Optimization of rice alpha-amylase production using temperature-sensitive mutants of Saccharomyces cerevisiae for the PHO regulatory system.
A typical example of rational system synthesis for bioproduction by the cultivation of microorganisms harboring a recombinant plasmid was studied. First, two temperature-controllable expression systems for a foreign gene in Saccharomyces cerevisiae were constructed. The promoter of the PHO84 gene, which encodes an inorganic phosphate (Pi) transporter of S. cerevisiae and is controlled by Pi concentration through the PHO regulatory system, was used. And two temperature-sensitive mutants of S. cerevisiae for the PHO regulatory system were used as the host with rice alpha-amylase expression vector. One was temperature-sensitive pho81 mutant, SH3337, and the other was temperature-sensitive pho80 mutant, YKU107. When the strain SH3337 is cultivated at a lower temperature, the rice alpha-amylase gene connected downstream of the PHO84 promoter is expressed, but at a higher temperature, it is not. Conversely, with the strain YKU107, the transcription of the rice alpha-amylase gene is induced at a higher temperature and repressed at a lower one. The optimal cultivation temperature strategies for maximum production of rice alpha-amylase in batch cultures of these two recombinant strains were then determined by the Maximum Principle using the relationships of the specific growth and specific production rates to the cultivation temperature. The optimal strategies were easy to realize and were shown to be effective for maximum product. Finally, under these maximum alpha-amylase production strategies, the alpha-amylase activities and alpha-amylase yields in terms of glucose consumption for these recombinant strains were calculated on the basis of experimental data and compared for various operation times. The study demonstrates how a foreign gene expression system can be synthesized using a temperature-sensitive mutant with a given objective and under given constraints.
Uchiyama
K
K
Department of Biotechnology, Faculty of Engineering, Osaka University, Japan.
Ohtani
T
T
Morimoto
M
M
Shioya
S
S
Suga
K
K
Harashima
S
S
Oshima
Y
Y
D001709:Biotechnology / Q000379:methods; D005809:Genes, Regulator; D008432:Mathematical Computing; D009154:Mutation; D012275:Oryza / Q000201:enzymology*; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012680:Sensitivity and Specificity; D013696:Temperature; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000235:genetics
D010940:Plant Proteins; D000516:alpha-Amylases