A key aspect of sustainable bioeconomy is the recirculation of renewable,agricultural waste streams as substrates for microbial production of high-value compounds.One approach is the bioconversion of corn stover,an ab...A key aspect of sustainable bioeconomy is the recirculation of renewable,agricultural waste streams as substrates for microbial production of high-value compounds.One approach is the bioconversion of corn stover,an abundant maize crop byproduct,using the fungal maize pathogen Ustilago maydis.U.maydis is already used as a unicellular biocatalyst in the production of several industrially-relevant compounds using plant biomass hydrolysates.In this study,we demonstrate that U.maydis can grow using untreated corn stover as its sole carbon source.We developed a small-scale bioreactor platform to investigate U.maydis processing of corn stover,combining online monitoring of fungal growth and metabolic activity profiles with biochemical analyses of the pre-and post-fermentation residues.Our results reveal that U.maydis primarily utilizes soluble sugars i.e.,glucose,sucrose and fructose present in corn stover,with only limited exploitation of the abundant lignocellulosic carbohydrates.Thus,we further explored the biotechnological potential of enhancing U.maydis´lignocellulosic utilization.Additive performance improvements of up to 120%were achieved when using a maize mutant with increased biomass digestibility,co-fermentation with a commercial cellulolytic enzyme cocktail,and exploiting engineered fungal strains expressing diverse lignocellulose-degrading enzymes.This work represents a key step towards scaling up the production of sustainable compounds from corn stover using U.maydis and provides a tool for the detailed monitoring of the fungal processing of plant biomass substrates.展开更多
A deep-sequencing approach was pursued utilizing 454 and Illumina sequencing methods to discover new genes involved in xyloglucan biosynthesis, cDNA sequences were generated from developing nasturtium （Tropaeolum ma...A deep-sequencing approach was pursued utilizing 454 and Illumina sequencing methods to discover new genes involved in xyloglucan biosynthesis, cDNA sequences were generated from developing nasturtium （Tropaeolum majus） seeds, which produce large amounts of non-fucosylated xyloglucan as a seed storage polymer. In addition to known xyloglucan biosynthetic genes, a previously uncharacterized putative xyloglucan galactosyltransferase was iden- tified. Analysis of an Arabidopsis thaliana mutant line defective in the corresponding ortholog （AT5G62220） revealed that this gene shows no redundancy with the previously characterized xyloglucan galactosyltransferase, MUR3, but is required for galactosyl-substitution of xyloglucan at a different position. The gene was termed XLT2 for Xyloglucan L-side chain galactosylTransferase position 2. It represents an enzyme in the same subclade of glycosyltransferase family 47 as MUR3. A double mutant defective in both MUR3 （mur3.1） and XLT2 led to an Arabidopsis plant with xyloglucan that consists essentially of only xylosylated glucosyl units, with no further substitutions.展开更多
Plant cell walls, like a multitude of other biological materials, are natural fiber-reinforced composite materials. Their mechanical properties are highly dependent on the interplay of the stiff fibrous phase and the ...Plant cell walls, like a multitude of other biological materials, are natural fiber-reinforced composite materials. Their mechanical properties are highly dependent on the interplay of the stiff fibrous phase and the soft matrix phase and on the matrix deformation itself. Using specific Arabidopsis thaliana mutants, we studied the mechanical role of the matrix assembly in primary cell walls of hypocotyls with altered xyloglucan and pectin composition. Standard microtensile tests and cyclic loading protocols were performed on tour1 hypocotyls with affected RGII borate diester cross-links and a hindered xyloglucan fucosylation as well as qua2 exhibiting 50% less homogalacturonan in comparison to wild-type. As a control, wild-type plants （Col-0） and mur2 exhibiting a specific xyloglucan fucosylation and no differences in the pectin network were utilized. In the standard tensile tests, the ultimate stress levels （-tensile strength） of the hypocotyls of the mutants with pectin alterations （mur1, qua2） were rather unaffected, whereas their tensile stiffness was noticeably reduced in comparison to Col-0. The cyclic loading tests indicated a stiffening of all hypocotyls after the first cycle and a plastic deformation during the first straining, the degree of which, however, was much higher for tour1 and qua2 hypocotyls. Based on the mechanical data and current cell wall models, it is assumed that folded xyloglucan chains between cellulose fibrils may tend to unfold during straining of the hypocotyls. This response is probably hindered by geometrical constraints due to pectin rigidity.展开更多
