AnUstilago maydischassis for itaconic acid production without by‐products

Abstract

Summary 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.

Development and Application of CRISPR/Cas in Microbial Biotechnology

“…In CHAnGE, guide sequence and the homologous recombination (HDR) template were arranged and synthesized in a single oligonucleotide, and a oligonucleotide library of 24,765 unique guide sequences targeting 6,459 ORFs was synthesized on a chip and then assembled into a vector [pCRCT, harboring iCas9, tracrRNA expression cassettes and a promoter for sgRNA expression, as reported in HI-CRISPR system (Bao et al, 2015)] to build a pool of plasmids. This plasmid pool was then used to create a genome-wide gene disruption collection, in which more than 98% of target sequences were efficiently edited with an average Increased by 3-fold with a specific titer of octadocanol at 10.3 mg/g DCW Kaczmarzyk et al, 2018 Ustilago maydis CRISPR/Cas9 Itaconic acid cyp3, MEL, UA, and P ria1 ::P etef Increased by 10.2-fold with a yield at 19.4 g/L and further enhanced to 53.5 g/L under optimized medium Becker et al, 2019 frequency of 82% (Bao et al, 2018). In parallel, Jakociunas et al employed error-prone PCR to generate DNA mutant libraries as donor, and used Cas9-mediated genome integration to introduce mutations at single-or multi-loci with efficiencies reaching 98-99%, for robust directed evolution (Jakociunas et al, 2018b).…”

Consolidated Bioprocessing of Cellulose to Itaconic Acid by a Co-Culture of Trichoderma Reesei and Ustilago Maydis.

“…Hence, an RQ above 1 in presence of CaCO 3 indicates that itaconic acid production is ongoing, while a drop of RQ close to 1 indicates cessation of itaconic acid production. During the experiment, RQ values well above the theoretically expected values have been measured, because the used strain still produces considerable amounts of reduced by-products such as glycolipids, which result in an increased RQ (17,48).…”

“…maydis has already proven suitable for SSF-based itaconic acid production, however reaching only very low yields despite application of high cellulase loadings of 0.1 g Protein /g Cellulose or 26 lter paper units (FPU)/g Cellulose (15). Recently, itaconic acid production with U. maydis and other Ustilaginaceae such as U. cynodontis has been improved considerably by genetic engineering (16)(17)(18)(19). This, along with advances in bioprocess design enabled by the yeast morphology (19)(20)(21), has considerably enhanced the yield, titer, and rate of itaconic acid production by Ustilago.…”

Polycistronic gene expression in the model micro-organismUstilago maydis

“…Besides its role as a model system, U. maydis is currently being developed as a production chassis for a wide range of biotechnological relevant compounds. This includes itaconic acid as a chemical platform molecule for biofuels, ustilagic acid and mannosylerythritol lipids (MEL) glycolipids as biosurfactants, and various antibody formats as valuable proteins (Teichmann et al, 2010;Feldbrügge et al, 2013;Sarkari et al, 2014;Terfrüchte et al, 2014;Becker et al, 2019;Stoffels et al, 2020). Furthermore, strains were generated to utilise cellobiose, xylose and polygalacturonic acid as a carbon source in the yeast phase, so that plant cell wall components including pectin can be used as starting point for sustainable production (Geiser et al, 2016;Müller et al, 2018;Stoffels et al, 2020).…”