(A) study on the production of microbial insecticide by recombinant escherichia coli

Abstract

880-01Physiological studies on the expression of Bacillus thuringiensis subsp. tenebrionis gene in recombinant Escherichia coli 537 were carried out to find optimal culture condition. It was necessary to shift culture temperature from 30℃ to 42℃ to express the insecticidal protein gene. Plasmid stability was dependent on cultivation time and temperature. Cells lost plasmids more quickly at higher temperature and as cultivation was prolonged. The optimal cultivation strategy was growing cells for 7 hours at 30℃ followed by 8 hours at 42℃. Soybean hydrolysate was apparently effective in improving insecticidal protein production. After induction, the content of insecticidal protein was 35.02% of total cellular protein, which was 5.4 times higher than that before induction. When yeast extract was used as a nitrogen source, the largest amount of insecticidal protein was obtained. The optimal cultivation pH for the production of insecticidal protein was 6.5. The optimal concentrations of amino acids were 0.1, 0.1, and 0.01 g/L for threonine, leucine, and tryptophan, respectively. Batch and fed-batch fermentations were carried out with different levels of glucose and nitrogen sources. Cell growth was severely repressed by acetate, when glucose concentration was high and yeast extract was used as a nitrogen source in modified M9 medium. Formation of acetate was reduced by using some amino acids instead of yeast extract.