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The Design of FluxML: A Universal Modeling Language for ¹³C Metabolic Flux Analysis.

ABSTRACT: ¹³C metabolic flux analysis (MFA) is the method of choice when a detailed inference of intracellular metabolic fluxes in living organisms under metabolic quasi-steady state conditions is desired. Being continuously developed since two decades, the technology made major contributions to the quantitative characterization of organisms in all fields of biotechnology and health-related research. ¹³C MFA, however, stands out from other "-omics sciences," in that it requires not only experimental-analytical data, but also mathematical models and a computational toolset to infer the quantities of interest, i.e., the metabolic fluxes. At present, these models cannot be conveniently exchanged between different labs. Here, we present the implementation-independent model description language FluxML for specifying ¹³C MFA models. The core of FluxML captures the metabolic reaction network together with atom mappings, constraints on the model parameters, and the wealth of data configurations. In particular, we describe the governing design processes that shaped the FluxML language. We demonstrate the utility of FluxML to represent many contemporary experimental-analytical requirements in the field of ¹³C MFA. The major aim of FluxML is to offer a sound, open, and future-proof language to unambiguously express and conserve all the necessary information for model re-use, exchange, and comparison. Along with FluxML, several powerful computational tools are supplied for easy handling, but also to maintain a maximum of flexibility. Altogether, the FluxML collection is an "all-around carefree package" for ¹³C MFA modelers. We believe that FluxML improves scientific productivity as well as transparency and therewith contributes to the efficiency and reproducibility of computational modeling efforts in the field of ¹³C MFA.

SUBMITTER: Beyß M

PROVIDER: S-EPMC6543931 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundBioprocessing offers a sustainable and green approach to manufacture various chemicals and materials. Development of bioprocesses requires transforming common producer strains to cell factories. 13C metabolic flux analysis (13C-MFA) can be applied to identify relevant targets to accomplish the desired phenotype, which has become one of the major tools to support systems metabolic engineering. In this research, we applied 13C-MFA to identify bottlenecks in the bioconversion of glycerol into acetol by Escherichia coli. Valorization of glycerol, the main by-product of biodiesel, has contributed to the viability of biofuel economy.ResultsWe performed 13C-MFA and measured intracellular pyridine nucleotide pools in a first-generation acetol producer strain (HJ06) and a non-producer strain (HJ06C), and identified that engineering the NADPH regeneration is a promising target. Based on this finding, we overexpressed nadK encoding NAD kinase or pntAB encoding membrane-bound transhydrogenase either individually or in combination with HJ06, obtaining HJ06N, HJ06P and HJ06PN. The step-wise approach resulted in increasing the acetol titer from 0.91 g/L (HJ06) to 2.81 g/L (HJ06PN). To systematically characterize and the effect of mutation(s) on the metabolism, we also examined the metabolomics and transcriptional levels of key genes in four strains. The pool sizes of NADPH, NADP+ and the NADPH/NADP+ ratio were progressively increased from HJ06 to HJ06PN, demonstrating that the sufficient NADPH supply is critical for acetol production. Flux distribution was optimized towards acetol formation from HJ06 to HJ06PN: (1) The carbon partitioning at the DHAP node directed gradually more carbon from the lower glycolytic pathway through the acetol biosynthetic pathway; (2) The transhydrogenation flux was constantly increased. In addition, 13C-MFA showed the rigidity of upper glycolytic pathway, PP pathway and the TCA cycle to support growth. The flux patterns were supported by most metabolomics data and gene expression profiles.ConclusionsThis research demonstrated how 13C-MFA can be applied to drive the cycles of design, build, test and learn implementation for strain development. This succeeding engineering strategy can also be applicable for rational design of other microbial cell factories.

Project description:BackgroundThe fungus Ashbya gossypii is an important industrial producer of the vitamin riboflavin. Using this microbe, riboflavin is manufactured in a two-stage process based on a rich medium with vegetable oil, yeast extract and different precursors: an initial growth and a subsequent riboflavin production phase. So far, our knowledge on the intracellular metabolic fluxes of the fungus in this complex process is limited, but appears highly relevant to better understand and rationally engineer the underlying metabolism. To quantify intracellular fluxes of growing and riboflavin producing A. gossypii, studies with different 13C tracers were embedded into a framework of experimental design, isotopic labeling analysis by MS and NMR techniques, and model-based data processing. The studies included the use 13C of yeast extract, a key component used in the process.ResultsDuring growth, the TCA cycle was found highly active, whereas the cells exhibited a low flux through gluconeogenesis as well as pentose phosphate pathway. Yeast extract was the main carbon donor for anabolism, while vegetable oil selectively contributed to the proteinogenic amino acids glutamate, aspartate, and alanine. During the subsequent riboflavin biosynthetic phase, the carbon flux through the TCA cycle remained high. Regarding riboflavin formation, most of the vitamin's carbon originated from rapeseed oil (81?±?1%), however extracellular glycine and yeast extract also contributed with 9?±?0% and 8?±?0%, respectively. In addition, advanced yeast extract-based building blocks such as guanine and GTP were directly incorporated into the vitamin.ConclusionIntracellular carbon fluxes for growth and riboflavin production on vegetable oil provide the first flux insight into a fungus on complex industrial medium. The knowledge gained therefrom is valuable for further strain and process improvement. Yeast extract, while being the main carbon source during growth, contributes valuable building blocks to the synthesis of vitamin B2. This highlights the importance of careful selection of the right yeast extract for a process based on its unique composition.

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The Design of FluxML: A Universal Modeling Language for ¹³C Metabolic Flux Analysis.

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The Design of FluxML: A Universal Modeling Language for 13C Metabolic Flux Analysis.

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OmicsDI is part of the ELIXIR infrastructure

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The Design of FluxML: A Universal Modeling Language for ¹³C Metabolic Flux Analysis.