Cell Factory• Cells capable of secreting valuable proteins• GRAS (Generally Recognized as Safe)microorganism• Escherichia coli• Lactobacillus spp.
Problem• Which of the 8 secretory proteins of B. subtilisNZ8900 is/are found in large amounts in thegrowth medium?
Methodology1. 8 genes cloned using SURE systemoverexpression vector pNZ8902 or pNZ89012. Genes were fused to C-terminal 6His-tagencoding sequences for imunnodetection3. Bacterial cultures grown to midexponentialphase and gene expression was induced withsubtilin4. Testing protein production (whole cell extract;extracellular; membrane; cytoplasmic) andMicroarray analyses
Conclusion• Data reveal possible bottlenecks in theprotein overproduction process• Recombinations in the genes controlling theexpression of XynA and NprE would producevaluable proteins in high amounts fit forindustrial production
Microarray Analyses• measure the expression levels of largenumbers of genes simultaneously orto genotype multiple regions of a genome
General Effects of Overproduction• Downregulation of the sdpABC (sporulating delayprotein) operon– Role in apoptosis– Kills non-sporulating siblings and feeding on the deadcells under conditions of nutrient limitation• Upregulation of class I heat-shock genes codingfor molecular chaperon groES and groEL– Protects the cell from toxic accumulation of mis- orunfolded protein