Oudemansin and 9-methoxystrobilurin derivatives with antimalarial activity from cultures of the basidiomycete Favolaschia minutissima: assignments of the absolute configurations of the isoprene-derived units.

Five undescribed polyketide metabolites, oudemansins E (1), M (2), P (3), and Q (4), and 9-methoxystrobilurin I (5), were isolated from cultures of basidiomycete Favolaschia minutissima TBRC-BCC 19434. A γ-lactone derivative (6) of noroudemansin A (8), which was previously reported as a semisynthetic compound, was also isolated. The absolute configuration of the isoprene-derived moiety of the known cometabolite 9-methoxystrobilurin E (9) was determined to be 2'R,6'S by comparison of the experimental and calculated ECD data, which was correlated to the new derivative 1. These compounds exhibited antimalarial activity against Plasmodium falciparum K1 (multidrug-resistant strain). A putative minor natural product, namely 9-methoxystrobilurin P (13), was prepared by semisynthesis, which exhibited significant antimalarial activity (IC50 0.086 µM).

Enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli.

BACKGROUND: Escherichia coli has two L-cysteine biosynthetic pathways; one is synthesized from O-acetyl L-serine (OAS) and sulfate by L-cysteine synthase (CysK), and another is produced via S-sulfocysteine (SSC) from OAS and thiosulfate by SSC synthase (CysM). SSC is converted into L-cysteine and sulfite by an uncharacterized reaction. As thioredoxins (Trx1 and Trx2) and glutaredoxins (Grx1, Grx2, Grx3, Grx4, and NrdH) are known as reductases of peptidyl disulfides, overexpression of such reductases might be a good way for improving L-cysteine production to accelerate the reduction of SSC in E. coli. RESULTS: Because the redox enzymes can reduce the disulfide that forms on proteins, we first tested whether these enzymes catalyze the reduction of SSC to L-cysteine. All His-tagged recombinant enzymes, except for Grx4, efficiently convert SSC into L-cysteine in vitro. Overexpression of Grx1 and NrdH enhanced a 15-40% increase in the E. coliL-cysteine production. On the other hand, disruption of the cysM gene cancelled the effect caused by the overexpression of Grx1 and NrdH, suggesting that its improvement was due to the efficient reduction of SSC under the fermentative conditions. Moreover, L-cysteine production in knockout mutants of the sulfite reductase genes (ΔcysI and ΔcysJ) and the L-cysteine synthase gene (ΔcysK) each decreased to about 50% of that in the wild-type strain. Interestingly, there was no significant difference in L-cysteine production between wild-type strain and gene deletion mutant of the upstream pathway of sulfite (ΔcysC or ΔcysH). These results indicate that sulfite generated from the SSC reduction is available as the sulfur source to produce additional L-cysteine molecule. It was finally found that in the E. coliL-cysteine producer that co-overexpress glutaredoxin (NrdH), sulfite reductase (CysI), and L-cysteine synthase (CysK), there was the highest amount of L-cysteine produced per cell. CONCLUSIONS: In this work, we showed that Grx1 and NrdH reduce SSC to L-cysteine, and the generated sulfite is then utilized as the sulfur source to produce additional L-cysteine molecule through the sulfate pathway in E. coli. We also found that co-overexpression of NrdH, CysI, and CysK increases L-cysteine production. Our results propose that the enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from SSC is a novel method for improvement of L-cysteine production.

The L-cysteine/L-cystine shuttle system provides reducing equivalents to the periplasm in Escherichia coli.

Intracellular thiols like L-cysteine and glutathione play a critical role in the regulation of cellular processes. Escherichia coli has multiple L-cysteine transporters, which export L-cysteine from the cytoplasm into the periplasm. However, the role of L-cysteine in the periplasm remains unknown. Here we show that an L-cysteine transporter, YdeD, is required for the tolerance of E. coli cells to hydrogen peroxide. We also present evidence that L-cystine, a product from the oxidation of L-cysteine by hydrogen peroxide, is imported back into the cytoplasm in a manner dependent on FliY, the periplasmic L-cystine-binding protein. Remarkably, this protein, which is involved in the recycling of the oxidized L-cysteine, is also found to be important for the hydrogen peroxide resistance of this organism. Furthermore, our analysis of the transcription of relevant genes revealed that the transcription of genes encoding FliY and YdeD is highly induced by hydrogen peroxide rather than by L-cysteine. These findings led us to propose that the inducible L-cysteine/L-cystine shuttle system plays an important role in oxidative stress tolerance through providing a reducing equivalent to the periplasm in E. coli.

The outer membrane TolC is involved in cysteine tolerance and overproduction in Escherichia coli.

L-cysteine is an important amino acid in terms of its industrial applications. We previously found marked production of L-cysteine directly from glucose in recombinant Escherichia coli cells by the combination of enhancing biosynthetic activity and weakening the degradation pathway. Further improvements in L-cysteine production are expected to use the amino acid efflux system. Here, we identified a novel gene involved in L-cysteine export using a systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection). Among the 3,985 nonessential gene mutants, tolC-disrupted cells showed hypersensitivity to L-cysteine relative to wild-type cells. Gene expression analysis revealed that the tolC gene encoding the outer membrane channel is essential for L-cysteine tolerance in E. coli cells. However, L-cysteine tolerance is not mediated by TolC-dependent drug efflux systems such as AcrA and AcrB. It also appears that other outer membrane porins including OmpA and OmpF do not participate in TolC-dependent L-cysteine tolerance. When a low-copy-number plasmid carrying the tolC gene was introduced into E. coli cells with enhanced biosynthesis, weakened degradation, and improved export of L-cysteine, the transformants exhibited more L-cysteine tolerance and production than cells carrying the vector only. We concluded that TolC plays an important role in L: -cysteine tolerance probably due to its export ability and that TolC overexpression is effective for L-cysteine production in E. coli.