An Ustilago maydis chassis for itaconic acid production without by-products

Ustilago maydis is a promising yeast for the production of a range of valuable metabolites, including itaconate, malate, glycolipids and triacylglycerols. However, wild‐type strains generally produce a potpourri of all of these metabolites, which hinders efficient production of single target chemicals. In this study, the diverse by‐product spectrum of U. maydis was reduced through strain engineering using CRISPR/Cas9 and FLP/FRT, greatly increasing the metabolic flux into the targeted itaconate biosynthesis pathway. With this strategy, a marker‐free chassis strain could be engineered, which produces itaconate from glucose with significantly enhanced titre, rate and yield. The lack of by‐product formation not only benefited itaconate production, it also increases the efficiency of downstream processing improving cell handling and product purity

Marker-free genome editing in <i>Ustilago trichophora</i> with the CRISPR-Cas9 technology

<p>In this communication, we report the adaptation of the CRISPR-Cas9 technology in <i>Ustilago trichophora</i> prototrophic wild-type isolate obtained from its natural host <i>Echinochloa crus-galli</i>. The established CRISPR vector and method enable a rapid and marker-free introduction of Cas9-induced non-homologous end-joining (NHEJ) dependent mutation at the targeted gene. Moreover, the method allows a specific modification of the chromosomal DNA sequence by Cas9-induced homologous recombination using short DNA repair templates. The results demonstrate the applicability of the CRISPR-Cas9 technology in <i>U. trichophora</i> for both gene knock-out by the NHEJ pathway and specific gene modification by templated genome editing, paving the way for rapid metabolic engineering of this <i>Ustilago</i> species for industrial applications.</p