Engineering microbial cells with metal chelating hydroxylated unnatural amino acids for removable of synthetic pollutants from water.

Lead (Pb2+) is a well-known heavy metal and toxic synthetic industrial pollutant in the ecosystem and causes severe threats to living organisms. It is paramount to develop a sustainable microbial engineering approach to remove synthetic pollutants from the environment. Genetic code engineering is emerging as an important microbial engineering tool in biosciences to biosynthesis congener protein production beyond the canonical set of natural molecules and expand the chemistries of living cells. Here, we prepare cells expressing unnatural amino acid encoded congener proteins for effectively removable toxic synthetic industrial pollutants (Pb2+) with high binding efficiency. Native and the developed congener proteins expressing cells adapted the Langmuir and Sips adsorption model that recommends uniform adsorption with Pb2+ ions. This could be due to a more significant number of functional groups on the protein surface. Fluorescence spectroscopic, field emission scanning electron microscope, X-ray photoelectron spectroscopic analysis, and protein-metal molecular stimulation coordination allowed us to explore the role of hydroxylation on Pb2+ adsorption. The bioreactor filled with immobilized protein-containing active granules showed >90% of lead removal in the contaminated water samples. The desorption of bound Pb2+ from GFP and its variants were studied by varying the pH to reuse the proteins for subsequent usage. We observed that about 70% of the GFP and its variants could be recycled and >75% of fluorescence efficiency could be recovered. Among all the variants, GFPHPDP exhibits high affinity and maintains the reusability efficiency in 7 consecutive cycles. These results suggest that genetic code engineering of cells encoding unnatural amino acids could be a next-generation microbial engineering tool for manipulating and developing the microbial strain's selective and effective removal of synthetic pollutants from the environment.