Nucleotide sugar transporters （NSTs） are antiporters comprising a gene family that plays a fundamental role in the biosynthesis of complex cell wall polysaccharides and glycoproteins in plants. However, due to the l...Nucleotide sugar transporters （NSTs） are antiporters comprising a gene family that plays a fundamental role in the biosynthesis of complex cell wall polysaccharides and glycoproteins in plants. However, due to the limited number of related mutants that have observable phenotypes, the biological function（s） of most NSTs in cell wall biosynthesis and assembly have remained elusive. Here, we report the characterization of AtUTr7 from Arabidopsis （Arabidopsis thaliana （L.） Heynh.）, which is homologous to multi-specific UDP-sugar transporters from Drosophila melanogaster, humans, and Caenorhabditis elegans. We show that AtUTr7 possesses the common structural characteristics conserved among NSTs. Using a green fluorescent protein （GFP） tagged version, we demonstrate that AtUTr7 is localized in the Golgi apparatus. We also show that AtUTr7 is widely expressed, especially in the roots and in specific floral organs. Additionally, the results of an in vitro nucleotide sugar transport assay carried out with a tobacco and a yeast expression system suggest that AtUTr7 is capable of transferring UDP-Gal and UDP-GIc, but not a range of other UDP- and GDP-sugars, into the Golgi lumen. Mutants lacking expression of AtUTr7 exhibited an early proliferation of lateral roots as well as distorted root hairs when cultivated at high sucrose concentrations. Furthermore, the distribution of homogalacturonan with a low degree of methyl esterification differed in lateral root tips of the mutant compared to wild-type plants, although additional analytical procedures revealed no further differences in the composition of the root cell walls. This evidence suggests that the transport of UDP-Gal and UDP-GIc into the Golgi under conditions of high root biomass production plays a role in lateral root and root hair development.展开更多
Oligosaccharide Mass Profiling （OLIMP） allows a fast and sensitive assessment of cell wall polymer structure when coupled with Matrix Assisted Laser Desorption Ionisation Time Of Flight Mass Spectrometry （MALDI-TOF...Oligosaccharide Mass Profiling （OLIMP） allows a fast and sensitive assessment of cell wall polymer structure when coupled with Matrix Assisted Laser Desorption Ionisation Time Of Flight Mass Spectrometry （MALDI-TOF MS）. The short time required for sample preparation and analysis makes possible the study of a wide range of plant organs, revealing a high degree of heterogeneity in the substitution pattern of wall polymers such as the cross-linking glycan xyloglucan and the pectic polysaccharide homogalacturonan. The high sensitivity of MALDI-TOF allows the use of small amounts of samples, thus making it possible to investigate the wall structure of single cell types when material is collected by such methods as laser micro-dissection. As an example, the analysis of the xyloglucan structure in the leaf cell types outer epidermis layer, entire epidermis cell layer, palisade mesophyll cells, and vascular bundles were investigated. OLIMP is amenable to in situ wall analysis, where wall polymers are analyzed on unprepared plant tissue itself without first isolating cell walls. In addition, OLIMP enables analysis of wall polymers in Golgi-enriched fractions, the location of nascent matrix polysaccharide biosynthesis, enabling separation of the processes of wall biosynthesis versus post-deposition apoplastic metabolism. These new tools will make possible a semi-quantitative analysis of the cell wall at an unprecedented level.展开更多
Dear Editor, Plant cells are encased by cell walls--rigid yet highly dynamic composite structures, which consist of cellulose, hemicelluloses, pectic polysaccharides, glycoproteins, and the polyphenol lignin （Somerv...Dear Editor, Plant cells are encased by cell walls--rigid yet highly dynamic composite structures, which consist of cellulose, hemicelluloses, pectic polysaccharides, glycoproteins, and the polyphenol lignin （Somerville et al., 2004）. Biosynthesis of the wall polysaccharides is facilitated by donor- and acceptor- substrate-specific glycosyltransferases catalyzing the transfer of the sugar moiety from a nucleotide sugar to form a specific glycosidic linkage. Glycosyltransferases have been grouped into families based on primary structure, one of which is fam- ily GT77 （www.cazy.org/GT77_all.html）.展开更多
基金Open Access funding enabled and organized by Projekt DEALfunded by the NRW Strategic Project Bioeconomy Science Center(BioSC),Boost Fund 2.0(“NextVegOil”2021_04)+1 种基金The scientific activities of the BioSC were financially supported by the Ministry of Culture and Science within the framework of the NRW Strategieprojekt BioSC(No.313/323-400-00213)Additional funding was provided by Germany’s Federal Ministry of Education and Research(BMBF)grant“Cornwall,”031B1303A and Deutsche Forschun-gsgemeinschaft(DFG,German Research Foundation)under Germany’s Excel-lence Strategy-EXC 2048/1-Project ID:390686111 to M.Pa.,and Marie Curie PIOF-GA-2013-623553 to V.R.
