The switch from the traditional fossil-based industry to a green and sustainable bioeconomy demands the complete utilization of renewable feedstocks. Many currently used bioconversion hosts are unable to utilize major components of plant biomass, warranting the identification of microorganisms with broader catabolic capacity and characterization of their unique biochemical pathways. d-Galacturonic acid is a plant component of bioconversion interest and is the major backbone sugar of pectin, a plant cell wall polysaccharide abundant in soft and young plant tissues. The red basidiomycete and oleaginous yeast Rhodosporidium toruloides has been previously shown to utilize a range of sugars and aromatic molecules. Using state-of-the-art functional genomic methods and physiological and biochemical assays, we elucidated the molecular basis underlying the efficient metabolism of d-galacturonic acid. This study identified an efficient pathway for uronic acid conversion to guide future engineering efforts and represents the first detailed metabolic analysis of pectin metabolism in a basidiomycete fungus.

The corn smut fungus Ustilago maydis requires the unfolded protein response (UPR) to maintain homeostasis of the endoplasmic reticulum (ER) during the biotrophic interaction with its host plant Zea mays (maize). Crosstalk between the UPR and pathways controlling pathogenic development is mediated by protein-protein interactions between the UPR regulator Cib1 and the developmental regulator Clp1. Cib1/Clp1 complex formation results in mutual modification of the connected regulatory networks thereby aligning fungal proliferation in planta , efficient effector secretion with increased ER stress tolerance and long-term UPR activation in planta . Here we address UPR-dependent gene expression and its modulation by Clp1 using combinatorial RNAseq/ChIPseq analyses. We show that increased ER stress resistance is connected to Clp1-dependent alterations of Cib1 phosphorylation, protein stability and UPR gene expression. Importantly, we identify by deletion screening of UPR core genes the signal peptide peptidase Spp1 as a novel key factor that is required for establishing a compatible biotrophic interaction between U . maydis and its host plant maize. Spp1 is dispensable for ER stress resistance and vegetative growth but requires catalytic activity to interfere with the plant defense, revealing a novel virulence specific function for signal peptide peptidases in a biotrophic fungal/plant interaction.

25Biorefining of renewable feedstocks is one of the most promising routes to 26 replace fossil-based products. Since many common fermentation hosts, such as 27Saccharomyces cerevisiae, are naturally unable to convert many component plant cell 28 wall polysaccharides, the identification of organisms with broad catabolism capabilities 29 represents an opportunity to expand the range of substrates used in fermentation 30 biorefinery approaches. The red basidiomycete yeast Rhodosporidium toruloides is a 31 promising and robust host for lipid and terpene derived chemicals. Previous studies 32 demonstrated assimilation of a range of substrates, from C5/C6-sugars to aromatic 33 molecules similar to lignin monomers. In the current study, we analyzed R. toruloides 34 potential to assimilate D-galacturonic acid, a major sugar in many pectin-rich agricultural 35 waste streams, including sugar beet pulp and citrus peels. D-galacturonic acid is not a 36 preferred substrate for many fungi, but its metabolism was found to be on par with 37 D-glucose and D-xylose in R. toruloides. A genome-wide analysis by combined 38RNAseq/RB-TDNAseq revealed those genes with high relevance for fitness on 39 D-galacturonic acid. While R. toruloides was found to utilize the same non-40 phosphorylative catabolic pathway known from ascomycetes, the maximal velocities of 41 several enzymes exceeded those previously reported. In addition, an efficient 42 downstream glycerol catabolism and a novel transcription factor were found to be 43 important for D-galacturonic acid utilization. These results set the basis for use of 44 R. toruloides as a potential host for pectin-rich waste conversions and demonstrate its 45 suitability as a model for metabolic studies in basidiomycetes. 46 functional genomic methods, physiological and biochemical assays, we elucidated the 57 molecular basis underlying the efficient metabolism of D-galacturonic acid. This study 58 identifies an efficient pathway for uronic acid conversion to guide future engineering 59 efforts, and represents the first detailed metabolic analysis of pectin metabolism in a 60 basidiomycete fungus. 61 D-galUA with D-glucose and D-xylose 126Since it was known that R. toruloides can utilize both D-glc and D-xyl (18), we 127 tested how the assimilation of D-galUA would compare to these rates and whether 128 growth inhibition would be visible in mixed substrate cultures. To this end, R. toruloides 129 IFO 0880 was grown in 200 µl volume cultures with 50 mM each of these sugars as sole 130 carbon source as well as in cultures in which D-galUA was mixed with either D-glc or D-131 xyl in a 1:1 ratio. Surprisingly, despite a slightly slower acceleration-phase on D-galUA 132 compared to D-glc in the first 24 hours, culture densities of R. toruloides reached almost 133 similar final ODs (Fig. 1A). Moreover, D-galUA was completely consumed by that time, 134 while total consumption of D-glc required about 70 hours (Fig. 1B). With this rate, growth 135 on D-galUA was faster than on D-xyl as the sole carbon so...