo , Glicosídeos/biossíntese , Malonil Coenzima A/metabolismo , Engenharia Metabólica , Plantas/química , Acetil-CoA Carboxilase/metabolismo , Ciclo do Ácido Cítrico , Polifenóis/biossíntese , Ácido Pirúvico/metabolismo
7.
Biotechnol Bioeng ; 116(6): 1380-1391, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30684355

RESUMO

In recent years microorganisms have been engineered towards synthesizing interesting plant polyphenols such as flavonoids and stilbenes from glucose. Currently, the low endogenous supply of malonyl-CoA, indispensable for plant polyphenol synthesis, impedes high product titers. Usually, limited malonyl-CoA availability during plant polyphenol production is avoided by supplementing fatty acid synthesis-inhibiting antibiotics such as cerulenin, which are known to increase the intracellular malonyl-CoA pool as a side effect. Motivated by the goal of microbial polyphenol synthesis being independent of such expensive additives, we used rational metabolic engineering approaches to modulate regulation of fatty acid synthesis and flux into the tricarboxylic acid cycle (TCA cycle) in Corynebacterium glutamicum strains capable of flavonoid and stilbene synthesis. Initial experiments showed that sole overexpression of genes coding for the native malonyl-CoA-forming acetyl-CoA carboxylase is not sufficient for increasing polyphenol production in C. glutamicum. Hence, the intracellular acetyl-CoA availability was also increased by reducing the flux into the TCA cycle through reduction of citrate synthase activity. In defined cultivation medium, the constructed C. glutamicum strains accumulated 24 mg·L -1 (0.088 mM) naringenin or 112 mg·L -1 (0.49 mM) resveratrol from glucose without supplementation of phenylpropanoid precursor molecules or any inhibitors of fatty acid synthesis.


Assuntos
Corynebacterium glutamicum , Malonil Coenzima A , Engenharia Metabólica/métodos , Compostos Fitoquímicos , Polifenóis , Reatores Biológicos , Citrato (si)-Sintase/metabolismo , Ciclo do Ácido Cítrico/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Flavanonas , Malonil Coenzima A/análise , Malonil Coenzima A/genética , Malonil Coenzima A/metabolismo , Compostos Fitoquímicos/análise , Compostos Fitoquímicos/metabolismo , Polifenóis/análise , Polifenóis/metabolismo , Resveratrol
8.
Biotechnol J ; 14(9): e1800428, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30318833

RESUMO

With modern genetic engineering tools, high number of potentially improved production strains can be created in a short time. This results in a bottleneck in the succeeding step of bioprocess development, which can be handled by accelerating quantitative microbial phenotyping. Miniaturization and automation are key technologies to achieve this goal. In this study, a novel workflow for repeated low-volume sampling of BioLector-based cultivation setups is presented. Six samples of 20 µL each can be taken automatically from shaken 48-well microtiter plates without disturbing cell population growth. The volume is sufficient for quantification of substrate and product concentrations by spectrophotometric-based enzyme assays. From transient concentration data and replicate cultures, valid performance indicators (titers, rates, yields) are determined through process modeling and random error propagation analysis. Practical relevance of the workflow is demonstrated with a set of five genome-reduced Corynebacterium glutamicum strains that are engineered for Sec-mediated heterologous cutinase secretion. Quantitative phenotyping of this strain library led to the identification of a strain with a 1.6-fold increase in cutinase yield. The prophage-free strain carries combinatorial deletions of three gene clusters (Δ3102-3111, Δ3263-3301, and Δ3324-3345) of which the last two likely contain novel target genes to foster rational engineering of heterologous cutinase secretion in C. glutamicum.


Assuntos
Reatores Biológicos/microbiologia , Biomassa , Corynebacterium glutamicum/genética , Engenharia Genética/métodos , Família Multigênica/genética , Fenótipo
9.
Anal Bioanal Chem ; 409(9): 2309-2326, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28116490

RESUMO

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.

10.
Appl Microbiol Biotechnol ; 100(4): 1871-1881, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26610800

RESUMO

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 ß-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,ß-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, ß-oxidative deacetylation pathway, which is essential for the utilization of phenylpropanoids in C. glutamicum.


Assuntos
Derivados de Benzeno/metabolismo , Cinamatos/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Redes e Vias Metabólicas/genética , Família Multigênica , Carbono/metabolismo , Metabolismo Energético , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica
11.
Biotechnol Bioeng ; 113(3): 661-74, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26375179

RESUMO

Corynebacterium glutamicum is the major workhorse for the microbial production of several amino and organic acids. As long as these derive from tricarboxylic acid cycle intermediates, the activity of anaplerotic reactions is pivotal for a high biosynthetic yield. To determine single anaplerotic activities (13) C-Metabolic Flux Analysis ((13) C-MFA) has been extensively used for C. glutamicum, however with different network topologies, inconsistent or poorly determined anaplerotic reaction rates. Therefore, in this study we set out to investigate whether a focused isotopomer model of the anaplerotic node can at all admit a unique solution for all fluxes. By analyzing different scenarios of active anaplerotic reactions, we show in full generality that for C. glutamicum only certain anaplerotic deletion mutants allow to uniquely determine the anaplerotic fluxes from (13) C-isotopomer data. We stress that the result of this analysis for different assumptions on active enzymes is directly transferable to other compartment-free organisms. Our results demonstrate that there exist biologically relevant metabolic network topologies for which the flux distribution cannot be inferred by classical (13) C-MFA.


Assuntos
Aminoácidos/metabolismo , Isótopos de Carbono/metabolismo , Ácidos Carboxílicos/metabolismo , Corynebacterium glutamicum/metabolismo , Análise do Fluxo Metabólico , Redes e Vias Metabólicas
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