Fungal NRPS-PKS Hybrid Enzymes Biosynthesize New γ-Lactam Compounds, Taslactams A-D, Analogous to Actinomycete Proteasome Inhibitors.

Proteasome inhibitors with γ-lactam structure, such as lactacystin and salinosporamide A, have been isolated from actinomycetes and have attracted attention as lead compounds for anticancer drugs. Previously, we identified a unique enzyme TAS1, which is the first reported fungal NRPS-PKS hybrid enzyme, from the filamentous fungus Pyricularia oryzae for the biosynthesis of a mycotoxin tenuazonic acid, a tetramic acid compound without γ-lactam structure. Homologues of TAS1 have been identified in several fungal genomes and classified into four groups (A-D). Here, we show that the group D TAS1 homologues from two filamentous fungi can biosynthesize γ-lactam compounds, taslactams A-D, with high similarity to actinomycete proteasome inhibitors. One of the γ-lactam compounds, taslactam C, showed potent proteasome inhibitory activity. In contrast to actinomycete γ-lactam compounds which require multiple enzymes for biosynthesis, the TAS1 homologue alone was sufficient for the biosynthesis of the fungal γ-lactam compounds.

Expression of bacterial tyrosine ammonia-lyase creates a novel p-coumaric acid pathway in the biosynthesis of phenylpropanoids in Arabidopsis.

Some flavonoids are considered as beneficial compounds because they exhibit anticancer or antioxidant activity. In higher plants, flavonoids are secondary metabolites that are derived from phenylpropanoid biosynthetic pathway. A large number of phenylpropanoids are generated from p-coumaric acid, which is a derivative of the primary metabolite, phenylalanine. The first two steps in the phenylpropanoid biosynthetic pathway are catalyzed by phenylalanine ammonia-lyase and cinnamate 4-hydroxylase, and the coupling of these two enzymes forms a rate-limiting step in the pathway. For the generation of p-coumaric acid, the conversion from phenylalanine to p-coumaric acid that is catalyzed by two enzymes can be theoretically performed by a single enzyme, tyrosine ammonia-lyase (TAL) that catalyzes the conversion of tyrosine to p-coumaric acid in certain bacteria. To modify the p-coumaric acid pathway in plants, we isolated a gene encoding TAL from a photosynthetic bacterium, Rhodobacter sphaeroides, and introduced the gene (RsTAL) in Arabidopsis thaliana. Analysis of metabolites revealed that the ectopic over-expression of RsTAL leads to higher accumulation of anthocyanins in transgenic 5-day-old seedlings. On the other hand, 21-day-old seedlings of plants expressing RsTAL showed accumulation of higher amount of quercetin glycosides, sinapoyl and p-coumaroyl derivatives than control. These results indicate that ectopic expression of the RsTAL gene in Arabidopsis enhanced the metabolic flux into the phenylpropanoid pathway and resulted in increased accumulation of flavonoids and phenylpropanoids.

Identification and functional analysis of novel (p)ppGpp synthetase genes in Bacillus subtilis.

Bacterial alarmone (p)ppGpp, is a global regulator responsible for the stringent control. Two homologous (p)ppGpp synthetases, RelA and SpoT, have been identified and characterized in Escherichia coli, whereas Gram-positive bacteria such as Bacillus subtilis have been thought to possess only a single RelA-SpoT enzyme. We have now identified two genes, yjbM and ywaC, in B. subtilis that encode a novel type of alarmone synthetase. The predicted products of these genes are relatively small proteins ( approximately 25 kDa) that correspond to the (p)ppGpp synthetase domain of RelA-SpoT family members. A database survey revealed that genes homologous to yjbM and ywaC are conserved in certain bacteria belonging to Firmicutes or Actinobacteria phyla but not in other phyla such as Proteobacteria. We designated the proteins as small alarmone synthetases (SASs) to distinguish them from RelA-SpoT proteins. The (p)ppGpp synthetase function of YjbM and YwaC was confirmed by genetic complementation analysis and by in vitro assay of enzyme activity. Molecular genetic analysis also revealed that ywaC is induced by alkaline shock, resulting in the transient accumulation of ppGpp. The SAS proteins thus likely function in the biosynthesis of alarmone with a mode of action distinct from that of RelA-SpoT homologues.