Atypical yeasts identified as Saccharomyces cerevisiae by MALDI-TOF MS and gene sequencing are the main responsible of fermentation of chicha, a traditional beverage from Peru.

Chicha is a drink prepared in several Andean countries from Inca's times by maize fermentation. Currently this fermentation is carried out in familiar artesanal "chicherías" that make one of the most known types of chicha, the "chicha de jora". In this study we isolate and identify the yeasts mainly responsible of the fermentation process in this type of chicha in 10 traditional "chicherías" in Cusco region in Peru. We applied by first time MALDI-TOF MS analysis for the identification of yeast of non-clinic origin and the results showed that all of yeast strains isolated belong to the species Saccharomyces cerevisiae. These results agree with those obtained after the analysis of the D1/D2 and 5.8S-ITS regions. However the chicha strains have a phenotypic profile that differed in more than 40% as compared to that of current S. cerevisiae strains. To the best of our knowledge this is the first report concerning the yeasts involved in chicha fermentation.

Proteinase K-catalyzed synthesis of linear and star oligo(L-phenylalanine) conjugates.

Chemoenzymatic synthesis of peptides is a green and clean chemical reaction that offers high yields without using organic synthesis and serves as an alternative to traditional peptide synthesis methods. This report describes the chemoenzymatic synthesis of oligo(L-phenylalanine) mediated by proteinase K from Tritirachium album, which is one of the most widely used proteases in molecular biological studies. The synthesized linear oligo-phenylalanine showed a unique self-assembly in aqueous solutions. To further functionalize linear oligo(L-phenylalanine) as a low-molecular-weight gelator, it was cosynthesized with tris(2-aminoethyl)amine to obtain star-oligo(L-phenylalanine), which was bioconjugated to demonstrate its self-assembly into fluorescent fibers. The self-assembled fibers of star-oligo(L-phenylalanine) formed fibrous networks with various branching ratios, which depended on the molecular weights and molecular aspect ratios of star-oligo(L-phenylalanine). This is the first study to demonstrate that proteinase K is a suitable enzyme for chemoenzymatic cosynthesis of oligopeptides and star-shaped heteropeptides.

A novel Kluyveromyces marxianus strain with an inducible flocculation phenotype.

Flocculation is a very useful phenotype for industrial yeast strains, since it facilitates cell harvest and represents an easy way of cell immobilization in continuous fermentation processes. The present work represents the first time that an inducible flocculation phenotype has been generated in a non flocculent strain of Kluyveromyces marxianus. This was accomplished by expressing Saccharomyces cerevisiae FLO5 gene in K. marxianus CECT 11769 strain. The FLO 5 gene was placed under the control of an EPG promoter, not repressed by glucose and induced by anoxia. Our experimental approach successfully generated two novel K. marxianus flocculent phenotypes: one inducible and one constitutive. The constitutive phenotype originated from deletions in the FLO5 promoter region, indicating the existence of putative upstream repressor site involved in oxygen regulation of the EPG1 promoter. The novel strains here generated had a unique set of characteristics that provided an advantage, over the wild-type strain, for the industrial co-production of ethanol and polygalacturonase.

Construction of a novel Pichia pastoris strain for production of xanthophylls.

In this study, we used the yeast carotenogenic producer Pichia pastoris Pp-EBIL strain, which has been metabolically engineered, by heterologously expressing ß-carotene-pathway enzymes to produce ß-carotene, as a vessel for recombinant astaxanthin expression. For this purpose, we designed new P. pastoris recombinant-strains harboring astaxanthin-encoding genes from carotenogenic microorganism, and thus capable of producing xanthophyllic compounds. We designed and constructed a plasmid (pGAPZA-WZ) containing both the ß-carotene ketolase (crtW) and ß-carotene hydroxylase (crtZ) genes from Agrobacterium aurantiacum, under the control of the GAP promoter and containing an AOX-1 terminator. The plasmid was then integrated into the P. pastoris Pp-EBIL strain genomic DNA, producing clone Pp-EBILWZ. The recombinant P. pastoris (Pp-EBILWZ) cells exhibited a strong reddish carotenoid coloration and were confirmed, by HPLC, to produce not only the previous described carotenoids lycopene and ß-carotene, but also de novo synthesized astaxanthin.

Influence of nutritional and environmental factors on ethanol and endopolygalacturonase co-production by Kluyveromyces marxianus CCEBI 2011.

Ethanol and endopolygalacturonase (endoPG) are simultaneously produced by the yeast Kluyveromyces marxianus CCEBI 2011. The aim of this study was to determine the optimal combination of seven environmental and nutritional variables, as well as the influence of each one, with respect to the fermentation process in yeast cultures in which sugarcane juice was the substrate. Simplex sequential optimization showed that after 15 runs the optimal conditions were: pH, 4.6; temperature, 31 ºC; total reducing sugars (TRS), 125 g/l; (NH(4))(2)SO(4), 2.48 g/l; (NH(4))(2)HPO(4), 2.73 g/l; CaCl(2), 0.33 g/l and MgSO(4)·7H(2)O, 0.54 g/l. Under these conditions, the ethanol concentration was 47.6 g/l and endoPG concentration was 9.8 U/ml, which represented increases of 22% and 10%, respectively, over the concentrations obtained under suboptimal conditions. Temperature and (NH(4))(2)SO(4) supplementation were the most significant factors influencing the co-production process.

Influence of nutritional and environmental factors on ethanol and endopolygalacturonase co-production by Kluyveromyces marxianus CCEBI 2011

Ethanol and endopolygalacturonase (endoPG) are simultaneously produced by the yeast Kluyveromyces marxianus CCEBI 2011. The aim of this study was to determine the optimal combination of seven environmental and nutritional variables, as well as the influence of each one, with respect to the fermentation process in yeast cultures in which sugarcane juice was the substrate. Simplex sequential optimization showed that after 15 runs the optimal conditions were: pH, 4.6; temperature, 31 ºC; total reducing sugars (TRS), 125 g/l; (NH(4))(2)SO(4), 2.48 g/l; (NH(4))(2)HPO(4), 2.73 g/l; CaCl(2), 0.33 g/l and MgSO(4)·7H(2)O, 0.54 g/l. Under these conditions, the ethanol concentration was 47.6 g/l and endoPG concentration was 9.8 U/ml, which represented increases of 22% and 10%, respectively, over the concentrations obtained under suboptimal conditions. Temperature and (NH(4))(2)SO(4) supplementation were the most significant factors influencing the co-production process (AU)

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