Fumarate production with Rhizopus oryzae: utilising the Crabtree effect to minimise ethanol by-product formation
Authors: Swart Reuben M. , le Roux Francois , Naude Andre , de Jongh Nicolaas W. , Nicol Willie 
Published: February 2020

Abstract
Background
The four-carbon dicarboxylic acids of the tricarboxylic acid cycle (malate, fumarate and succinate) remain promising bio-based alternatives to various precursor chemicals derived from fossil-based feed stocks. The double carbon bond in fumarate, in addition to the two terminal carboxylic groups, opens up an array of downstream reaction possibilities, where replacement options for petrochemical derived maleic anhydride are worth mentioning. To date the most promising organism for producing fumarate is Rhizopus oryzae (ATCC 20344, also referred to as Rhizopus delemar) that naturally excretes fumarate under nitrogen-limited conditions. Fumarate excretion in R. oryzae is always associated with the co-excretion of ethanol, an unwanted metabolic product from the fermentation. Attempts to eliminate ethanol production classically focus on enhanced oxygen availability within the mycelium matrix. In this study our immobilised R. oryzae process was employed to investigate and utilise the Crabtree characteristics of the organism in order to establish the limits of ethanol by-product formation under growth and non-growth conditions.

Results
All fermentations were performed with either nitrogen excess (growth phase) or nitrogen limitation (production phase) where medium replacements were done between the growth and the production phase. Initial experiments employed excess glucose for both growth and production, while the oxygen partial pressure was varied between a dissolved oxygen of 18.4% and 85%. Ethanol was formed during both growth and production phases and the oxygen partial pressure had zero influence on the response. Results clearly indicated that possible anaerobic zones within the mycelium were not responsible for ethanol formation, hinting that ethanol is formed under fully aerobic conditions as a metabolic overflow product. For Crabtree-positive organisms like Saccharomyces cerevisiae ethanol overflow is manipulated by controlling the glucose input to the fermentation. The same strategy was employed for R. oryzae for both growth and production fermentations. It was shown that all ethanol can be eliminated during growth for a glucose addition rate of $$0.07\,\hbox {g}\,\hbox {L}^{-1}\,\hbox {h}^{-1}$$0.07gL-1h-1. The production phase behaved in a similar manner, where glucose addition of $$0.197\,\hbox {g}\,\hbox {L}^{-1}\,\hbox {h}^{-1}$$0.197gL-1h-1 resulted in fumarate production of $$0.150\,\hbox {g}\,\hbox {L}^{-1}\,\hbox {h}^{-1}$$0.150gL-1h-1 and a yield of $$0.802\,\hbox {g}\,\hbox {g}^{-1}$$0.802gg-1 fumarate on glucose. Further investigation into the effect of glucose addition revealed that ethanol overflow commences at a glucose addition rate of $$0.395\,\hbox {g}\,\hbox {g}^{-1}\,\hbox {h}^{-1}$$0.395gg-1h-1 on biomass, while the maximum glucose uptake rate was established to be between 0.426 and $$0.533\,\hbox {g}\,\hbox {g}^{-1}\,\hbox {h}^{-1}$$0.533gg-1h-1.

Conclusions
The results conclusively prove that R. oryzae is a Crabtree-positive organism and that the characteristic can be utilised to completely discard ethanol by-product formation. A state referred to as “homofumarate production” was illustrated, where all carbon input exits the cell as either fumarate or respiratory $$\hbox {CO}_{2}$$CO2. The highest biomass-based “homofumarate production”: rate of $$0.243\,\hbox {g}\,\hbox {g}^{-1}\,\hbox {h}^{-1}$$0.243gg-1h-1 achieved a yield of $$0.802\,\hbox {g}\,\hbox {g}^{-1}$$0.802gg-1 on glucose, indicating the bounds for developing an ethanol free process. The control strategy employed in this study in conjunction with the uncomplicated scalability of the immobilised process provides new direction for further developing bio-fumarate production.