Biosynthetic Engineering and Green Manufacturing

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-Ybtresin, and (3) the commercial metal

scavengers. The system is designedsuch that valves and be used to

redirect the stream flow inorder to compare the

metal removal efficiency of each of the

materials.

XAD + Cu XAD-Ybt + Cu

AcknowledgmentsWe 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 (mahmoudk@buffalo.edu)Advising Faculty: Dr. Blaine PfeiferSchool 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 applicationswith various metals. Yersiniabactin (Ybt) is a siderophore that comes from thebacteria Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica.

Yersiniabactin-Fe

Fe3+

Success established a production platform independent of handling the native Y. pestispathogen 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 coliGenetic Transfer

The biosynthetic process required expressionof genes encoding two high molecular weightproteins (HMWP1 and HMWP2) to form a

mixed nonribosomal peptide synthetase-polyketidesynthase complex utilizing three cysteines, amalonyl-CoA unit, and a salicylate starter unit inaddition to S-adenosylmethionine (SAM) and NADPH(Figure 1). Endogenous salicylate production allowed forsuccessful heterologous biosynthesis as the remainingsubstrates and cofactors were native to E. coli metabolism.

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Initial Concentration of Copper (mg/L)

XAD Ybt-XAD

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1st extraction 2nd extraction 3rd extraction

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XAD Ybt-XAD

+ ZinconBorate buffer

Measure using (ICP-MS)

Metal solution (50 ppb)

Shake for 30 min.

Or

XAD resinXAD resin

Ybt

ybtE HMWP1

pBP205

HMWP2 ybtU

pBP198E. coli

irp9

pCDF-irp9

3. Extraction

4. Concentrate

5. Final product

1. Growth

2. Gene expression

Without pre-treatment column

water

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Backwash with pH=12

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With activated carbon pre-treatment column

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activated carbon

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1,10-phenanthroline assay

Yersiniabactin (Ybt) is a mixed nonribosomal peptide-polyketide naturalproduct natively produced by the pathogen Yersinia pestis. This pathway has

been engineered for expression and biosynthesis using Escherichia coli as aheterologous host. The biosynthetic process for Ybt formation has been improved tthrough the incorporation of a dedicated step to eliminate the need for exogenous

precursor provision. Produced Ybt were tested in applications that highlight the metal chelatingnature of the compound. More specifically, the compound is being tested for industrial wastewaterheavy metal removal and rust removal the goal of aiding the environmental and economic outcomes.