Production of abscisic acid in the oleaginous yeast Yarrowia lipolytica.

Abscisic acid (ABA) is a phytohormone with applications in agriculture and human health. ABA can be produced by Botrytis cinerea, a plant pathogenic filamentous fungus. However, the cultivation process is lengthy and strain improvement by genetic engineering is difficult. Therefore, we engineered the oleaginous yeast Yarrowia lipolytica as an alternative host for ABA production. First, we expressed five B. cinerea genes involved in ABA biosynthesis (BcABA1,BcABA2,BcABA3,BcABA4 and BcCPR1) in a Y. lipolytica chassis with optimized mevalonate flux. The strain produced 59.2 mg/L of ABA in small-scale cultivation. Next, we expressed an additional copy of each gene in the strain, but only expression of additional copy of BcABA1 gene increased the ABA titer to 168.5 mg/L. We then integrated additional copies of the mevalonate pathway and ABA biosynthesis encoding genes, and we expressed plant ABA transporters resulting in an improved strain producing 263.5 mg/L and 9.1 mg/g dry cell weight (DCW) ABA. Bioreactor cultivation resulted in a specific yield of 12.8 mg/g DCW ABA; however, surprisingly, the biomass level obtained in bioreactors was only 10.5 g DCW/L, with a lower ABA titer of 133.6 mg/L. While further optimization is needed, this study confirms Y. lipolytica as a potential alternative host for the ABA production.

Association of vitamin D receptor genetic variants with bone mineral density and inflammatory markers in rheumatoid arthritis.

BACKGROUND AND AIMS: Vitamin D receptor (VDR) genetic variants are considered to have a role in the pathogenesis of rheumatoid arthritis (RA). This study examines an association of FokI, BsmI, ApaI and TaqI with RA, as well as with bone mineral density (RA with normal bone mineral density, RA-NBMD; RA with associated osteopenia, RA-OSTP; and RA with associated osteoporosis, RA-OP) and inflammatory markers. MATERIALS AND METHODS: VDR genetic variants were tested in 248 subjects using the PCR-RFLP method. RESULTS: Significant differences were observed in the distribution of FokI genotypes between RA patients (p < 0.001), or subgroups (RA-NBMD, RA-OSTP, RA-OP) (p = 0.035, p = 0.02, p < 0.001, respectively) and controls. Prevalence of FokI f allele was significantly higher in RA group (p < 0.001) and subgroups (p = 0.003, p = 0.021, p < 0.001, respectively) compared to controls. An increased susceptibility to RA-OSTP was revealed in BsmI/ApaI Ba (AC) haplotype carriers (p = 0.012). A significantly higher erythrocyte sedimentation rate values were obtained in FokI FF compared to Ff + ff carriers (54.57 ± 23.73 vs. 22.83 ± 12.42; p < 0.001) within the RA-NBMD subgroup. CONCLUSION: The results of the study indicate an association of RA with FokI genetic variant and increased susceptibility to RA in f allele carriers, as well as to RA-OSTP in BsmI/ApaI Ba (AC) haplotype carriers.

Engineering the Yeast Saccharomyces cerevisiae for the Production of L-(+)-Ergothioneine.

L-(+)-Ergothioneine (ERG) is an unusual, naturally occurring antioxidant nutraceutical that has been shown to help reduce cellular oxidative damage. Humans do not biosynthesise ERG, but acquire it from their diet; it exploits a specific transporter (SLC22A4) for its uptake. ERG is considered to be a nutraceutical and possible vitamin that is involved in the maintenance of health, and seems to be at too low a concentration in several diseases in vivo. Ergothioneine is thus a potentially useful dietary supplement. Present methods of commercial production rely on extraction from natural sources or on chemical synthesis. Here we describe the engineering of the baker's yeast Saccharomyces cerevisiae to produce ergothioneine by fermentation in defined media. After integrating combinations of ERG biosynthetic pathways from different organisms, we screened yeast strains for their production of ERG. The highest-producing strain was also engineered with known ergothioneine transporters. The effect of amino acid supplementation of the medium was investigated and the nitrogen metabolism of S. cerevisiae was altered by knock-out of TOR1 or YIH1. We also optimized the media composition using fractional factorial methods. Our optimal strategy led to a titer of 598 ± 18 mg/L ergothioneine in fed-batch culture in 1 L bioreactors. Because S. cerevisiae is a GRAS ("generally recognized as safe") organism that is widely used for nutraceutical production, this work provides a promising process for the biosynthetic production of ERG.

Penicillium arizonense, a new, genome sequenced fungal species, reveals a high chemical diversity in secreted metabolites.

A new soil-borne species belonging to the Penicillium section Canescentia is described, Penicillium arizonense sp. nov. (type strain CBS 141311T = IBT 12289T). The genome was sequenced and assembled into 33.7 Mb containing 12,502 predicted genes. A phylogenetic assessment based on marker genes confirmed the grouping of P. arizonense within section Canescentia. Compared to related species, P. arizonense proved to encode a high number of proteins involved in carbohydrate metabolism, in particular hemicellulases. Mining the genome for genes involved in secondary metabolite biosynthesis resulted in the identification of 62 putative biosynthetic gene clusters. Extracts of P. arizonense were analysed for secondary metabolites and austalides, pyripyropenes, tryptoquivalines, fumagillin, pseurotin A, curvulinic acid and xanthoepocin were detected. A comparative analysis against known pathways enabled the proposal of biosynthetic gene clusters in P. arizonense responsible for the synthesis of all detected compounds except curvulinic acid. The capacity to produce biomass degrading enzymes and the identification of a high chemical diversity in secreted bioactive secondary metabolites, offers a broad range of potential industrial applications for the new species P. arizonense. The description and availability of the genome sequence of P. arizonense, further provides the basis for biotechnological exploitation of this species.