Identification , functional expression in Saccharomyces cerevisiae and characterization of a Penicillium chrysogenum high-affinity L-arabinose transporter
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PROVIDER: PRJNA412825 | ENA |
REPOSITORIES: ENA
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Project description:L-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Especially at low concentrations of L-arabinose, its uptake via the Gal2 galactose transporter is an important rate-controlling step in the complete conversion of these feedstocks by engineered, pentose-metabolizing Saccharomyces cerevisiae strains. Chemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least three-fold higher in L-arabinose-limited cultures than in glucose-limited and ethanol-limited cultures. Of five genes that showed an over 30-fold higher transcript level in arabinose-grown cultures, only one (Pc20g01790) restored growth on L-arabinose upon expression in an engineered L-arabinose-fermenting S. cerevisiae strain in which GAL2 had been deleted. Sugar-transport assays indicated that Pc20g01790this transporter, designated as PcAraT, encodes functions as a high-affinity (Km = 0.13 mM) L-arabinose-proton symporter that does not transport xylose or glucose. An L-arabinose-metabolizing S. cerevisiae strain co-expressing Pc20g01790PcAraT and GAL2 showed lower residual substrate concentrations in L-arabinose-limited chemostat cultures (4 mg L-1) than a congenic strain in which L-arabinose import exclusively depended on Gal2 (1.8 g L-1). Inhibition of L-arabinose transport by these sugars was less pronounced than observed with Gal2. A hexose-phosphorylation-deficient, L-arabinose-metabolizing S. cerevisiae strain expressing PcAraT Pc20g0190 grew on 20 g L-1 L-arabinose in the presence of 20 g L-1 glucose, which completely inhibited growth on L-arabinose of a congenic strain dependent on L-arabinose transport via Gal2. Its high affinity and specificity for L-L-arabinose, combined with limited sensitivity to inhibition by glucose and D-D-xylose make PcAraT/ Pc20g01790 a valuable transporter gene for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.
2017-10-03 | GSE104491 | GEO
Project description:The recent discovery of a velvet complex containing several regulators of secondary metabolism in the model fungus Aspergillus nidulans raises the question whether similar type complexes direct fungal development in genera other than Aspergillus. Penicillium chrysogenum is the industrial producer of the antibiotic penicillin, whose biosynthetic regulation is barely understood. Here we provide a functional analysis of two major homologues of the velvet complex in P. chrysogenum, that we have named PcvelA and PclaeA. Data from array analysis using a ?PcvelA deletion strain indicate a significant role of PcvelA on the expression of biosynthesis and developmental genes, including PclaeA. Northern hybridization and HPLC quantifications of penicillin titres clearly show that both PcvelA and PclaeA play a major role in penicillin biosynthesis. Both regulators are further involved in different and distinct developmental processes. While PcvelA deletion leads to light independent conidial formation, dichotomous branching of hyphae and pellet formation in shaking cultures, a ?PclaeA strain shows a severe impairment in conidiophore formation in both the light and dark. Bimolecular fluorescence complementation assays finally provide evidence for a velvet-like complex in Penicillium chrysogenum, with structurally conserved components that have distinct developmental roles, illustrating the functional plasticity of these regulators within filamentous ascomycetes. Transcriptomes of PcvelA- and PclaeA- deletion mutants were compared with expression data from recipient strain deltaPcku70 and reference strain P2niaD18 as a control
2010-06-17 | E-GEOD-18585 | biostudies-arrayexpress
Project description:The recent discovery of a velvet complex containing several regulators of secondary metabolism in the model fungus Aspergillus nidulans raises the question whether similar type complexes direct fungal development in genera other than Aspergillus. Penicillium chrysogenum is the industrial producer of the antibiotic penicillin, whose biosynthetic regulation is barely understood. Here we provide a functional analysis of two major homologues of the velvet complex in P. chrysogenum, that we have named PcvelA and PclaeA. Data from array analysis using a ΔPcvelA deletion strain indicate a significant role of PcvelA on the expression of biosynthesis and developmental genes, including PclaeA. Northern hybridization and HPLC quantifications of penicillin titres clearly show that both PcvelA and PclaeA play a major role in penicillin biosynthesis. Both regulators are further involved in different and distinct developmental processes. While PcvelA deletion leads to light independent conidial formation, dichotomous branching of hyphae and pellet formation in shaking cultures, a ΔPclaeA strain shows a severe impairment in conidiophore formation in both the light and dark. Bimolecular fluorescence complementation assays finally provide evidence for a velvet-like complex in Penicillium chrysogenum, with structurally conserved components that have distinct developmental roles, illustrating the functional plasticity of these regulators within filamentous ascomycetes.
