According to the World Health Organization, Tuberculosis (TB) is the second leading cause of death by a single infectious disease behind COVID-19. Despite a century of effort, the current TB vaccine does not effectively prevent pulmonary TB, promote herd immunity, or prevent transmission. Therefore, we seek to develop a genetic prophylaxis for TB. We have determined D-cycloserine to be the optimal target for this approach due to its relatively short six-enzyme biosynthetic pathway. D-CS is a second-line antibiotic for TB that inhibits bacterial cell wall synthesis. The first committed step towards D-CS synthesis is catalyzed by the L-serine-O-acetyltransferase (DcsE) which converts L-serine and acetyl-CoA to O-acetyl-L-serine (L-OAS). To test if the D-CS pathway could be an effective prophylaxis for TB in human cells, we endeavored to express DcsE in human cells and test its functionality. We overexpressed DcsE tagged with FLAG and GFP in A549 lung cancer cells as determined using fluorescence microscopy. We observed that purified DcsE catalyzed the synthesis of L-OAS as observed by HPLC-MS. Therefore, DcsE synthesized in human cells is a functional enzyme capable of converting L-serine and acetyl-CoA to L-OAS demonstrating the first step towards D-CS production in human cells.

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.

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Tuberculosis (TB) is the second leading cause of death by a single infectious disease behind COVID-19. Despite a century of effort, the current TB vaccine does not effectively prevent pulmonary TB, promote herd immunity, or prevent transmission. Therefore, alternative approaches are needed. We seek to develop a cell therapy that produces an effective antibiotic in response to TB infection. D-cycloserine (D-CS) is a second-line antibiotic for TB that inhibits bacterial cell wall synthesis. We have determined D-CS to be the optimal candidate for anti-TB cell therapy due to its effectiveness against TB, relatively short biosynthetic pathway, and its low-resistance incidence. The first committed step towards D-CS synthesis is catalyzed by the L-serine-O-acetyltransferase (DcsE) which converts L-serine and acetyl-CoA to O-acetyl-L-serine (L-OAS). To test if the D-CS pathway could be an effective prophylaxis for TB, we endeavored to express functional DcsE in A549 cells as a human pulmonary model. We observed DcsE-FLAG-GFP expression using fluorescence microscopy. DcsE purified from A549 cells catalyzed the synthesis of L-OAS as observed by HPLC–MS. Therefore, human cells synthesize functional DcsE capable of converting L-serine and acetyl-CoA to L-OAS demonstrating the first step towards D-CS production in human cells.