文摘A key aspect of sustainable bioeconomy is the recirculation of renewable,agricultural waste streams as substrates for microbial production of high-value compounds.One approach is the bioconversion of corn stover,an abundant maize crop byproduct,using the fungal maize pathogen Ustilago maydis.U.maydis is already used as a unicellular biocatalyst in the production of several industrially-relevant compounds using plant biomass hydrolysates.In this study,we demonstrate that U.maydis can grow using untreated corn stover as its sole carbon source.We developed a small-scale bioreactor platform to investigate U.maydis processing of corn stover,combining online monitoring of fungal growth and metabolic activity profiles with biochemical analyses of the pre-and post-fermentation residues.Our results reveal that U.maydis primarily utilizes soluble sugars i.e.,glucose,sucrose and fructose present in corn stover,with only limited exploitation of the abundant lignocellulosic carbohydrates.Thus,we further explored the biotechnological potential of enhancing U.maydis´lignocellulosic utilization.Additive performance improvements of up to 120%were achieved when using a maize mutant with increased biomass digestibility,co-fermentation with a commercial cellulolytic enzyme cocktail,and exploiting engineered fungal strains expressing diverse lignocellulose-degrading enzymes.This work represents a key step towards scaling up the production of sustainable compounds from corn stover using U.maydis and provides a tool for the detailed monitoring of the fungal processing of plant biomass substrates.
文摘A deep-sequencing approach was pursued utilizing 454 and Illumina sequencing methods to discover new genes involved in xyloglucan biosynthesis, cDNA sequences were generated from developing nasturtium （Tropaeolum majus） seeds, which produce large amounts of non-fucosylated xyloglucan as a seed storage polymer. In addition to known xyloglucan biosynthetic genes, a previously uncharacterized putative xyloglucan galactosyltransferase was iden- tified. Analysis of an Arabidopsis thaliana mutant line defective in the corresponding ortholog （AT5G62220） revealed that this gene shows no redundancy with the previously characterized xyloglucan galactosyltransferase, MUR3, but is required for galactosyl-substitution of xyloglucan at a different position. The gene was termed XLT2 for Xyloglucan L-side chain galactosylTransferase position 2. It represents an enzyme in the same subclade of glycosyltransferase family 47 as MUR3. A double mutant defective in both MUR3 （mur3.1） and XLT2 led to an Arabidopsis plant with xyloglucan that consists essentially of only xylosylated glucosyl units, with no further substitutions.
文摘Plant cell walls, like a multitude of other biological materials, are natural fiber-reinforced composite materials. Their mechanical properties are highly dependent on the interplay of the stiff fibrous phase and the soft matrix phase and on the matrix deformation itself. Using specific Arabidopsis thaliana mutants, we studied the mechanical role of the matrix assembly in primary cell walls of hypocotyls with altered xyloglucan and pectin composition. Standard microtensile tests and cyclic loading protocols were performed on tour1 hypocotyls with affected RGII borate diester cross-links and a hindered xyloglucan fucosylation as well as qua2 exhibiting 50% less homogalacturonan in comparison to wild-type. As a control, wild-type plants （Col-0） and mur2 exhibiting a specific xyloglucan fucosylation and no differences in the pectin network were utilized. In the standard tensile tests, the ultimate stress levels （-tensile strength） of the hypocotyls of the mutants with pectin alterations （mur1, qua2） were rather unaffected, whereas their tensile stiffness was noticeably reduced in comparison to Col-0. The cyclic loading tests indicated a stiffening of all hypocotyls after the first cycle and a plastic deformation during the first straining, the degree of which, however, was much higher for tour1 and qua2 hypocotyls. Based on the mechanical data and current cell wall models, it is assumed that folded xyloglucan chains between cellulose fibrils may tend to unfold during straining of the hypocotyls. This response is probably hindered by geometrical constraints due to pectin rigidity.
