Regulatory Gene Effects On Recombinant Human Growth Hormone Production By Bacillus Subtilis

• Main Author: Sahin, Merve
• Other Authors: Calik, Pinar
• Format: Others
• Language: Eng
• Published: METU 2010
• Subjects: TA Bioengineering 164; Bacillus subtilis pH Regulatory genes Recombinant human growth hormone
• Online Access: http://etd.lib.metu.edu.tr/upload/12612312/index.pdf

In this study, regulatory gene effects on recombinant human growth hormone (rhGH) production by Bacillus subtilis were investigated. For this purpose, firstly Bacillus strains, which are deficient in abrB, aprE, degQ, degS, degU, scoC, sinI, sinR, and spo0A genes, were selected according to the regulatory gene network of aprE gene (serine alkaline protease gene of B. subtilis) since due to the degQ promoter and the pre-signal sequence of subC gene cloned in front of the hGH gene, hGH is produced by mimicking the serine alkaline protease synthesis. R-Bacillus strains were constructed by transformation of pMK4::pre(subC)::hGH plasmid to the selected strains. Thereafter, by the laboratory scale experiments, strains having the highest hGH production capacity were determined as scoC, aprE, sinR, and degU knockout strains. Using these strains, fermentation experiments were carried out in pilot-scale bioreactor in defined medium. Effect of pH control was also investigated and the highest cell and hGH concentration was obtained by scoC knockout strain in pH controlled operation as 1.62 kg m-3 and 126 g m-3, respectively. By this strain, the overall product and cell yield on total substrate were found as 16.12 g kg-1 and 0.15 g g-1, respectively. Furthermore, the highest total protease activity was attained by degU knockout strain as 65 U cm-3. On the other hand, maximum total organic acid secretion was determined as 1.31 kg m-3 in aprE knockout strain.