Metabolic flux analysis in Ashbya gossypii using 13C-labeled yeast extract: industrial riboflavin production under complex nutrient conditions

Abstract

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.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-1003-y) contains supplementary material, which is available to authorized users.

Fermentation of plant-based milk alternatives for improved flavour and nutritional value

“…Due to the enormous progress in the field, quantitative systems biology studies of mixed-culture fermentations of plant-based milk alternatives could become a next level of research to better understand the underlying physiological, cellular and molecular processes. The system to be studied is admittedly complex, but seminal studies on similarly complex fermentation processes involving cocoa fermentation (Adler et al 2013), oil-based riboflavin production (Schwechheimer et al 2018), growth on substrate mixtures (Schilling et al 2007) and under environmental changes (Hou et al 2000; Kohajdová et al 2006; Kohlstedt et al 2014; Wittmann et al 2007) are encouraging success stories, which demonstrate the power of systems biology to shed more light on this subject and provide valuable guidance for improvement. It can be expected that similar systems level studies, which unravel genomics, transcriptomics, proteomics, metabolomics, and fluxomics in multi-omics approaches, will significantly contribute to a better understanding of plant material fermentation and advance rational designs and improvements in this field.…”

Production of riboflavin and related cofactors by biotechnological processes

“…Despite the fact that industrial RF synthesis is exclusively achieved by fermentation, most of the strains used in the industrial field were isolated by mutagenesis, which made their genomic background ambiguous. Researchers have tried to identify the genes and genetic elements responsible for the RF overproduction phenotype through different techniques, including transcriptome analysis [117] and screening of transposon-tagged mutants [103], as well as the more recently reported integrated whole-genome and transcriptome sequence analysis [118] and metabolic flux analysis [145,146]. Novel "omics" techniques such as metabolomics may prove useful in revealing further details of the flavin overproduction mechanism.…”

Production of Vitamin B2 (Riboflavin) by Microorganisms: An Overview

“…However, for overproduction, A. gossypii prefers the use of plant oils (corn or soybean), which are obtained as decomposing fatty acids and glycerol by extracellular lipases. In A. gossypii , fatty acids are oxidized into acetyl-CoA (ß-oxidation pathway), then converted into malate through the glyoxylate shunt to enter gluconeogenesis and serve together with the immediate precursor GTP as carbon donors for riboflavin ( Schwechheimer et al, 2018 ). Industrial waste materials, such as oil discharged by oil refinery plants, grape-must, beet molasses, peanut seed cake, and whey, have also been employed in riboflavin production but with limited success.…”