ABSTRACT
BACKGROUND: The fermentation of sugars to alcohols by microbial systems underpins many biofuel initiatives. Short chain alcohols, like n-butanol, isobutanol and isopropanol, offer significant advantages over ethanol in terms of fuel attributes. However, production of ethanol from resistant Saccharomyces cerevisiae strains is significantly less complicated than for these alternative alcohols. RESULTS: In this study, we have transplanted an n-butanol synthesis pathway largely from Clostridial sp. to the genome of an S. cerevisiae strain. Production of n-butanol is only observed when additional genetic manipulations are made to restore any redox imbalance and to drive acetyl-CoA production. We have used this butanol production strain to address a key question regarding the sensitivity of cells to short chain alcohols. In the past, we have defined specific point mutations in the translation initiation factor eIF2B based upon phenotypic resistance/sensitivity to high concentrations of exogenously added n-butanol. Here, we show that even during endogenous butanol production, a butanol resistant strain generates more butanol than a butanol sensitive strain. CONCLUSION: These studies demonstrate that appreciable levels of n-butanol can be achieved in S. cerevisiae but that significant metabolic manipulation is required outside of the pathway converting acetyl-CoA to butanol. Furthermore, this work shows that the regulation of protein synthesis by short chain alcohols in yeast is a critical consideration if higher yields of these alcohols are to be attained.
ABSTRACT
Following the purification of an odorant-binding protein (OBP) from rabbit nasal mucosa, we have identified, purified and partially characterized two additional OBPs from the nasal tissue of the same animal species. OBP-II is a monomer of 21 kDa and isoelectric point 4.2; OBP-III is a dimer with subunits of 23 kDa and isoelectric point 4.8. Like OBP-I, both these new members bind the odorant 2-isobutyl-3-methoxypyrazine. The partial amino acid sequences of the three OBPs, determined by Edman degradation, confirm that they are members of the OBP family, but reveal poor similarity between them. However, higher similarity is found between each OBP and other members of the lipocalin family. In particular, OBP-I is most similar to bovine OBP (55% identity in the N-terminal region), OBP-II is > 50% identical, limited to its first 18 amino acids, to mouse OBP-I and porcupine OBP-II, while OBP-III shares 26 out of the first 40 amino acids with major urinary protein (MUP) 4, a member of the mouse salivary proteins. The possible role of these proteins in olfactory transduction is also discussed.
Subject(s)
Nasal Mucosa/chemistry , Receptors, Odorant/isolation & purification , Amino Acid Sequence , Animals , Chromatography, Ion Exchange/methods , Electrophoresis, Polyacrylamide Gel/methods , Molecular Sequence Data , Molecular Weight , Olfactory Mucosa/chemistry , Protein Binding , Pyrazines/metabolism , Rabbits , Receptors, Odorant/analysis , Sequence Alignment , Sequence Analysis , Sequence Homology, Amino AcidABSTRACT
2-Isobutyl-3-methoxypyrazine, a potent bell-pepper odorant, binds to cow olfactory mucosa homogenate. The receptor is saturable in the micromolar range and is competitively inhibited by other bell-pepper odourants, but not by other pyrazines of different odours. Other tissues do not bind 2-isobutyl-3-methoxypyrazine at a significant extent. We suggest that this receptor is involved in odour discrimination.
