A comparative genomics study of 23 Aspergillus species from section Flavi

Section Flavi encompasses both harmful and beneficial Aspergillus species, such as Aspergillus oryzae, used in food fermentation and enzyme production, and Aspergillus flavus, food spoiler and mycotoxin producer. Here, we sequence 19 genomes spanning section Flavi and compare 31 fungal genomes including 23 Flavi species. We reassess their phylogenetic relationships and show that the closest relative of A. oryzae is not A. flavus, but A. minisclerotigenes or A. aflatoxiformans and identify high genome diversity, especially in sub-telomeric regions. We predict abundant CAZymes (598 per species) and prolific secondary metabolite gene clusters (73 per species) in section Flavi. However, the observed phenotypes (growth characteristics, polysaccharide degradation) do not necessarily correlate with inferences made from the predicted CAZyme content. Our work, including genomic analyses, phenotypic assays, and identification of secondary metabolites, highlights the genetic and metabolic diversity within section Flavi.Peer reviewe

Production of protocatechuic acid from p-hydroxyphenyl (H) units and related aromatic compounds using an aspergillus niger cell factory

Protocatechuic acid (3,4-dihydroxybenzoic acid) is a chemical building block for polymers and plastics. In addition, protocatechuic acid has many properties of great pharmaceutical interest. Much research has been performed in creating bacterial protocatechuic acid production strains, but no protocatechuic acid-producing fungal cell factories have been described. The filamentous fungus Aspergillus niger can produce protocatechuic acid as an intermediate of the benzoic acid metabolic pathway. Recently, the p-hydroxybenzoate-m-hydroxylase (phhA) and protocatechuate 3,4-dioxy-genase (prcA) of A. niger have been identified. It has been shown that the prcA deletion mutant is still able to grow on protocatechuic acid. This led to the identification of an alternative pathway that converts protocatechuic acid to hydroxyquinol (1,3,4-trihydrox-ybenzene). However, the gene involved in the hydroxylation of protocatechuic acid to hydroxyquinol remained unidentified. Here, we describe the identification of protocatechuate hydroxylase (decarboxylating) (PhyA) by using whole-genome transcriptome data. The identification of phyA enabled the creation of a fungal cell factory that is able to accumulate protocatechuic acid from benzyl alcohol, benzaldehyde, benzoic acid, caffeic acid, cinnamic acid, cinnamyl alcohol, m-hydroxybenzoic acid, p-hydroxybenzyl alcohol, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, p-anisyl alcohol, p-anisaldehyde, p-anisic acid, p-coumaric acid, and protocatechuic aldehyde. IMPORTANCE Aromatic compounds have broad applications and are used in many industries, such as the cosmetic, food, fragrance, paint, plastic, pharmaceutical, and polymer industries. The majority of aromatic compounds are synthesized from fossil sources, which are becoming limited. Plant biomass is the most abundant renewable resource on Earth and can be utilized to produce chemical building blocks, fuels, and bioplastics through fermentations with genetically modified microorganisms. Therefore, knowledge about the metabolic pathways and the genes and enzymes involved is essential to create efficient strategies for producing valuable aromatic compounds such as protocatechuic acid. Protocatechuic acid has many pharmaceutical properties but also can be used as a chemical building block to produce polymers and plastics. Here, we show that the fungus Aspergillus niger can be engineered to produce protocatechuic acid from plant-derived aromatic compounds and con-tributes to creating alternative methods for the production of platform chemicals

Production of protocatechuic acid from p-hydroxyphenyl (H) units and related aromatic compounds using an aspergillus niger cell factory

