Establishing a versatile Golden Gate cloning system for genetic engineering in fungi

Terfrüchte, Marius; Joehnk, Bastian; Fajardo-Somera, Rosa A.; Braus, Gerhard H.; Riquelme, Meritxell; Schipper, Kerstin; Feldbrügge, Michael

doi:10.1016/j.fgb.2013.10.012

Cited by 98 publications

(103 citation statements)

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“…This strain (34) enables the induction of filamentous growth without prior mating, thereby allowing the analysis of the yeast as well as the filamentous growth stage. Deletion mutants lacking one, two, three, or all four chitinase genes (Table 1) were generated by gene replacement via homologous recombination (21,23,24). Chitinase deletions, even in the quadruple mutant, were readily obtained, and recombination rates of 30 to 50% are indicative of nonessential functions of the enzymes.…”

Section: Resultsmentioning

confidence: 99%

Chitinases Are Essential for Cell Separation in Ustilago maydis

et al. 2015

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Chitin is an essential component of the fungal cell wall, providing rigidity and stability. Its degradation is mediated by chitinases and supposedly ensures the dynamic plasticity of the cell wall during growth and morphogenesis. Hence, chitinases should be particularly important for fungi with dramatic morphological changes, such as Ustilago maydis. This smut fungus switches from yeast to filamentous growth for plant infection, proliferates as a mycelium in planta, and forms teliospores for spreading. Here, we investigate the contribution of its four chitinolytic enzymes to the different morphological changes during the complete life cycle in a comprehensive study of deletion strains combined with biochemical and cell biological approaches. Interestingly, two chitinases act redundantly in cell separation during yeast growth. They mediate the degradation of remnant chitin in the fragmentation zone between mother and daughter cell. In contrast, even the complete lack of chitinolytic activity does not affect formation of the infectious filament, infection, biotrophic growth, or teliospore germination. Thus, unexpectedly we can exclude a major role for chitinolytic enzymes in morphogenesis or pathogenicity of U. maydis. Nevertheless, redundant activity of even two chitinases is essential for cell separation during saprophytic growth, possibly to improve nutrient access or spreading of yeast cells by wind or rain.

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Section: Resultsmentioning

confidence: 99%

Chitinases Are Essential for Cell Separation in Ustilago maydis

et al. 2015

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“…The PCR products were purified by standard procedures (e.g., SureClean, Bioline; JetSorb, Genomed). To generate destination vectors containing the gene activation constructs, BsaI-mediated Golden Gate reaction mixtures containing the two respective PCR products (flanks), a storage vector, and the destination vector (pDestI/ pUMa1467) were made as described elsewhere (40). Plasmids pStor1_2-4 h (pUMa1507 [40]) and pStorI_2-5n (pUMa2326, see below) served as storage vectors harboring a hygromycin resistance (HygR)/P otef and a nourseothricin resistance (NatR)/P oma resistance cassette module, respectively, for constitutive expression of the target gene.…”

Section: Methodsmentioning

confidence: 99%

“…For that purpose, the 2.5-kb backbone of pUMa1778 (pStorI_2-3 h [40]) was isolated by SfiI restriction, and the 1.4-kb NatR cassette of pUMa326 (pMF3-4n [39]) was generated by restriction with XbaI/SfiI. Both parts were combined with a PCR fragment containing P oma in a three-fragment ligation.…”

Section: Methodsmentioning

confidence: 99%

“…The genome is sequenced and manually annotated (29). Numerous molecular tools have been developed over the last few years, including resistance markers, protein tags (39), and efficient strain generation by Golden Gate cloning (40). In addition, in bioreactors, the fungus confers a remarkably high resistance to shearing forces and to impurities in the fermentation broth, like those observed after biomass pretreatment (26,41).…”

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Activating Intrinsic Carbohydrate-Active Enzymes of the Smut Fungus Ustilago maydis for the Degradation of Plant Cell Wall Components

