Search for transcription factors affecting productivity of the polyketide FR901512 in filamentous fungal sp. No. 14919 and identification of Drf1, a novel negative regulator of the biosynthetic gene cluster.

In order to increase secondary metabolite production in filamentous fungi, a transcription factor gene in the biosynthetic gene cluster and global regulator genes such as laeA are considered plausible as targets for overexpression by genetic modification. In this study, we examined these overexpression effect in fungal sp. No. 14919 that produces FR901512, an HMG-CoA reductase inhibitor. Resultantly, the productivity was improved at 1.7-1.8 fold by overexpressing frlE, a transcription factor gene in the biosynthetic gene cluster, whereas productivity did not change by overexpression of laeA and veA. Furthermore, we searched for extra transcription factors affecting the productivity by transcriptome analysis between wild-type strain and highly productive UV mutants. After verifying productivity decrease by overexpression, Drf1, a novel transcription factor encoded by drf1 was identified as the negative regulator. Because each frlE product (FrlE) and Drf1 worked on the same cluster in positive and negative regulatory manners, their network was analyzed.

Biosynthesis of Novel Statins by Combining Heterologous Genes from Xylaria and Aspergillus.

For many secondary metabolites, heterologous synthesis is the definitive step to determine their required biosynthetic genes. Using a multivector expression system in Saccharomyces cerevisiae, we reconstituted not only two natural statins from two fungal species, i.e., lovastatin from Aspergillus terreus and FR901512 from Xylaria grammica, but also new statin structures by mixing their genes. Combinatorial gene exchange experiments revealed the functional promiscuity of two polyketide synthases in A. terreus, lovB, and lovF; they could synthesize FR901512 with Xylaria genes. Key structure determinants of statins are essential accessory genes that are irreplaceable across species.

Knockout of the SREBP system increases production of the polyketide FR901512 in filamentous fungal sp. No. 14919 and lovastatin in Aspergillus terreus ATCC20542.

In the production of useful microbial secondary metabolites, the breeding of strains is generally performed by random mutagenesis. However, because random mutagenesis introduces many mutations into genomic DNA, the causative mutations leading to increased productivity are mostly unknown. Therefore, although gene targeting is more efficient for breeding than random mutagenesis, it is difficult to apply. In this study, a wild-type strain and randomly mutagenized strains of fungal sp. No. 14919, a filamentous fungus producing the HMG-CoA reductase inhibitor polyketide FR901512, were subjected to point mutation analysis based on whole genome sequencing. Among the mutated genes found, mutation of the sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) had a positive effect on increasing FR901512 productivity. By complementing the SCAP gene in the SCAP-mutated strain, productivity was decreased to the level of the SCAP-intact strain. Conversely, when either the SCAP or SREBP gene was deleted, the productivity was significantly increased. By genomic transcriptional analysis, the expression levels of three enzymes in the ergosterol biosynthesis pathway were shown to be decreased by SCAP mutation. These findings led to the hypothesis that raw materials of polyketides, such as acetyl-CoA and malonyl-CoA, became more available for FR901512 biosynthesis due to depression in sterol biosynthesis caused by knockout of the SREBP system. This mechanism was confirmed in Aspergillus terreus producing the polyketide lovastatin, which is structurally similar to FR901512. Thus, knockout of the SREBP system should be considered significant for increasing the productivities of polyketides, such as HMG-CoA reductase inhibitors, by filamentous fungi.

Genome Sequence of the Fungal Strain 14919 Producing 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase Inhibitor FR901512.

Fungal strain 14919 was originally isolated from a soil sample collected at Mt. Kiyosumi, Chiba Prefecture, Japan. It produces FR901512, a potent and strong 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor. The genome sequence of fungal strain 14919 was determined and annotated to improve the productivity of FR901512.

AxyR, an AraC family transcriptional activator of the xylanase 3 gene, requires xylo-oligosaccharides as a cofactor for DNA binding in Paenibacillus sp. strain W-61.

The xylanolytic bacterium Paenibacillus sp. strain W-61 encodes three extracellular xylanase genes, xyn1, xyn3, and xyn5. In this study, we identified a transcriptional activator required for transcription of the xyn3 gene in strain W-61. The activator, AxyR, contained the highly homologous AraC-type DNA binding domain and required xylobiose, xylotriose, or xylotetraose as cofactor for binding to the xyn3 promoter region.

Cell surface xylanases of the glycoside hydrolase family 10 are essential for xylan utilization by Paenibacillus sp. W-61 as generators of xylo-oligosaccharide inducers for the xylanase genes.

Paenibacillus sp. W-61 is capable of utilizing water-insoluble xylan for carbon and energy sources and has three xylanase genes, xyn1, xyn3, and xyn5. Xyn1, Xyn3, and Xyn5 are extracellular enzymes of the glycoside hydrolase (GH) families 11, 30, and 10, respectively. Xyn5 contains several domains including those of carbohydrate-binding modules (CBMs) similar to a surface-layer homologous (SLH) protein. This study focused on the role of Xyn5, localized on the cell surface, in water-insoluble xylan utilization. Electron microscopy using immunogold staining revealed Xyn5 clusters over the entire cell surface. Xyn5 was bound to cell wall fractions through its SLH domain. A Deltaxyn5 mutant grew poorly and produced minimal amounts of Xyn1 and Xyn3 on water-insoluble xylan. A Xyn5 mutant lacking the SLH domain (Xyn5DeltaSLH) grew poorly, secreting Xyn5DeltaSLH into the medium and producing minimal Xyn1 and Xyn3 on water-insoluble xylan. A mutant with an intact xyn5 produced Xyn5 on the cell surface, grew normally, and actively synthesized Xyn1 and Xyn3 on water-insoluble xylan. Quantitative reverse transcription-PCR showed that xylobiose, generated from water-insoluble xylan decomposition by Xyn5, is the most active inducer for xyn1 and xyn3. Luciferase assays using a Xyn5-luciferase fusion protein suggested that xylotriose is the best inducer for xyn5. The cell surface Xyn5 appears to play two essential roles in water-insoluble xylan utilization: (i) generation of the xylo-oligosaccharide inducers of all the xyn genes from water-insoluble xylan and (ii) attachment of the cells to the substrate so that the generated inducers can be immediately taken up by cells to activate expression of the xyn system.