23 research outputs found
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Genomewide and Enzymatic Analysis Reveals Efficient d-Galacturonic Acid Metabolism in the Basidiomycete Yeast Rhodosporidium toruloides.
Biorefining of renewable feedstocks is one of the most promising routes to replace fossil-based products. Since many common fermentation hosts, such as Saccharomyces cerevisiae, are naturally unable to convert many component plant cell wall polysaccharides, the identification of organisms with broad catabolism capabilities represents an opportunity to expand the range of substrates used in fermentation biorefinery approaches. The red basidiomycete yeast Rhodosporidium toruloides is a promising and robust host for lipid- and terpene-derived chemicals. Previous studies demonstrated assimilation of a range of substrates, from C5/C6 sugars to aromatic molecules similar to lignin monomers. In the current study, we analyzed the potential of R. toruloides to assimilate d-galacturonic acid, a major sugar in many pectin-rich agricultural waste streams, including sugar beet pulp and citrus peels. d-Galacturonic acid is not a preferred substrate for many fungi, but its metabolism was found to be on par with those of d-glucose and d-xylose in R. toruloides A genomewide analysis by combined transcriptome sequencing (RNA-seq) and RB-TDNA-seq revealed those genes with high relevance for fitness on d-galacturonic acid. While R. toruloides was found to utilize the nonphosphorylative catabolic pathway known from ascomycetes, the maximal velocities of several enzymes exceeded those previously reported. In addition, an efficient downstream glycerol catabolism and a novel transcription factor were found to be important for d-galacturonic acid utilization. These results set the basis for use of R. toruloides as a potential host for pectin-rich waste conversions and demonstrate its suitability as a model for metabolic studies with basidiomycetes.IMPORTANCE 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
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Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides.
BACKGROUND:Rhodosporidium toruloides has emerged as a promising host for the production of bioproducts from lignocellulose, in part due to its ability to grow on lignocellulosic feedstocks, tolerate growth inhibitors, and co-utilize sugars and lignin-derived monomers. Ent-kaurene derivatives have a diverse range of potential applications from therapeutics to novel resin-based materials. RESULTS:The Design, Build, Test, and Learn (DBTL) approach was employed to engineer production of the non-native diterpene ent-kaurene in R. toruloides. Following expression of kaurene synthase (KS) in R. toruloides in the first DBTL cycle, a key limitation appeared to be the availability of the diterpene precursor, geranylgeranyl diphosphate (GGPP). Further DBTL cycles were carried out to select an optimal GGPP synthase and to balance its expression with KS, requiring two of the strongest promoters in R. toruloides, ANT (adenine nucleotide translocase) and TEF1 (translational elongation factor 1) to drive expression of the KS from Gibberella fujikuroi and a mutant version of an FPP synthase from Gallus gallus that produces GGPP. Scale-up of cultivation in a 2 L bioreactor using a corn stover hydrolysate resulted in an ent-kaurene titer of 1.4 g/L. CONCLUSION:This study builds upon previous work demonstrating the potential of R. toruloides as a robust and versatile host for the production of both mono- and sesquiterpenes, and is the first demonstration of the production of a non-native diterpene in this organism
Prospects for utilizing microbial consortia for lignin conversion
Naturally occurring microbial communities are able to decompose lignocellulosic biomass through the concerted production of a myriad of enzymes that degrade its polymeric components and assimilate the resulting breakdown compounds by members of the community. This process includes the conversion of lignin, the most recalcitrant component of lignocellulosic biomass and historically the most difficult to valorize in the context of a biorefinery. Although several fundamental questions on microbial conversion of lignin remain unanswered, it is known that some fungi and bacteria produce enzymes to break, internalize, and assimilate lignin-derived molecules. The interest in developing efficient biological lignin conversion approaches has led to a better understanding of the types of enzymes and organisms that can act on different types of lignin structures, the depolymerized compounds that can be released, and the products that can be generated through microbial biosynthetic pathways. It has become clear that the discovery and implementation of native or engineered microbial consortia could be a powerful tool to facilitate conversion and valorization of this underutilized polymer. Here we review recent approaches that employ isolated or synthetic microbial communities for lignin conversion to bioproducts, including the development of methods for tracking and predicting the behavior of these consortia, the most significant challenges that have been identified, and the possibilities that remain to be explored in this field
Conversion of poplar biomass into high-energy density tricyclic sesquiterpene jet fuel blendstocks
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Conversion of depolymerized sugars and aromatics from engineered feedstocks by two oleaginous red yeasts.