Protocatechuic acid (3,4-dihydroxybenzoic acid) is a chemical building block for polymers and plastics. In addition, protocatechuic acid has many properties of great pharmaceutical interest. Much research has been performed in creating bacterial protocatechuic acid production strains, but no protocatechuic acid-producing fungal cell factories have been described. The filamentous fungus Aspergillus niger can produce protocatechuic acid as an intermediate of the benzoic acid metabolic pathway. Recently, the p-hydroxybenzoate-m-hydroxylase (phhA) and protocatechuate 3,4-dioxy-genase (prcA) of A. niger have been identified. It has been shown that the prcA deletion mutant is still able to grow on protocatechuic acid. This led to the identification of an alternative pathway that converts protocatechuic acid to hydroxyquinol (1,3,4-trihydrox-ybenzene). However, the gene involved in the hydroxylation of protocatechuic acid to hydroxyquinol remained unidentified. Here, we describe the identification of protocatechuate hydroxylase (decarboxylating) (PhyA) by using whole-genome transcriptome data. The identification of phyA enabled the creation of a fungal cell factory that is able to accumulate protocatechuic acid from benzyl alcohol, benzaldehyde, benzoic acid, caffeic acid, cinnamic acid, cinnamyl alcohol, m-hydroxybenzoic acid, p-hydroxybenzyl alcohol, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, p-anisyl alcohol, p-anisaldehyde, p-anisic acid, p-coumaric acid, and protocatechuic aldehyde. IMPORTANCE Aromatic compounds have broad applications and are used in many industries, such as the cosmetic, food, fragrance, paint, plastic, pharmaceutical, and polymer industries. The majority of aromatic compounds are synthesized from fossil sources, which are becoming limited. Plant biomass is the most abundant renewable resource on Earth and can be utilized to produce chemical building blocks, fuels, and bioplastics through fermentations with genetically modified microorganisms. Therefore, knowledge about the metabolic pathways and the genes and enzymes involved is essential to create efficient strategies for producing valuable aromatic compounds such as protocatechuic acid. Protocatechuic acid has many pharmaceutical properties but also can be used as a chemical building block to produce polymers and plastics. Here, we show that the fungus Aspergillus niger can be engineered to produce protocatechuic acid from plant-derived aromatic compounds and con-tributes to creating alternative methods for the production of platform chemicals

A novel Zn2Cys6 transcription factor BcGaaR regulates D-galacturonic acid utilization in Botrytis cinerea

Summary: D-galacturonic acid (GalA) is the most abundant monosaccharide component of pectin. Previous transcriptome analysis in the plant pathogenic fungus Botrytis cinerea identified eight GalA-inducible genes involved in pectin decomposition, GalA transport and utilization. Co-expression of these genes indicates that a specific regulatory mechanism occurs in B. cinerea. In this study, promoter regions of these genes were analysed and eight conserved sequence motifs identified. The Bclga1 promoter, containing all these motifs, was functionally analysed and the motif designated GalA Responsive Element (GARE) was identified as the crucial cis-regulatory element in regulation of GalA utilization in B. cinerea. Yeast one-hybrid screening with the GARE motif led to identification of a novel Zn2Cys6 transcription factor (TF), designated BcGaaR. Targeted knockout analysis revealed that BcGaaR is required for induction of GalA-inducible genes and growth of B. cinerea on GalA. A BcGaaR-GFP fusion protein was predominantly localized in nuclei in mycelium grown in GalA. Fluorescence in nuclei was much stronger in mycelium grown in GalA, as compared to fructose and glucose. This study provides the first report of a GalA-specific TF in filamentous fungi. Orthologs of BcGaaR are present in other ascomycete fungi that are able to utilize GalA, including Aspergillus spp., Trichoderma reesei and Neurospora crassa. Short Abstract: The transcriptional regulator that controls D-galacturonic acid (GalA) utilization in fungi was unknown. We identified in Botrytis cinerea a novel Zn2Cys6 transcription factor (TF), designated BcGaaR, which is required for the induction of GalA-utilization genes. The BcGaaR protein was predominantly localized in nuclei in cultures grown on GalA, but remained in the cytoplasm in cultures grown on glucose. This study provides the first report of a GalA-specific TF in filamentous fungi. Orthologs of BcGaaR are present in other ascomycete fungi that are able to utilize GalA, including Aspergillus spp., Trichoderma reesei and Neurospora crassa

Vanillic acid and methoxyhydroquinone production from guaiacyl units and related aromatic compounds using Aspergillus niger cell factories

