Mayooreshwar Rajankar

I am a Systems and Synthetic Biologist with Keen interest in method development to inquire cell metabolism for strain design for chemical production. We apply state of the art molecular techniques to tinker cells genetic makeup and observe its consequential prototypes . We use genome scale systems biology approach to investigate the metabolism of engineered strains and iterate the process to propose and validate experimental strategies.A step wise engineering principle broken down into Design, Build, Test and Analyze was used. E.coli strains were designed for the production of two molecules, Violacein and Poly(R)-hydroxybutyrate (PHB), belonging to two very different classes A step wise engineering principle broken down into Design, Build, Test and Analyze was used. E.coli strains were designed for the production of two molecules, Violacein and Poly(R)-hydroxybutyrate (PHB), belonging to two very different classes

Violacein is a bacterial bis-indole pigment of clinical significance having antibacterial, antitumoral, trypanocidal and antiprotozoan properties. It requires a five gene operon (~ 8 Kbp) from Chromobaterium violaceium to synthesize violacein from two molecules of L-tryptophan. Since violacein is low yield-high value product use of plasmid for expression of violacein producing enzyme is feasible solution. To improve the violacein yield knockouts were generated using λ Red recombinase system to remove bottlenecks towards violacein production in E.coli metabolism.

PHB is the naturally most abundant among polyhydroxyalkanoate (PHA) class of bio-polymers produced by microorganism and plants during nitrogen deprivation and physiological stress. Naturally PHB biosynthesis operon consists of three genes, PhaA PhaB and PhaC. The three enzymes catalyze the conversion of acetyl-CoA into PHB. (R)-3-hydroxybutyrate-CoA (3HB), which is the monomer for PHB is generated through two routes in E.coli metabolism. One of the routes is from central metabolism Glycolysis -> Acetyl-CoA-> 3HB and another route is from fatty acid degradation. Since E.coli can produce 3HB, instead of introducing entire PHB producing operon, in this study, enzymes augmenting the incomplete PHB producing pathway were introduced in E.coli. Here we have used the synthetic genetic circuit, constructed using two enzymes, including Propionate-CoA Transferase (pct_ap) from Acetobacter pasteurianus which transfers CoA group from acyl-CoA (mostly Butanoyl-CoA) to acetate and Polyhydroxyalkonate polymerase (phaC_cv) from C. violaceium, which polymerizes the monomer into PHB. PHB is a low value-high yield molecule, and the precursor for PHB originates from central carbon metabolism. Therefore PHB production does not require knockout strategy rather it requires more stable expression for large volume culture without specialized media and antibiotic selection. To achieve this we have genome engineered the PHB producing genetic circuit (4.4 Kbp) into E.coli genome using Tn7 transposon based system.

Selected publication

Rajankar, M. P , Sapna Ravindranathan , P.R Rajamohanan and Anu Raghunathan Absolute Quantitation Of Poly(R)-3-Hydroxybutyric Acid Using Spectrofluorometry In Recombinant Escherichia Coli. Oxford Academic-Biology Methods and Protocols. doi:10.1093/biomethods/bpy007

Immanuel, S. R. C., Banerjee, D., Rajankar, M. P. & Raghunathan, A. Integrated constraints based analysis of an engineered violacein pathway in Escherichia coli. Biosystems 171, 10–19 (2018).