Biotransformation using halotolerant yeast in seawater: a sustainable strategy to produce R-(-)-phenylacetylcarbinol.

Acyloin condensation between benzaldehyde and decarboxylated pyruvate results in the production of R-(-)-phenylacetylcarbinol, a chiral precursor of the drug ephedrine. Huge research efforts have been made to improve the conditions of this reaction and to avoid the generation of by-products. Recently, we reported the advantages of using whole cells of the yeast Debaryomyces etchellsii as biocatalysts for this purpose. In this work, a new strategy, which fulfills green chemistry principles, is proposed and is based on using seawater as a gentle solvent. We demonstrate that, under these conditions, several improvements can be made compared to employing freshwater: (1) the conversion of the starting material in (R)-PAC is higher and with a minimum production of by-products; (2) it is possible to increase at least twofold the benzaldehyde load in the reaction medium; (3) cells can maintain their activity after several recycling rounds, which makes (R)-PAC production an easy and economical process.

The C-terminal region of the Hot1 transcription factor binds GGGACAAA-related sequences in the promoter of its target genes.

Response to hyperosmotic stress in the yeast Saccharomyces cerevisiae involves the participation of the general stress response mediated by Msn2/4 transcription factors and the HOG pathway. One of the transcription factors activated through this pathway is Hot1, which contributes to the control of the expression of several genes involved in glycerol synthesis and flux, or in other functions related to adaptation to adverse conditions. This work provides new data about the interaction mechanism of this transcription factor with DNA. By means of one-hybrid and electrophoretic mobility assays, we demonstrate that the C-terminal region, which corresponds to amino acids 610-719, is the DNA-binding domain of Hot1. We also describe how this domain recognizes sequence 5'-GGGACAAA-3' located in the promoter of gene STL1. The bioinformatics analysis carried out in this work allowed the identification of identical or similar sequences (with up to two mismatches) in the promoter of other Hot1 targets, where central element GGACA was quite conserved among them. Finally, we found that small variations in the sequence recognized by Hot1 may influence its ability to recognize its targets in vivo.

A sequence element downstream of the yeast HTB1 gene contributes to mRNA 3' processing and cell cycle regulation.

Histone mRNAs accumulate in the S phase and are rapidly degraded as cells progress into the G(2) phase of the cell cycle. In Saccharomyces cerevisiae, fusion of the 3' untranslated region and downstream sequences of the yeast histone gene HTB1 to a neomycin phosphotransferase open reading frame is sufficient to confer cell cycle regulation on the resulting chimera gene (neo-HTB1). We have identified a sequence element, designated the distal downstream element (DDE), that influences both the 3'-end cleavage site selection and the cell cycle regulation of the neo-HTB1 mRNA. Mutations in the DDE, which is located approximately 110 nucleotides downstream of the HTB1 gene, lead to a delay in the accumulation of the neo-HTB1 mRNA in the S phase and a lack of mRNA turnover in the G(2) phase. The DDE is transcribed as part of the primary transcript and binds a protein factor(s). Maximum binding is observed in the S phase of the cell cycle, and mutations that affect the turnover of the HTB1 mRNA alter the binding activity. While located in the same general region, mutations that affect 3'-end cleavage site selection act independently from those that alter the cell cycle regulation.