Itaconic acid production by co-feeding of Ustilago maydis: A combined approach of experimental data, design of experiments, and metabolic modeling.

Itaconic acid is a platform chemical with a range of applications in polymer synthesis and is also discussed for biofuel production. While produced in industry from glucose or sucrose, co-feeding of glucose and acetate was recently discussed to increase itaconic acid production by the smut fungus Ustilago maydis. In this study, we investigate the optimal co-feeding conditions by interlocking experimental and computational methods. Flux balance analysis indicates that acetate improves the itaconic acid yield up to a share of 40% acetate on a carbon molar basis. A design of experiment results in the maximum yield of 0.14 itaconic acid per carbon source from 100 g L - 1 $\,\text{g L}{}^{-1}$ glucose and 12 g L - 1 $\,\text{g L}{}^{-1}$ acetate. The yield is improved by around 22% when compared to feeding of glucose as sole carbon source. To further improve the yield, gene deletion targets are discussed that were identified using the metabolic optimization tool OptKnock. The study contributes ideas to reduce land use for biotechnology by incorporating acetate as co-substrate, a C2-carbon source that is potentially derived from carbon dioxide.

Co-feeding enhances the yield of methyl ketones.

The biotechnological production of methyl ketones is a sustainable alternative to fossil-derived chemical production. To date, the best host for microbial production of methyl ketones is a genetically engineered Pseudomonas taiwanensis VLB120 ∆6 pProd strain, achieving yields of 101 mgg-1 on glucose in batch cultivations. For competitiveness with the petrochemical production pathway, however, higher yields are necessary. Co-feeding can improve the yield by fitting the carbon-to-energy ratio to the organism and the target product. In this work, we developed co-feeding strategies for P. taiwanensis VLB120 ∆6 pProd by combined metabolic modeling and experimental work. In a first step, we conducted flux balance analysis with an expanded genome-scale metabolic model of iJN1463 and found ethanol as the most promising among five cosubstrates. Next, we performed cultivations with ethanol and found the highest reported yield in batch production of methyl ketones with P. taiwanensis VLB120 to date, namely, 154 mg g-1 methyl ketones. However, ethanol is toxic to the cell, which reflects in a lower substrate consumption and lower product concentrations when compared to production on glucose. Hence, we propose cofeeding ethanol with glucose and find that, indeed, higher concentrations than in ethanol-fed cultivation (0.84 g Laq-1 with glucose and ethanol as opposed to 0.48 g Laq-1 with only ethanol) were achieved, with a yield of 85 mg g-1. In a last step, comparing experimental with computational results suggested the potential for improving the methyl ketone yield by fed-batch cultivation, in which cell growth and methyl ketone production are separated into two phases employing optimal ethanol to glucose ratios. ONE-SENTENCE SUMMARY: By combining computational and experimental work, we demonstrate that feeding ethanol in addition to glucose improves the yield of biotechnologically produced methyl ketones.