biosynthetic engineering and green manufacturing
TRANSCRIPT
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 ([email protected])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
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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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Co
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Time (min)
XAD Ybt-XAD
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Co
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Initial Concentration of Copper (mg/L)
XAD Ybt-XAD
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1st extraction 2nd extraction 3rd extraction
Co
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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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Co
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Time (min)
Backwash with pH=12
Our resin
Commercial scavenger
With activated carbon pre-treatment column
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Time (min)
Our resin
Commercial scavenger
water
activated carbon
535.1
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Ab
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A.U
.)
m/z
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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.