No abstract

Two respiratory-deficient nuclear petites, FY23 delta pet191 and FY23 delta cox5a, of the yeast Saccharomyces cerevisiae were generated using polymerase-chain-reaction-mediated gene disruption, and their respective ethanol tolerance and productivity assessed and compared to those of the parental grande, FY23WT, and a mitochondrial petite, FY23 rho(0). Batch culture studies demonstrated that the parental strain was the most tolerant to exogenously added ethanol with an inhibition constant, Ki, of 2.3% (w/v) and a specific rate of ethanol production, qp, of 0.90 g ethanol g dry cells-1 h-1. FY23 rho(0) was the most sensitive to ethanol, exhibiting a Ki of 1.71% (w/v) and qp of 0.87 ethanol g dry cells-1 h-1. Analyses of the ethanol tolerance of the nuclear petites demonstrate that functional mitochondria are essential for maintaining tolerance to the toxin with the 100% respiratory-deficient nuclear petite, FY23 delta pet191, having a Ki of 2.14% (w/v) and the 85% respiratory-deficient FY23 delta cox5a, having a Ki of 1.94% (w/v). The retention of ethanol tolerance in the nuclear petites as compared to that of FY23 rho(0) is mirrored by the ethanol productivities of these nuclear mutants, being respectively 43% and 30% higher than that of the respiratory-sufficient parent strain. This demonstrates that, because of their respiratory deficiency, the nuclear petites are not subject to the Pasteur effect and so exhibit higher rates of fermentation.

The genetic engineering of yeasts used in commercial processes can be both time-consuming and laborious. This is because industrial yeasts possess largely uncharacterised genomes, which frequently carry at least tzvo copies of any gene. Such strains are usually devoid of auxotrophic or other genetic markers and this requires the incorporation of positively selectable (and often heterologous) genes into plasmids or other transforming DNA molecules. In this paper, we demonstrate that multiple gene deletions may be readily performed in industrial yeasts. Using a specially designed loxPkanMX4 gene replacement cassette, we deleted the tzvo PET191 alleles essential to respiration in the diploid, high alcohol-producing, wine yeast, Kl. The tivo integrated deletion cassettes, which rendered the respiratory-deficient mutant, K2Apetl91ab, resistant to the antibiotic geneticin were then excised from the genome folloiving the expression of a ere recombinase gene harboured on the multi-copy plasmid YEP351-cre-cyh. This plasmid ivas maintained in the mutant under the selective pressure of the antibiotic cycloheximide and then removed when both genes had been successfully deleted. Batch fermentations were performed in homebrew style for strains Kl and JCZApetl91ab and revealed a 40% higher volumetric ethanol production rate and a 9% higher ethanol ceiling for the mutant. Tltis demonstrates that, because of their respiratory deficiency, nuclear petites are not siibject to the Pasteur effect and so exhibit higher rates of fermentation. Furthermore, nuclear petites cannot metabolise the product of fermentation, ethanol, allowing higher ethanol titres to be achieved. We believe that the method of strain manipulation demonstrated here ivill be of interest to scientists in the alcoholic beverages industry, who wish to delete genes in production yeast strains, while simultaneously ensuring the removal of all foreign coding sequences.

The design of controllers for a continuous selection technique (BOICS; Brown and Oliver, 1982) is considered. This technique is used to obtain microbial mutants that are tolerant to extreme environmental stress. Applications of BOICS have been hampered by the problem of controller design. In this paper, a modified implementation of BOICS is considered which has a number of practical advantages. A model-based approach to controller design is taken. The case in which the stress is due to an inhibitory substance in the growth environment is considered. The analysis is intended to be applicable to any reasonable combination of organism and inhibitor. Conventional linear and time-invariant controllers are considered. Guidelines for the selection of controller parameters' values are suggested. The application of these guidelines requires that certain process parameters' values be identified. Methods by which these parameters' values can be identified are suggested. Simulation results indicate that the resulting controllers perform satisfactorily. This is confirmed by experimental data from a model selection experiment. A recipe for the design of controllers is a necessary part of a protocol for BOICS. It is hoped that the solution to the controller design problem that is offered in this paper will encourage further applications for the technique.

Researchers are becoming increasingly concerned that the confidentiality of their novel biomolecule sequences is being jeopardised, particularly when these sequences are either submitted to sequence databases or uploaded as query terms onto internet-based bioinformatic software suites. The researcher's fears stem from the fact that the actual uploading of their sequences acts as a novelty destroying prior disclosure or publication, and that this may subsequently preclude valid patent protection for the sequences. This article addresses the key issues involved in the analyses of biomolecules, highlighting potential risks taken by many researchers in regard to patent protection and suggests possible ways in which these risks may be mitigated.