Use of an ethanol sensor for feedback control of growth and expression of TBV25H in Saccharomyces cerevisiae.

A process for production of a malaria transmission blocking vaccine candidate under the control of the ADH2 promoter in Saccharomyces cerevisiae was developed. Monitoring and controlling the ethanol concentration during the process is essential for successful expression of the recombinant protein. A simple sensor accomplishing this task has been developed, the principle of its operation is the following: air-flow through silicone tubing submerged in the media picks up ethanol, which is detected by an alcohol sensor that relays a signal to a controller regulating the amount of ethanol added to the culture. The sensor was used successfully in high cell density cultures of various scales.

Engineering Pichia pastoris for biocatalysis: co-production of two active enzymes.

High levels of active glycolate oxidase from spinach (GO) and active catalase T from Saccharomyces cerevisiae (catT) have been co-produced in the methylotrophic yeast Pichia pastoris (Pp). In sequential rounds of transformation using two selectable markers, multiple copies of the genes encoding GO and catT were integrated into the Pp chromosome under control of the methanol inducible alcohol oxidase I promoter, resulting in a strain designated MSP8.6. MSP8.6 is a second-generation biocatalyst used for the conversion of glycolate to glyoxylate in the presence of a reaction component which inhibits endogenous Pp catalase. This work demonstrates a significant advance in the utility of recombinant Pp for commercial bioprocess development.

High-level production of spinach glycolate oxidase in the methylotrophic yeast Pichia pastoris: engineering a biocatalyst.

Glycolate oxidase (GO) is a flavo-enzyme that catalyzes the oxidation of glycolate, and is useful for the biocatalytic production of glyoxylate. We have produced high levels of spinach GO in the methylotrophic yeast Pichia pastoris (Pp), by chromosomal integration of multiple copies of an expression cassette containing the GO coding sequence under control of the methanol-inducible alcohol oxidase I promoter. Under fermentation conditions, greater than 250 units of GO per gram of cells (wet weight) was obtained, corresponding to roughly 20-30% of soluble cell protein. This recombinant Pp strain was used as a whole-cell biocatalyst for conversion of glycolic acid to glyoxylic acid.

Preparative isolation of plasmid DNA with sedimentation field flow fractionation.

Sedimentation field flow fractionation (SFFF) can be used to isolate plasmids preparatively from crude cellular lysates. Total purification time is about 3/4 day, including lysate preparation. The purity and yield of plasmids isolated by SFFF appear to be at least equivalent to those prepared by traditional methods. Molecular-weight data also are supplied rapidly by SFFF without the need for standards.

Thermophilic anaerobic bacteria which ferment hemicellulose: characterization of organisms and identification of plasmids.

Seven thermophilic anaerobic bacteria which ferment xylan were isolated from natural geothermal features in the western United States. Typically, these strains were Gram-negative non-sporeforming rods with an unusual double-layered cell wall which resembled that observed in Thermobacteroides acetoethylicus. The strains differed from known thermophilic anaerobes in their ability to utilize a very wide variety of carbohydrates and in their ability to grow in a chemically-defined medium and/or at pH 3.5. Four of the strains contained cryptic plasmids of 1.2 or 1.5 X 10(6) daltons. The taxonomic characteristics of the strains are discussed in terms of their relatedness to those of Thermoanaerobium, Thermoanaerobacter, and Thermobacteroides species.