文摘Nucleotide sugar transporters （NSTs） are antiporters comprising a gene family that plays a fundamental role in the biosynthesis of complex cell wall polysaccharides and glycoproteins in plants. However, due to the limited number of related mutants that have observable phenotypes, the biological function（s） of most NSTs in cell wall biosynthesis and assembly have remained elusive. Here, we report the characterization of AtUTr7 from Arabidopsis （Arabidopsis thaliana （L.） Heynh.）, which is homologous to multi-specific UDP-sugar transporters from Drosophila melanogaster, humans, and Caenorhabditis elegans. We show that AtUTr7 possesses the common structural characteristics conserved among NSTs. Using a green fluorescent protein （GFP） tagged version, we demonstrate that AtUTr7 is localized in the Golgi apparatus. We also show that AtUTr7 is widely expressed, especially in the roots and in specific floral organs. Additionally, the results of an in vitro nucleotide sugar transport assay carried out with a tobacco and a yeast expression system suggest that AtUTr7 is capable of transferring UDP-Gal and UDP-GIc, but not a range of other UDP- and GDP-sugars, into the Golgi lumen. Mutants lacking expression of AtUTr7 exhibited an early proliferation of lateral roots as well as distorted root hairs when cultivated at high sucrose concentrations. Furthermore, the distribution of homogalacturonan with a low degree of methyl esterification differed in lateral root tips of the mutant compared to wild-type plants, although additional analytical procedures revealed no further differences in the composition of the root cell walls. This evidence suggests that the transport of UDP-Gal and UDP-GIc into the Golgi under conditions of high root biomass production plays a role in lateral root and root hair development.
文摘Oligosaccharide Mass Profiling （OLIMP） allows a fast and sensitive assessment of cell wall polymer structure when coupled with Matrix Assisted Laser Desorption Ionisation Time Of Flight Mass Spectrometry （MALDI-TOF MS）. The short time required for sample preparation and analysis makes possible the study of a wide range of plant organs, revealing a high degree of heterogeneity in the substitution pattern of wall polymers such as the cross-linking glycan xyloglucan and the pectic polysaccharide homogalacturonan. The high sensitivity of MALDI-TOF allows the use of small amounts of samples, thus making it possible to investigate the wall structure of single cell types when material is collected by such methods as laser micro-dissection. As an example, the analysis of the xyloglucan structure in the leaf cell types outer epidermis layer, entire epidermis cell layer, palisade mesophyll cells, and vascular bundles were investigated. OLIMP is amenable to in situ wall analysis, where wall polymers are analyzed on unprepared plant tissue itself without first isolating cell walls. In addition, OLIMP enables analysis of wall polymers in Golgi-enriched fractions, the location of nascent matrix polysaccharide biosynthesis, enabling separation of the processes of wall biosynthesis versus post-deposition apoplastic metabolism. These new tools will make possible a semi-quantitative analysis of the cell wall at an unprecedented level.
文摘Dear Editor, Plant cells are encased by cell walls--rigid yet highly dynamic composite structures, which consist of cellulose, hemicelluloses, pectic polysaccharides, glycoproteins, and the polyphenol lignin （Somerville et al., 2004）. Biosynthesis of the wall polysaccharides is facilitated by donor- and acceptor- substrate-specific glycosyltransferases catalyzing the transfer of the sugar moiety from a nucleotide sugar to form a specific glycosidic linkage. Glycosyltransferases have been grouped into families based on primary structure, one of which is fam- ily GT77 （www.cazy.org/GT77_all.html）.
