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RNAseq data for P. putida with vanillate
Illumina sequencing reads from RNA sequencing of vanillate-utilizing strains of Pseudomonas putida, described in Evolution and engineering of pathways for aromatic O-demethylation in Pseudomonas putida KT2440 by A. Bleem, et al. (2024)
Comparison of native and heterologous pathways for aromatic O-demethylation in Pseudmonas putida KT2440 enabled by adaptive laboratory evolution
Comparison of native and heterologous pathways for aromatic O-demethylation in Pseudomonas putida KT2440 enabled by adaptive laboratory evolution
A Multiomic Approach to Understand How Pleurotus eryngii Transforms Non-Woody Lignocellulosic Material
Pleurotus eryngii is a grassland-inhabiting fungus of biotechnological interest due to its ability to colonize non-woody lignocellulosic material. Genomic, transcriptomic, exoproteomic, and metabolomic analyses were combined to explain the enzymatic aspects underlaying wheat–straw transformation. Up-regulated and constitutive glycoside–hydrolases, polysaccharide–lyases, and carbohydrate–esterases active on polysaccharides, laccases active on lignin, and a surprisingly high amount of constitutive/inducible aryl–alcohol oxidases (AAOs) constituted the suite of extracellular enzymes at early fungal growth. Higher enzyme diversity and abundance characterized the longer-term growth, with an array of oxidoreductases involved in depolymerization of both cellulose and lignin, which were often up-regulated since initial growth. These oxidative enzymes included lytic polysaccharide monooxygenases (LPMOs) acting on crystalline polysaccharides, cellobiose dehydrogenase involved in LPMO activation, and ligninolytic peroxidases (mainly manganese-oxidizing peroxidases), together with highly abundant H2O2-producing AAOs. Interestingly, some of the most relevant enzymes acting on polysaccharides were appended to a cellulose-binding module. This is potentially related to the non-woody habitat of P. eryngii (in contrast to the wood habitat of many basidiomycetes). Additionally, insights into the intracellular catabolism of aromatic compounds, which is a neglected area of study in lignin degradation by basidiomycetes, were also provided. The multiomic approach reveals that although non-woody decay does not result in dramatic modifications, as revealed by detailed 2D-NMR and other analyses, it implies activation of the complete set of hydrolytic and oxidative enzymes characterizing lignocellulose-decaying basidiomycetes.
RNA-seq studying the catabolism of β-5 linked aromatics by Novosphingobium aromaticivorans
RNA-seq comparison of transcript abundance in a PDC-producing N. aromaticivorans strain (12444PDC) grown in the presence of dehydrodiconiferyl alcohol (DC-A) and glucose, vanillin and glucose, ferulic acid and glucose, or glucose alone
Effect of Cultivation Conditions on the Bioconversion of 4-Hydroxybenzoic Acid in Two White-Rot Fungi
White-rot fungi (WRF) are the most efficient organisms for lignin degradation in nature. A recent study has also shown the ability of WRF in funneling lignin-derived aromatics, including 4-hydroxybenzoic acid (4HBA) and vanillic acid, to central metabolism. However, it is unknown how the cultivation conditions affect the conversion of lignin-derived aromatic compounds. To address this, we performed multi-omic analyses and tracked the conversion of 4HBA in different cultivation conditions in two white-rot fungi: Trametes versicolor and Ceriporiopsis subvermispora. Specifically, we evaluated the effect of static and agitation cultivation conditions in the absence and the presence of antioxidants on fungal performance. We found that the comparison between static and agitation has the higher number of differentially expressed genes or protein abundances at the transcriptomic and proteomic levels, respectively, compared to the presence or the absence of antioxidants. Gene mapping on the enzymatic steps of the proposed 4HBA conversion pathway revealed that agitation causes upregulation of several oxidase genes such as aldehyde dehydrogenases, aldehyde oxidases, and hydroxylases in both fungi. Time-course experiments including 4HBA and cellobiose as carbon sources showed that T. versicolor exhibited higher 4HBA conversion levels in agitation (~110% more conversion, compared to static cultivation conditions) and in the presence of antioxidants (~10% more conversion, compared to the treatments without antioxidants). Interestingly, these trends were not found in C. subvermispora, suggesting different carbon metabolism in both species. Our findings are fundamental to better understand carbon sequestration from lignin in WRF, which will be key for future applications in the lignin valorization field.
The Catabolism of Lignin-Derived p-Methoxylated Aromatic Compounds by Rhodococcus jostii RHA1
Emergent strategies to valorize lignin, an abundant but underutilized aromatic biopolymer, include tandem processes that integrate chemical depolymerization and biological catalysis. To date, aromatic monomers from C-O bond cleavage of lignin have been converted to bioproducts, but the presence of recalcitrant C-C bonds in lignin limits the product yield. A promising chemocatalytic strategy that overcomes this limitation involves phenol methyl protection and autoxidation. Incorporating this into a tandem process requires microbial cell factories able to transform the p-methoxylated products in the resulting methylated lignin stream. In this study, we assessed the ability of Rhodococcus jostii RHA1 to catabolize the major aromatic products in a methylated lignin stream and elucidated the pathways responsible for this catabolism. RHA1 grew on a methylated pine lignin stream, catabolizing the major aromatic monomers: p-methoxybenzoate (p-MBA), veratrate, and veratraldehyde. Bioinformatic analyses suggested that a cytochrome P450, PbdA, and its cognate reductase, PbdB, are involved in p-MBA catabolism. Gene deletion studies established that both pbdA and pbdB are essential for growth on p-MBA and several derivatives. Furthermore, a deletion mutant of a candidate p-hydroxybenzoate (p-HBA) hydroxylase, ..delta..pobA, did not grow on p-HBA. Veratraldehyde and veratrate catabolism required both vanillin dehydrogenase (Vdh) and vanillate O-demethylase (VanAB), revealing previously unknown roles of these enzymes. Finally, a ..delta..pcaL strain grew on neither p-MBA nor veratrate, indicating they are catabolized through the ..beta..-ketoadipate pathway. This study expands our understanding of the bacterial catabolism of aromatic compounds and facilitates the development of biocatalysts for lignin valorization.
