Riboflavin production with Ashbya gossypii: a 13C high-resolution metabolic network analysis under industrial process conditions

The fungus Ashbya gossypii is an important industrial producer of riboflavin, i.e. vitamin B2. Here, we developed and then used a highly sophisticated set-up of parallel 13C tracer studies with labeling analysis by GC/MS, LC/MS, 1D, and 2D NMR to resolve carbon fluxes and obtain a detailed picture of the underlying metabolism in the overproducing strain A. gossypii B2 during growth and riboflavin production from a complex industrial medium using vegetable oil as carbon source. Glycine was exclusively used as carbon-two – but not carbon-one (C1) – donor of the vitamin’s pyrimidine ring due to the proven absence of a functional glycine cleavage system. Yeast extract was the main carbon source during growth, while still contributing 8 % overall carbon to riboflavin. Overall carbon flux from rapeseed oil into riboflavin equaled 80 %. Transmembrane formate flux simulations revealed that the C1-supply displayed a severe bottleneck during the initial riboflavin production, which was overcome in later phases of the cultivation by intrinsic formate accumulation. The transiently limiting C1-pool was successfully replenished by time-resolved feeding of formate or serine. This increased the intracellular precursor availability and resulted in a riboflavin titer increase of 45 %. This study is the first that successfully sheds light on carbon fluxes during the growth and riboflavin production phase by use of 13C tracers and a complementary platform of analytical techniques.Der Pilz Ashbya gossypii ist ein wichtiger industrieller Produzent für Riboflavin. In dieser Studie wurde eine anspruchsvolle Kombination an parallelen 13C Tracerexperimenten entwickelt und durchgeführt, wobei die Markierungen mittels GC/MS, LC/MS, 1D und 2D NMR analysiert wurden, um die Kohlenstoffflüsse in dem Überproduzenten A. gossypii B2 sowohl im Wachstum als auch während der Riboflavinproduktion auf Komplexmedium mit Pflanzenöl als Kohlenstoffquelle, auflösen zu können. Glycin wurde ausschließlich als Zwei-Kohlenstoff-Donor – aber nicht C1-Donor – für den Pyrimidinring des Vitamins verwendet, was das Fehlen eines funktionalen Glycin-Decarboxylase-Komplexes bewies. Hefeextrakt (YE) war die wichtigste Kohlenstoffquelle während des Wachstums. Der Gesamtkohlenstofffluss von YE und Rüböl zu Riboflavin betrug entsprechend 8 % bzw. 80 %. Simulationen des transmembranen Formiatflusses zeigten, dass die C1-Bereitstellung während der frühen Riboflavinproduktion limitierend war, was in der späten Kultivierungsphase durch intrinsische Formiatbildung überwunden wurde. Der transient limitierende C1-Pool wurde erfolgreich durch die zeitaufgelöste Zugabe von Formiat oder Serin aufgefüllt. Dies steigerte den Produkttiter um 45 % durch erhöhte Verfügbarkeit von Vorläufermolekülen. Diese Studie ist die Erste, die Aufschluss über Stofflüsse während des Wachstums und der Riboflavinproduktion durch den Gebrauch von 13C Tracersubstanzen und komplementären analytischen Techniken gibt

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

Abstract Background The 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. Results During 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. Conclusion Intracellular 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

Improved riboflavin production with Ashbya gossypii from vegetable oil based on 13 C metabolic network analysis with combined labeling analysis by GC/MS, LC/MS, 1D, and 2D NMR

The fungus Ashbya gossypii is an important industrial producer of riboflavin, i.e. vitamin B-2. In order to meet the constantly increasing demands for improved production processes, it appears essential to better understand the underlying metabolic pathways of the vitamin. Here, we used a highly sophisticated set-up of parallel C-13 tracer studies with labeling analysis by GC/MS, LC/MS, 1D, and 2D NMR to resolve carbon fluxes in the overproducing strain A. gossypii B2 during growth and subsequent riboflavin production from vegetable oil as carbon source, yeast extract, and supplemented glycine. The studies provided a detailed picture of the underlying metabolism. Glycine was exclusively used as carbon-two donor of the vitamin's pyrimidine ring, which is part of its iso-alloxazine ring structure, but did not contribute to the carbon-one metabolism due to the proven absence of a functional glycine cleavage system. The pools of serine and glycine were closely connected due to a highly reversible serine hydroxymethyltransferase. Transmembrane formate flux simulations revealed that the one-carbon metabolism displayed a severe bottleneck during initial riboflavin production, which was overcome in later phases of the cultivation by intrinsic formate accumulation. The transiently limiting carbon-one pool was successfully replenished by time-resolved feeding of small amounts of formate and serine, respectively. This increased the intracellular availability of glycine, serine, and formate and resulted in a final riboflavin titer increase of 45%