In Escherichia coli , three additional proteins having l -cysteine desulfhydrase activity were identified as O -acetylserine sulfhydrylase-A, O -acetylserine sulfhydrylase-B, and MalY protein, in addition to tryptophanase and cystathionine β-lyase, which have been reported previously. The gene disruption for each protein was significantly effective for overproduction of l -cysteine and l -cystine. Growth phenotype and transcriptional analyses suggest that tryptophanase contributes primarily to l -cysteine degradation.

The keratinase produced by Bacillus licheniformis PWD-1 was induced by feather powder. Maximal enzyme production could be achieved by culturing in a medium containing 1% hammer-milled feather powder (100 mesh) at 45 degrees C for 30 h. Maximal growth of PWD-1 was achieved at 50 degrees C, and maximal enzyme induction was at 45 degrees C. The molecular mass and isoelectric point of this enzyme were 31.4 kDa and 8.5, respectively. This enzyme was stable from pH 5 to 12. The optimal reaction pHs for feather powder and casein were 8.5 and 10.5 to 11.5, respectively. The optimal reaction temperature was 50 degrees C to 55 degrees C. The relative activity of this enzyme toward casein, feather powder, keratin, elastin, and collagen was 100:52:41:18:7, and 100:56:32:3 for Suc-AAPL-pNA, Suc-AAPF-pNA, Suc-AAPM-pNA, and Suc-AAVA-pNA (Suc, succinyl; pNA, p-nitrophenylanilide).

The red carotenoid astaxanthin possesses higher antioxidant activity than other carotenoids and has great commercial potential for use in the aquaculture, pharmaceutical, and food industries. In this study, we produced astaxanthin in the budding yeast Saccharomyces cerevisiae by introducing the genes involved in astaxanthin biosynthesis of carotenogenic microorganisms. In particular, expression of genes of the red yeast Xanthophyllomyces dendrorhous encoding phytoene desaturase ( crtI product) and bifunctional phytoene synthase/lycopene cyclase ( crtYB product) resulted in the accumulation of a small amount of β-carotene in S. cerevisiae . Overexpression of geranylgeranyl pyrophosphate (GGPP) synthase from S. cerevisiae (the BTS1 gene product) increased the intracellular β-carotene levels due to the accelerated conversion of farnesyl pyrophosphate to GGPP. Introduction of the X. dendrorhous crtS gene, encoding astaxanthin synthase, assumed to be the cytochrome P450 enzyme, did not lead to astaxanthin production. However, coexpression of CrtS with X. dendrorhous CrtR, a cytochrome P450 reductase, resulted in the accumulation of a small amount of astaxanthin. In addition, the β-carotene-producing yeast cells transformed by the bacterial genes crtW and crtZ , encoding β-carotene ketolase and hydroxylase, respectively, also accumulated astaxanthin and its intermediates, echinenone, canthaxanthin, and zeaxanthin. Interestingly, we found that these ketocarotenoids conferred oxidative stress tolerance on S. cerevisiae cells. This metabolic engineering has potential for overproduction of astaxanthin and breeding of novel oxidative stress-tolerant yeast strains.

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.