An Optimized Ustilago maydis for Itaconic Acid Production at Maximal Theoretical Yield

Abstract

Ustilago maydis, a member of the Ustilaginaceae family, is a promising host for the production of several metabolites including itaconic acid. This dicarboxylate has great potential as a bio-based building block in the polymer industry, and is of special interest for pharmaceutical applications. Several itaconate overproducing Ustilago strains have been generated by metabolic and morphology engineering. This yielded stabilized unicellular morphology through fuz7 deletion, reduction of by-product formation through deletion of genes responsible for itaconate oxidation and (glyco)lipid production, and the overexpression of the regulator of the itaconate cluster ria1 and the mitochondrial tricarboxylate transporter encoded by mttA from Aspergillus terreus. In this study, itaconate production was further optimized by consolidating these different optimizations into one strain. The combined modifications resulted in itaconic acid production at theoretical maximal yield, which was achieved under biotechnologically relevant fed-batch fermentations with continuous feed.

Keywords: U. maydis; fungi; itaconic acid; metabolic engineering; yeast.

Figure 1

System Duetz^® cultivation of morphology-engineered U. maydis MB215 strains in modified Tabuchi medium (MTM) with 50 g L⁻¹ glucose and 100 mM (2-(N-morpholino) ethanesulfonic acid (MES), incubated in 24-well plates with a filling volume of 1.5 mL (shaking diameter = 50 mm, n = 300 rpm, T = 30 °C and Φ = 80%). (A) Concentrations of itaconate (continuous lines) and glucose (dotted lines), (B) optical density measured at a wavelength of 600 nm (OD_600, continuous lines) and pH (dotted lines) of U. maydis MB215 ∆cyp3 ∆MEL ∆UA ∆dgat ∆P_ria1::P_etef (▲) and the same strain with additional fuz7 deletion (●). Error bars indicate the standard error of the mean (n = 3).

Figure 2

System Duetz^® cultivation of U. maydis MB215 strains expressing mttA in MTM with 50 g L⁻¹ glucose and 100 mM MES, incubated in 24-well plates with a filling volume of 1.5 mL (shaking diameter = 50 mm, n = 300 rpm, T = 30 °C and Φ = 80%). Itaconate production (continuous lines) and glucose consumption (dotted lines) (A) are shown, as well as OD₆₀₀ (continuous lines) and pH (dotted lines) (B) of U. maydis MB215 ∆cyp3 ∆MEL ∆UA ∆dgat ∆P_ria1::P_etef ∆fuz7 (red) and five P_etefmttA transformants named K3 (grey), K8 (green), K9 (orange), K10 (pink), and K14 (black). Error bars indicate the standard error of the mean (n = 3).

Figure 3

System Duetz^® cultivation of six U. maydis MB215 mutants in MTM with 100 g L⁻¹ glucose and 66 g L⁻¹ CaCO₃, incubated in 24-well plates with a filling volume of 1.5 mL (shaking diameter = 50 mm, n = 300 rpm, T = 30 °C and Φ = 80%). Concentrations of itaconate (continuous lines) and glucose (dotted lines) (A) are shown, as well as OD₆₀₀ (B) of U. maydis MB215 ∆cyp3 ∆MEL ∆UA ∆dgat ∆P_ria1::P_etef ∆fuz7 (red) and five P_etefmttA transformants U. maydis MB215 ∆cyp3 ∆MEL ∆UA ∆dgat ∆P_ria1::P_etef ∆fuz7 P_etefmttA, K3 (grey), K8 (green), K9 (orange), K10 (pink), and K14 (black). Error bars indicate the standard error of the mean (n = 3).

Figure 4

Production parameters of U. maydis MB215 ∆cyp3 ∆MEL ∆UA ∆dgat ∆P_ria1::P_etef ∆fuz7 transformants with different mttA copy numbers incubated in MTM with 50 g L⁻¹ glucose and 100 mM MES (A) and with 100 g L⁻¹ glucose and 66 g L⁻¹ CaCO₃ (B). Error bars indicate the standard error of the mean (n = 3).

Figure 5

High-density pulsed fed-batch fermentation of U. maydis strain K14. (A) concentration of glucose (●) and OD₆₀₀ values (▲) and (B) concentration of itaconate (■) during fermentation in a bioreactor containing batch medium (200 g L⁻¹ glucose, 4 g L⁻¹ NH₄Cl, CaCO₃ as buffering agent, 30 °C, 1000 rpm, top pitched blade impeller, bottom Rushton impeller). Arrows indicate the addition of 80 g glucose + 50 g CaCO₃ (black arrow), 80 g glucose (grey arrow), or 50 g CaCO₃ (light grey arrow). Error bars indicate the deviation from the mean (n = 2).

Figure 6

High-density fed-batch fermentation with continuous feed of U. maydis strain K14. (A) concentration of glucose, itaconate and ammonium, and OD₆₀₀ values and (B) filling volume, CO₂ production and the added volumes of 5 M NaOH and 50% glucose during fermentation in a bioreactor containing batch medium with 120 g L⁻¹ glucose and 4 g L⁻¹ NH₄Cl. The pH was kept at 6.5 by automatic titration with NaOH. Cultures were fed with an additional 130 g glucose at a rate of 2.8 g h⁻¹ during the indicated time interval. Error bars indicate the deviation from the mean (n = 2).

Figure 7

Low-density fed-batch fermentation with continuous feed of U. maydis strain K14. (A) concentration of glucose, itaconate and ammonium, and OD₆₀₀ values and (B) filling volume, CO₂ production and the added volumes of 5 M NaOH and 50% glucose during fermentation in a bioreactor containing batch medium with 120 g L⁻¹ glucose, 0.8 g L⁻¹ NH₄Cl. The pH was kept at 6.5 by automatic titration with NaOH. Cultures were fed with an additional 130 g glucose at a rate of 0.75 g h⁻¹ during the indicated time interval. Error bars indicate the deviation from the mean (n = 2).