2010-06-17 | GSE18585 | GEO
Project description:Penicillium chrysogenum 4088766 Genome sequencing
| PRJNA210202 | ENA
Project description:Deletion of biosynthetic gene clusters in Penicillium chrysogenum DS68530
| PRJNA587307 | ENA
Project description:Background:l-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Its low-affinity uptake via the Saccharomyces cerevisiae Gal2 galactose transporter is inhibited by d-glucose. Especially at low concentrations of l-arabinose, uptake is an important rate-controlling step in the complete conversion of these feedstocks by engineered pentose-metabolizing S. cerevisiae strains. Results:Chemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least threefold higher in l-arabinose-limited cultures than in d-glucose-limited and ethanol-limited cultures. Of five genes, that encoded putative transport proteins and showed an over 30-fold higher transcript level in l-arabinose-grown cultures compared to d-glucose-grown cultures, only one (Pc20g01790) restored growth on l-arabinose upon expression in an engineered l-arabinose-fermenting S. cerevisiae strain in which the endogenous l-arabinose transporter, GAL2, had been deleted. Sugar transport assays indicated that this fungal transporter, designated as PcAraT, is a high-affinity (Km = 0.13 mM), high-specificity l-arabinose-proton symporter that does not transport d-xylose or d-glucose. An l-arabinose-metabolizing S. cerevisiae strain in which GAL2 was replaced by PcaraT showed 450-fold lower residual substrate concentrations in l-arabinose-limited chemostat cultures than a congenic strain in which l-arabinose import depended on Gal2 (4.2 × 10-3 and 1.8 g L-1, respectively). Inhibition of l-arabinose transport by the most abundant sugars in hydrolysates, d-glucose and d-xylose was far less pronounced than observed with Gal2. Expression of PcAraT in a hexose-phosphorylation-deficient, l-arabinose-metabolizing S. cerevisiae strain enabled growth in media supplemented with both 20 g L-1 l-arabinose and 20 g L-1 d-glucose, which completely inhibited growth of a congenic strain in the same condition that depended on l-arabinose transport via Gal2. Conclusion:Its high affinity and specificity for l-arabinose, combined with limited sensitivity to inhibition by d-glucose and d-xylose, make PcAraT a valuable transporter for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.
| S-EPMC5848512 | biostudies-literature
Project description:Penicillium chrysogenum NCPC10086 Genome sequencing and assembly
| PRJNA190642 | ENA
Project description:In this study, we investigated the role of L-arabinose transporter MtLat-1 in the induction of hemicellulase genes of M. thermophila. The deletion of MtLat-1 dramatically improved protein secretion and hemicellulase activities on arabinan. Comparative transcriptomic analysis indicated MtLat-1 repressed expression of hemcellulase genes. Furthermore, based on analyses of non-transporting MtLat-1 mutant, we found MtLat-1 in M. thermophila was involved in arabinose transportation, sensing, and tdownstream signalling cascades. Additionally, we also demonstrated Mtlat-1 expression is directly regulated by transcription factor MtAra-1 and MtAra-1 is involved in L-arabinose transporter and metabolism and arabinan hydrolysis.
2023-03-29 | GSE221464 | GEO
Project description:Characterization of the Ku70 homologue HdfA deletion in Penicillium chrysogenum
| PRJNA110889 | ENA