S Approach Prototype for Continues Copper Removal Heterologous Production Rust Removal Conclusion/Future Direction Biosynthetic Engineering and Green Manufacturing Applications for Siderophore Yersiniabactin . Introduction Metal solutions treated with XAD-Ybt resin were analyzed using ICP or a plate reader (as shown). This image is a photograph of our constructed prototype of our proposed wastewater treatment system for Precious Plate, Inc. to incorporate as a wastewater treatment system. The working prototype includes a wastewater source, a centrifugal pump, three packed columns containing (1) activated carbon, (2) Our novel XAD-Ybt resin, and (3) the commercial metal scavengers. The system is designed such that valves and be used to redirect the stream flow in order to compare the metal removal efficiency of each of the materials. XAD + Cu XAD-Ybt + Cu Acknowledgments We would like to thank Precious Plate, Inc. for working with us and allowing us to use their facilities to further our research in this area. Furthermore, we would like to thank NYSP2I and NSF-Icorps for providing funding to facilitate our research. Mahmoud Kamal Ahmadi ([email protected] ) Advising Faculty: Dr. Blaine Pfeifer School of Engineering and Applied Sciences, University at Buffalo Objectives Heterologous Production of Yersiniabactin Copper Removal Rust Removal Siderophores are strong iron chelating agents. Due to their high affinity for iron, they are promising for medicinal, industrial, and environmental applications with various metals. Yersiniabactin (Ybt) is a siderophore that comes from the bacteria Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica. Yersiniabactin-Fe Fe 3+ Success established a production platform independent of handling the native Y. pestis pathogen and capable of extensive engineering given the recombinant features of E. coli. Siderophore Fe(III) ion 1 2 3 4 Yersinia pestis Yersinia pestis Escherichia coli Genetic Transfer The biosynthetic process required expression of genes encoding two high molecular weight proteins (HMWP1 and HMWP2) to form a mixed nonribosomal peptide synthetase-polyketide synthase complex utilizing three cysteines, a malonyl-CoA unit, and a salicylate starter unit in addition to S-adenosylmethionine (SAM) and NADPH (Figure 1). Endogenous salicylate production allowed for successful heterologous biosynthesis as the remaining substrates and cofactors were native to E. coli metabolism. 0 10 20 30 40 50 60 20 40 60 90 120 Copper Removal (%) Time (min) XAD Ybt-XAD 0 10 20 30 40 50 60 70 80 0.25 1 2.5 5 10 Copper Removal (%) Initial Concentration of Copper (mg/L) XAD Ybt-XAD 0 10 20 30 40 50 60 70 80 90 100 1st extraction 2nd extraction 3rd extraction Copper Removal (%) XAD Ybt-XAD + Zincon Borate buffer Measure using (ICP-MS) Metal solution (50 ppb) Shake for 30 min . Or XAD resin XAD resin Ybt ybtE HMWP1 pBP205 HMWP2 ybtU pBP198 E. coli irp9 pCDF-irp9 3. Extraction 4. Concentrate 5. Final product 1. Growth 2. Gene expression Without pre-treatment column water 0 20 40 60 80 100 0 20 40 60 80 100 Copper Removal (%) Time (min) Backwash with pH=12 Our resin Commercial scavenger With activated carbon pre-treatment column 65 70 75 80 85 90 95 100 105 0 10 20 30 40 50 60 Copper Removal (%) Time (min) Our resin Commercial scavenger water activated carbon 535.1 0 50 100 150 200 500 510 520 530 540 550 Abundance (A.U.) m/z 0 10 20 30 40 50 60 Rust Removal (%) 1,10-phenanthroline assay Yersiniabactin (Ybt) is a mixed nonribosomal peptide-polyketide natural product natively produced by the pathogen Yersinia pestis. This pathway has been engineered for expression and biosynthesis using Escherichia coli as a heterologous host. The biosynthetic process for Ybt formation has been improved t through the incorporation of a dedicated step to eliminate the need for exogenous precursor provision. Produced Ybt were tested in applications that highlight the metal chelating nature of the compound. More specifically, the compound is being tested for industrial wastewater heavy metal removal and rust removal the goal of aiding the environmental and economic outcomes.