Background: The aromatic compounds vanillin and vanillic acid are important fragrances used in the food, beverage, cosmetic and pharmaceutical industries. Currently, most aromatic compounds used in products are chemically synthesized, while only a small percentage is extracted from natural sources. The metabolism of vanillin and vanillic acid has been studied for decades in microorganisms and many studies have been conducted that showed that both can be produced from ferulic acid using bacteria. In contrast, the degradation of vanillin and vanillic acid by fungi is poorly studied and no genes involved in this metabolic pathway have been identified. In this study, we aimed to clarify this metabolic pathway in Aspergillus niger and identify the genes involved. Results: Using whole-genome transcriptome data, four genes involved in vanillin and vanillic acid metabolism were identified. These include vanillin dehydrogenase (vdhA), vanillic acid hydroxylase (vhyA), and two genes encoding novel enzymes, which function as methoxyhydroquinone 1,2-dioxygenase (mhdA) and 4-oxo-monomethyl adipate esterase (omeA). Deletion of these genes in A. niger confirmed their role in aromatic metabolism and the enzymatic activities of these enzymes were verified. In addition, we demonstrated that mhdA and vhyA deletion mutants can be used as fungal cell factories for the accumulation of vanillic acid and methoxyhydroquinone from guaiacyl lignin units and related aromatic compounds. Conclusions: This study provides new insights into the fungal aromatic metabolic pathways involved in the degradation of guaiacyl units and related aromatic compounds. The identification of the involved genes unlocks new potential for engineering aromatic compound-producing fungal cell factories

Vanillic acid and methoxyhydroquinone production from guaiacyl units and related aromatic compounds using Aspergillus niger cell factories

Background: The aromatic compounds vanillin and vanillic acid are important fragrances used in the food, beverage, cosmetic and pharmaceutical industries. Currently, most aromatic compounds used in products are chemically synthesized, while only a small percentage is extracted from natural sources. The metabolism of vanillin and vanillic acid has been studied for decades in microorganisms and many studies have been conducted that showed that both can be produced from ferulic acid using bacteria. In contrast, the degradation of vanillin and vanillic acid by fungi is poorly studied and no genes involved in this metabolic pathway have been identified. In this study, we aimed to clarify this metabolic pathway in Aspergillus niger and identify the genes involved. Results: Using whole-genome transcriptome data, four genes involved in vanillin and vanillic acid metabolism were identified. These include vanillin dehydrogenase (vdhA), vanillic acid hydroxylase (vhyA), and two genes encoding novel enzymes, which function as methoxyhydroquinone 1,2-dioxygenase (mhdA) and 4-oxo-monomethyl adipate esterase (omeA). Deletion of these genes in A. niger confirmed their role in aromatic metabolism and the enzymatic activities of these enzymes were verified. In addition, we demonstrated that mhdA and vhyA deletion mutants can be used as fungal cell factories for the accumulation of vanillic acid and methoxyhydroquinone from guaiacyl lignin units and related aromatic compounds. Conclusions: This study provides new insights into the fungal aromatic metabolic pathways involved in the degradation of guaiacyl units and related aromatic compounds. The identification of the involved genes unlocks new potential for engineering aromatic compound-producing fungal cell factories

A comparative genomics study of 23 Aspergillus species from section Flavi

Section Flavi encompasses both harmful and beneficial Aspergillus species, such as Aspergillus oryzae, used in food fermentation and enzyme production, and Aspergillus flavus, food spoiler and mycotoxin producer. Here, we sequence 19 genomes spanning section Flavi and compare 31 fungal genomes including 23 Flavi species. We reassess their phylogenetic relationships and show that the closest relative of A. oryzae is not A. flavus, but A. minisclerotigenes or A. aflatoxiformans and identify high genome diversity, especially in sub-telomeric regions. We predict abundant CAZymes (598 per species) and prolific secondary metabolite gene clusters (73 per species) in section Flavi. However, the observed phenotypes (growth characteristics, polysaccharide degradation) do not necessarily correlate with inferences made from the predicted CAZyme content. Our work, including genomic analyses, phenotypic assays, and identification of secondary metabolites, highlights the genetic and metabolic diversity within section Flavi