Geiser

Reindl

Blank

et al. 2016

Appl Environ Microbiol

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The microbial conversion of plant biomass to valuable products in a consolidated bioprocess could greatly increase the ecologic and economic impact of a biorefinery. Current strategies for hydrolyzing plant material mostly rely on the external application of carbohydrate-active enzymes (CAZymes). Alternatively, production organisms can be engineered to secrete CAZymes to reduce the reliance on externally added enzymes. Plant-pathogenic fungi have a vast repertoire of hydrolytic enzymes to sustain their lifestyle, but expression of the corresponding genes is usually highly regulated and restricted to the pathogenic phase. Here, we present a new strategy in using the biotrophic smut fungus Ustilago maydis for the degradation of plant cell wall components by activating its intrinsic enzyme potential during axenic growth. This fungal model organism is fully equipped with hydrolytic enzymes, and moreover, it naturally produces value-added substances, such as organic acids and biosurfactants. To achieve the deregulated expression of hydrolytic enzymes during the industrially relevant yeast-like growth in axenic culture, the native promoters of the respective genes were replaced by constitutively active synthetic promoters. This led to an enhanced conversion of xylan, cellobiose, and carboxymethyl cellulose to fermentable sugars. Moreover, a combination of strains with activated endoglucanase and ␤-glucanase increased the release of glucose from carboxymethyl cellulose and regenerated amorphous cellulose, suggesting that mixed cultivations could be a means for degrading more complex substrates in the future. In summary, this proof of principle demonstrates the potential applicability of activating the expression of native CAZymes from phytopathogens in a biocatalytic process. IMPORTANCEThis study describes basic experiments that aim at the degradation of plant cell wall components by the smut fungus Ustilago maydis. As a plant pathogen, this fungus contains a set of lignocellulose-degrading enzymes that may be suited for biomass degradation. However, its hydrolytic enzymes are specifically expressed only during plant infection. Here, we provide the proof of principle that these intrinsic enzymes can be synthetically activated during the industrially relevant yeast-like growth. The fungus is known to naturally synthesize valuable compounds, such as itaconate or glycolipids. Therefore, it could be suited for use in a consolidated bioprocess in which more complex and natural substrates are simultaneously converted to fermentable sugars and to value-added compounds in the future. O ne central aim of a sustainable bioeconomy is the switch from fossil-to bio-based production of platform chemicals and other valuable substances. To date, the most promising feedstock for biorefineries is lignocellulosic nonfood plant biomass (1). Lignocellulose is a complex composite mainly consisting of cellulose, hemicelluloses, pectin, and lignin (2). The selective conversion of lignocellulosic biomass comprises different steps: pret...

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“…Gate procedure as described (Terfrüchte et al, 2014). In short, successive steps of 173 cleavage, using the BsaI restriction enzyme, and DNA ligation with T4 ligase generated 174 a plasmid with the gene replacement module flanked by homologous regions 500 bp in 175 length.…”

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confidence: 99%

The Functional Specialization of Exomer as a Cargo Adaptor During the Evolution of Fungi

2018

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30Yeast exomer is a heterotetrameric complex assembled at the trans-Golgi 31 network (TGN) that is required for the delivery of a distinct set of proteins to the plasma 32 membrane using ChAPs proteins Chs6 and Bch2 as dedicated cargo adaptors. Our 33 results show, however, a significant functional divergence between them, suggesting an 34 evolutionary specialization among the ChAPs proteins. Moreover, the characterization 35 of exomer mutants in several fungi indicates that exomer function as a cargo adaptor is 36 a late evolutionary acquisition associated with several gene duplications of the fungal 37

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Establishing a versatile Golden Gate cloning system for genetic engineering in fungi

Cited by 98 publications

References 45 publications

Chitinases Are Essential for Cell Separation in Ustilago maydis

Chitinases Are Essential for Cell Separation in Ustilago maydis

Activating Intrinsic Carbohydrate-Active Enzymes of the Smut Fungus Ustilago maydis for the Degradation of Plant Cell Wall Components

The Functional Specialization of Exomer as a Cargo Adaptor During the Evolution of Fungi

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