Aromatic amino acids are precursors of numerous plant secondary metabolites with diverse biological functions. Many of these secondary metabolites are already being used as active pharmaceutical or nutraceutical ingredients, and there are numerous exploratory studies of other compounds with promising applications. p-Coumaric acid is derived from aromatic amino acids and, besides being a valuable chemical building block, it serves as precursor for biosynthesis of many secondary metabolites, such as polyphenols, flavonoids, and some polyketides. Here we developed a p-coumaric acid-overproducing Saccharomyces cerevisiae platform strain. First, we reduced by-product formation by knocking out phenylpyruvate decarboxylase ARO10 and pyruvate decarboxylase PDC5. Second, different versions of feedback-resistant DAHP synthase and chorismate mutase were overexpressed. Finally, we identified shikimate kinase as another important flux-controlling step in the aromatic amino acid pathway by overexpressing enzymes from Escherichia coli, homologous to the pentafunctional enzyme Aro1p and to the bifunctional chorismate synthase-flavin reductase Aro2p. The highest titer of p-coumaric acid of 1.93 ± 0.26 g L(-1) was obtained, when overexpressing tyrosine ammonia-lyase TAL from Flavobacterium johnsoniaeu, DAHP synthase ARO4(K229L), chorismate mutase ARO7(G141S) and E. coli shikimate kinase II (aroL) in Δpdc5Δaro10 strain background. To our knowledge this is the highest reported titer of an aromatic compound produced by yeast. The developed S. cerevisiae strain represents an attractive platform host for production of p-coumaric-acid derived secondary metabolites, such as flavonoids, polyphenols, and polyketides.

Resveratrol is a natural antioxidant compound, used as food supplement and cosmetic ingredient. Microbial production of resveratrol has until now been achieved by supplementation of expensive substrates, p-coumaric acid or aromatic amino acids. Here we engineered the yeast Saccharomyces cerevisiae to produce resveratrol directly from glucose or ethanol via tyrosine intermediate. First we introduced the biosynthetic pathway, consisting of tyrosine ammonia-lyase from Herpetosiphon aurantiacus, 4-coumaryl-CoA ligase from Arabidopsis thaliana and resveratrol synthase from Vitis vinifera, and obtained 2.73 ± 0.05 mg L(-1) resveratrol from glucose. Then we over-expressed feedback-insensitive alleles of ARO4 encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate and ARO7 encoding chorismate mutase, resulting in production of 4.85 ± 0.31 mg L(-1) resveratrol from glucose as the sole carbon source. Next we improved the supply of the precursor malonyl-CoA by over-expressing a post-translational de-regulated version of the acetyl-CoA carboxylase encoding gene ACC1; this strategy further increased resveratrol production to 6.39 ± 0.03 mg L(-1). Subsequently, we improved the strain by performing multiple-integration of pathway genes resulting in resveratrol production of 235.57 ± 7.00 mg L(-1). Finally, fed-batch fermentation of the final strain with glucose or ethanol as carbon source resulted in a resveratrol titer of 415.65 and 531.41 mg L(-1), respectively.

Five sequential Cryptococcus neoformans isolates recovered from an AIDS patient with recurrent meningitis were analyzed. Four isolates were fluconazole susceptible, while the fifth isolate developed fluconazole resistance. Analysis of the 14-␣ lanosterol demethylase gene (ERG11) showed a point mutation in the resistant strain responsible for the amino acid substitution G484S.Mechanisms of azole resistance already described for yeasts include altered affinity of lanosterol 14-␣ demethylase (ERG11) to azole drugs due to target site mutation or its overexpression and decreased accumulation of drugs due to enhanced energydependent drug efflux (5, 13). Changes in the azole affinity of the lanosterol 14-␣ demethylase have already been related to low-level fluconazole resistance in Cryptococcus neoformans isolates (15). In addition, the decreased affinity of lanosterol 14-␣ demethylase for azole derivatives due to mutations that contributes to the increase in the MICs of fluconazole has been described for sequential clinical isolates of Candida albicans (5, 6). To elucidate if this mechanism could also be implicated in the resistance of C. neoformans to azole, we compared the ERG11 genomic sequence in five sequential isolates recovered from recurrent episodes of cryptococcal meningitis.Clinical case. The five strains of C. neoformans were isolated from a 33-year-old male patient who had been positive for human immunodeficiency virus since 1990 and was presenting advanced AIDS (CD4 ϩ count, Ͻ100 cells/mm 3 ) and recurrent cryptococcosis. The first episode of cryptococcosis was diagnosed in August 1997 at Hospital Fernandez, Buenos Aires, Argentina. During the following 15 months, four more episodes of cryptococcal meningitis were detected and documented by cultures recovered from cerebrospinal fluid. In the first episode the patient was treated with amphotericin B (AMB), and for the rest of the episodes a fluconazole therapy at different doses was always established, reaching a cumulative dose of 336 g at the moment of the fifth episode. The five isolates from each episode were identified as C. neoformans var. grubii by the following parameters: morphology, assimilation and fermentation of carbon and nitrogen compounds, and molecular taxonomy.Susceptibility testing was performed by microdilution and E-test methods. Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were used as quality control strains throughout the experiments (7). Microdilution method. The susceptibility testing followed the NCCLS recommendations (7) but included some modifications as previously described (11). Briefly, the susceptibility testing included RPMI medium supplemented with 2% glucose as the assay medium (RPMI-2% glucose), an inoculum size of 10 5 CFU/ml, flat-bottomed trays, spectrophotometric reading at 530 nm, incubation at 30°C, and shaking at 350 rpm for 48 h (11). The antifungal agents used in the study were as follows: AMB (Sigma Aldrich Quimica S.A., Madrid, Spain), 5-flucytosine (5FC) (Sigma Aldrich Quimica), fluconazole ...