Genetic and biochemical insights into the itaconate pathway of Ustilago maydis enable enhanced production

Geiser, Elena; Przybilla, Sandra; Engel, Meike; Kleineberg, Wiebke; Büttner, Linda; Sarikaya, Eda; Hartog, Tim den; Klankermayer, Jürgen; Leitner, Walter; Bölker, Michael; Blank, Lars M.; Wierckx, Nick

doi:10.1016/j.ymben.2016.10.006

Cited by 61 publications

(108 citation statements)

References 49 publications

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“…Even though production values both for malic acid, as well as itaconic acid were improved by ALE, the reached titers and production rates are still low compared to published values [16, 28, 40, 48, 53]. To further improve production properties, medium optimization was performed.…”

Section: Resultsmentioning

confidence: 99%

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Efficient itaconic acid production from glycerol with Ustilago vetiveriae TZ1

Zambanini

Tehrani

Geiser

et al. 2017

Biotechnol Biofuels

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BackgroundThe family of Ustilaginaceae is known for their capability to naturally produce industrially valuable chemicals from different carbon sources. Recently, several Ustilaginaceae were reported to produce organic acids from glycerol, which is the main side stream in biodiesel production.ResultsIn this study, we present Ustilago vetiveriae as new production organism for itaconate synthesis from glycerol. In a screening of 126 Ustilaginaceae, this organism reached one of the highest titers for itaconate combined with a high-glycerol uptake rate. By adaptive laboratory evolution, the production characteristics of this strain could be improved. Further medium optimization with the best single colony, U. vetiveriae TZ1, in 24-deep well plates resulted in a maximal itaconate titer of 34.7 ± 2.5 g L−1 produced at a rate of 0.09 ± 0.01 g L−1 h−1 from 196 g L−1 glycerol. Simultaneously, this strain produced 46.2 ± 1.4 g L−1 malate at a rate of 0.12 ± 0.00 g L−1 h−1. Due to product inhibition, the itaconate titer in NaOH-titrated bioreactor cultivations was lower (24 g L−1). Notably, an acidic pH value of 5.5 resulted in decreased itaconate production, however, completely abolishing malate production. Overexpression of ria1 or mtt1, encoding a transcriptional regulator and mitochondrial transporter, respectively, from the itaconate cluster of U. maydis resulted in a 2.0-fold (ria1) and 1.5-fold (mtt1) higher itaconate titer in comparison to the wild-type strain, simultaneously reducing malate production by 75 and 41%, respectively.ConclusionsThe observed production properties of U. vetiveriae TZ1 make this strain a promising candidate for microbial itaconate production. The outcome of the overexpression experiments, which resulted in reduced malate production in favor of an increased itaconate titer, clearly strengthens its potential for industrial itaconate production from glycerol as major side stream of biodiesel production.

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Section: Resultsmentioning

confidence: 99%

“…Recently, the itaconate production pathway in U. maydis has been clarified, allowing for targeted metabolic engineering of itaconate production in this host [27, 28]. …”

Section: Introductionmentioning

confidence: 99%

Efficient itaconic acid production from glycerol with Ustilago vetiveriae TZ1

Zambanini

Tehrani

Geiser

et al. 2017

Biotechnol Biofuels

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“…By comparison to the itaconate gene cluster of U. maydis MB215 (8) (GenBank Accession Number KT852988), the complete itaconate cluster was identified in all sequenced organisms except Pseudozyma tsukubaensis NBRC 1940, which did not contain rdo1 and cyp3 , which encode a putative dioxygenase and a monooxygenase involved in OH-paraconate production, respectively (10). The synteny of the itaconate cluster is preserved in the investigated Ustilaginaceae .…”

Section: Genome Announcementmentioning

confidence: 99%

Draft Genome Sequences of Itaconate-Producing Ustilaginaceae

et al. 2016

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“…U. maydis represents a promising organism for application in industrial itaconate production. Recent efforts have achieved considerable improvements in itaconate production with Ustilago , including characterization and upregulation of the itaconate gene cluster and associated pathway (Geiser et al , ,c, ), engineering of transporters involved in itaconate production (Hosseinpour Tehrani et al , ), and morphological and metabolic engineering of the acid‐tolerant U. cynodontis (Hosseinpour Tehrani et al , ). However, the potpourri of metabolites naturally synthesized by U. maydis results in suboptimal specificity, productivity and yield of itaconate as a product.…”

Section: Introductionmentioning

confidence: 99%

“…Regarding itaconate production, a combination of deleting cyp3, which encodes an itaconate oxidase that converts itaconate into 2-hydroxyparaconate, and overexpressing ria1, which encodes the itaconate gene cluster regulator, increased itaconate titres by nearly 4-fold (Geiser et al, 2016b). This also led to a strong decrease in malate production due to upregulation of mtt1 encoding a cis-aconitate/malate antiporter (Geiser et al, 2016b;Scarcia et al, 2019). MEL biosynthesis is mediated by a gene cluster comprising five genes, and single mutant strains U. maydis MB215 Δemt1, Δmac1 and Δmac2 completely lost their ability to produce MELs (Hewald et al, 2005(Hewald et al, , 2006.…”

Section: Introductionmentioning

confidence: 99%

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

Becker

Tehrani

Gauert

et al. 2019

Microbial Biotechnology

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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.

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Genetic and biochemical insights into the itaconate pathway of Ustilago maydis enable enhanced production

Cited by 61 publications

References 49 publications

Efficient itaconic acid production from glycerol with Ustilago vetiveriae TZ1

Efficient itaconic acid production from glycerol with Ustilago vetiveriae TZ1

Draft Genome Sequences of Itaconate-Producing Ustilaginaceae

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

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