Laboratory evolution in Novosphingobium aromaticivorans enables rapid catabolism of a model lignin-derived aromatic dimer
Lignin contains a variety of interunit linkages, leading to a range of potential decomposition products that can be used as carbon and energy sources by microbes. β-O-4 linkages are the most common in native lignin, and associated catabolic pathways have been well characterized. However, the fate of the mono-aromatic intermediates that result from β-O-4 dimer cleavage has not been fully elucidated. Here, we used experimental evolution to identify mutant strains of Novosphingobium aromaticivorans with improved catabolism of a model aromatic dimer containing a β-O-4 linkage, guaiacylglycerol-β-guaiacyl ether (GGE). We identified several parallel causal mutations, including a single nucleotide polymorphism in the promoter of an uncharacterized gene that roughly doubled the growth yield with GGE. We characterized the associated enzyme and demonstrated that it oxidizes an intermediate in GGE catabolism, β-hydroxypropiovanillone, to vanilloyl acetaldehyde. Identification of this enzyme and its key role in GGE catabolism furthers our understanding of catabolic pathways for lignin-derived aromatic compounds.
Study of the effect of the trancription factor SARO_RS14285 in aromatics degradation in Novosphingobium aromaticivorans
Novosphingobium aromaticivorans DSM12444 is a bacterium capable of catabolizing several lignin aromatics. A main degradation pathway for these compounds is the protocatechuate (PCA) meta-cleavage. Nevertheless, the transcriptional regulation of this pathway is still unknown. Immediately upstream of the genes encoding the enzymes for this pathway, the LysR-type transcription factor (LTTF) SARO_RS14285 was identified. To evaluate the functionality of this LTTF, a deletion mutant was constructed. A transcriptomic analysis of the mutant strain cultured in several aromatic compounds is included in this report. Overall design: RNA-seq profiling of the WT and the deletion mutant cultured in minimal medium with glucose and one of the following aromatics: protocatechuic acid (PCA), 4-coumaric acid (4-CA), vanillic acid (VA) or syringic acid (SA).
Biochemical parallels between catabolic pathways for lignin-associated aromatic dimers
Lignin is one of the most common biopolymers on Earth. In nature, lignin is primarily deconstructed by fungi into mixtures of aromatic compounds that are then assimilated by bacteria and fungi. Industrially, lignin is primarily generated as a byproduct of pulp and paper production and burned for process heat. However, if the appropriate assimilatory pathways were identified, deconstructed lignin could be funneled into value-added products using engineered bacteria. Foundational work has described pathways for assimilation of diverse monomeric aromatic compounds such as protocatechuate, ferulate, and syringate, as well as select dimers including those with β-O-4 and 5-5 interunit linkages. Recent advances have elucidated additional pathways for dimer assimilation, including pathways for new substrates as well as parallel pathways for previously characterized substrates. Comparing these dimer assimilation pathways can illuminate the underlying biochemical logic of assimilation for lignin-associated aromatic dimers and provide opportunities for metabolic engineering to enhance lignin valorization.
Biochemical and structural characterization of an aromatic ring–hydroxylating dioxygenase for terephthalic acid catabolism
Significance More than 400 million tons of plastic waste is produced each year, the overwhelming majority of which ends up in landfills. Bioconversion strategies aimed at plastics have emerged as important components of enabling a circular economy for synthetic plastics, especially those that exhibit chemically similar linkages to those found in nature, such as polyesters. The enzyme system described in this work is essential for mineralization of the xenobiotic components of poly(ethylene terephthalate) (PET) in the biosphere. Our description of its structure and substrate preferences lays the groundwork for in vivo or ex vivo engineering of this system for PET upcycling.
Cross-kingdom comparative genomics reveal the metabolic potential of fungi for lignin turnover in deadwood
Deadwood is a major carbon source in forests, and yet the fate of this carbon remains a gap in our understanding of global carbon cycling. Lignin, the most recalcitrant biopolymer in wood, is mainly decayed through extracellular enzymatic and chemical processes initiated by white-rot fungi. However, the intracellular conversion of lignin decay products has been overlooked in the fungal kingdom. Here we integrate comparative genomic and phylogenetic analyses to understand the distribution and evolution of enzymes responsible for modifying lignin-related aromatic compounds—such as decarboxylases, hydroxylases, dioxygenases and other downstream ring-cleavage enzymes—that funnel carbon to central metabolism across the bacterial and the fungal kingdoms. We demonstrate that specific fungal lineages conserve these enzyme families, and that the abilities to enzymatically depolymerize lignin and catabolize lignin-related aromatic compounds are not necessarily coupled. Our analyses also reveal an expanded substrate specificity of aromatic ring-cleavage enzymes during fungal evolution, as well as a clade of extracellular enzymes among them, broadening the spatial range of these biochemical capabilities. Together, our results highlight a large diversity of fungal enzymes and hosts that warrant further investigation for inclusion into carbon cycling models and biotechnological applications for the conversion of aromatic compounds.