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Biosynthetic Engineering and Green Manufacturing Applications for Siderophore Yersiniabactin

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