One of the requirements for efficient biological conversion of lignocellulose to bioproducts is the compatibility of biological catalysts with the processes employed to solubilize and depolymerize the lignocellulosic components. The red yeasts Rhodosporidium toruloides and Rhodotorula mucilaginosa were evaluated for their ability to assimilate sugars and aromatic compounds extracted from two engineered lines of Arabidopsis thaliana with modified lignin or the wild-type using ionic liquid, acid or alkaline pretreatments. Differential amounts of monomeric sugars, organic acids and, in the case of the engineered lines, either 4-hydroxybenzoic or protocatechuic acid were additionally released from the biomass and found to be tolerated and consumed by both microorganisms. Genetically-engineered strains of the two red yeasts successfully converted the depolymerized products into the biofuel precursor bisabolene when cultivated on hydrolysates or synthetic media containing specific sugars, acids and aromatics found in the hydrolysates
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Conversion of depolymerized sugars and aromatics from engineered feedstocks by two oleaginous red yeasts
One of the requirements for efficient biological conversion of lignocellulose to bioproducts is the compatibility of biological catalysts with the processes employed to solubilize and depolymerize the lignocellulosic components. The red yeasts Rhodosporidium toruloides and Rhodotorula mucilaginosa were evaluated for their ability to assimilate sugars and aromatic compounds extracted from two engineered lines of Arabidopsis thaliana with modified lignin or the wild-type using ionic liquid, acid or alkaline pretreatments. Differential amounts of monomeric sugars, organic acids and, in the case of the engineered lines, either 4-hydroxybenzoic or protocatechuic acid were additionally released from the biomass and found to be tolerated and consumed by both microorganisms. Genetically-engineered strains of the two red yeasts successfully converted the depolymerized products into the biofuel precursor bisabolene when cultivated on hydrolysates or synthetic media containing specific sugars, acids and aromatics found in the hydrolysates
Engineering transcriptional regulation of pentose metabolism in Rhodosporidium toruloides for improved conversion of xylose to bioproducts
Abstract Efficient conversion of pentose sugars remains a significant barrier to the replacement of petroleum-derived chemicals with plant biomass-derived bioproducts. While the oleaginous yeast Rhodosporidium toruloides (also known as Rhodotorula toruloides) has a relatively robust native metabolism of pentose sugars compared to other wild yeasts, faster assimilation of those sugars will be required for industrial utilization of pentoses. To increase the rate of pentose assimilation in R. toruloides, we leveraged previously reported high-throughput fitness data to identify potential regulators of pentose catabolism. Two genes were selected for further investigation, a putative transcription factor (RTO4_12978, Pnt1) and a homolog of a glucose transceptor involved in carbon catabolite repression (RTO4_11990). Overexpression of Pnt1 increased the specific growth rate approximately twofold early in cultures on xylose and increased the maximum specific growth by 18% while decreasing accumulation of arabitol and xylitol in fast-growing cultures. Improved growth dynamics on xylose translated to a 120% increase in the overall rate of xylose conversion to fatty alcohols in batch culture. Proteomic analysis confirmed that Pnt1 is a major regulator of pentose catabolism in R. toruloides. Deletion of RTO4_11990 increased the growth rate on xylose, but did not relieve carbon catabolite repression in the presence of glucose. Carbon catabolite repression signaling networks remain poorly characterized in R. toruloides and likely comprise a different set of proteins than those mainly characterized in ascomycete fungi