Engineering Prophylactic Biosynthesis of an Antituberculosis Antibiotic

Abstract

Tuberculosis is a deadly respiratory disease caused by the bacterium Mycobacterium tuberculosis which does not currently have a fully effective vaccine. The goal of this project is to develop an effective prophylaxis for tuberculosis using mammalian cells to biosynthesize the anti‐tuberculosis antibiotic, d‐cycloserine (dCS). We took two approaches to activate this biosynthetic pathway in mammalian cells. Initially, Chinese Hamster Ovarian cancer cells (CHO) were transfected with plasmids encoding all six dCS biosynthesis enzymes (dcsA‐G) and isolated total protein was tested for synthetic capability. The expression of the genes detected by fluorescence microscopy was low and the in‐vitro dCS biosynthesis from lysates was not detected by HPLC. Currently, individual enzymes are being optimized, starting with dcsE, which converts L‐serine to O‐acetyl‐L‐serine. To focus on dcsE, human lung epithelial cancer cells (A549) were transfected with a higher concentration of DNA. High dcsE expression was detected by fluorescence microscopy and a Western Blot was used to detect the presence of our GFP tagged dcsE protein. The Western Blot indicated that GFP dimerized and there is no dcsE tagged with GFP.