Cloning Of Promoters From Pseudomonas Maltophila, Applying And Microcalorimetric Study

Pseudomonas maltophila is a wide distributed gram negative bacteria, with a strong metabolic activities and adaptation ability. With its rich metabolism, the bacteria can produce important enzymes including protease, amylase, tyrosinase and lipase. In our laboratory, investigators have cloned Pseudomonas maltophila tyrosinase encoding mel gene into E. coli and constructed the engineering strain E. coli/pWSY. To increase melanin production, promoter activity fragments from P. maltophila were cloned and used to construct a high melanin production engineering strain E. coli/ pAWS. Moreover, promoter fragment was used in an attempt to regulate protease gene from Bacillus licheniformis in E. coli. Besides, 8 different promoter activity fragments from P maltophila were subjected to microcalorimetric measurement analysis and the results otained are satisfying.In this work, promoter probe pKK232-8 containing a promoterless cat gene, was used to clone fragments containing promoter activity from Pseudomonas matophila chromosomal DNA into E. coli. Transformants were subjected to chloramphencol resistance level, and the results revealed a high promoter activity of 1000ug/ml performed by pPASl recombinant plasmid holding a 1.4 kb promoter fragment. Hybridization experiments and SDS-PAGE have shown that pPASl fragment belong to Pseudomonas maltophila and give expression to an additional protein of 22Da.mel gene, a tyrosinase encoding gene on pWSY, was cleaved and subcloned at the downstream region of the pPASl promoter fragment on pKK232-8. Fermentation results revealed an increase in melanine production by 70.6% in E. coli/pPAS1 than in E. coli/pWSY.In further study, pPASl promoter fragment was used to regulate theexpression of Bicillus licheniformis protease gene in E. coli. The 1.4kb pPASl fragment was inserted in both direction at the upstream region of the protease gene on pAPR8-3. The results indicated that, pPAS1 fragment is unable to give expression to Bacillus licheniformis protease gene in E. coli and that, different protease genes have different expression models.The cloned promoters were used for microcalorimetric studies of promoter activity. The experiments were performed in isothermal microcalorimeter LKB 2277 and promoters of different activity level were used. The results obtained by calorimetric information showed that this method is a very sensitive method, and that different promoter DNA fragments in E. coli implied differents metabolism levels. Microcalorimetric method can be then a very sensitive shortcut tool for testing promoter function and foreign gene expression. These results also indicate that although calorimetry cannot identify the reaction system and the non- specificity of the reactions within the living cell, calorimetric studies on promoter activity is a breakthrough in specificity of solely reaction for microcalorimetric studies in microorganisms.