Contribution

To tackle multidrug-resistant bacteria, —we have identified a strategy based on siderophores— iron-chelating molecules used by bacteria, using the Trojan horse method. We selected aerobactin and its genes: iucA-B-C-D optimized for heterologous expression in Escherichia coli with the pBluescript plasmid. Totally [Fe]rocious's contribution to the iGEM community focused on Wet lab experiments involving the various enzymes responsible for aerobactin biosynthesis in the pathogen Klebsiella pneumoniae.

New Individual Parts

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Each component below plays a crucial role in aerobactin biosynthesis. A summary table and a more detailed overview describe the individual enzymes involved. The specific order of these enzymes within the pathway is illustrated below. The integrated pathway, composed of all the enzymes, is also presented.

Detailed Explanation

Part BBa_255DLKJB, referred to as IucD_EcoOpt, encodes the iucD gene, which catalyzes the first step in the biosynthesis of N6-acetyl-N6-hydroxy-L-lysine (ahLys), a critical component of dihydroxamate structure. It functions as an L-lysine N⁶-monooxygenase, hydroxylating the ε-amino group of lysine to produce N⁶-hydroxy-L-lysine [1,2]. This part was optimized for expression in Escherichia coli and used in the Wet Lab experiments of BBa_25ZGK3Y5 assembly.

Part BBa_253NP2T0, referred to as IucB_EcoOpt, encodes the iucB gene, which performs the second and final enzymatic step in ahLys biosynthesis. As an N⁶-acetyltransferase, it converts N⁶-hydroxy-L-lysine into N⁶-acetyl-N⁶-hydroxy-L-lysine [1,2]. This part was also optimized for expression in Escherichia coli and incorporated into the BBa_25ZGK3Y5 assembly.

Part BBa_256Z6XQY, referred to as IucA_EcoOpt, encodes the iucA gene, which initiates the core biosynthesis pathway of aerobactin. It functions as a citryl transferase, ligating citrate to a first molecule of N⁶-hydroxy-L-lysine to form N²-citryl-N⁶-hydroxy-lysine [1,2]. This part was codon-optimized for expression in Escherichia coli and used in the BBa_25ZGK3Y5 assembly.

Part BBa_25TEHQLR, referred to as IucC_EcoOpt, encodes the iucC gene, which acts as the second citryl transferase. Following the enzymatic activity of IucA, it catalyzes the addition of a second citrate fragment onto N⁶-acetyl-N⁶-hydroxy-lysine to yield the bis-citryl hydroxamate structure of aerobactin. This enzyme catalyse carries out the final biochemical reaction in the biosynthesis of the siderophore [1,2]. iucC is included in one of the gBlock (composite parts) used to produce the final assembly (BBa_25ZGK3Y5).

New Composite Parts

Part BBa_25ZGK3Y5, named IucABCD_3FragmentAssembly_EcoOpt, encapsulates the four enzymes needed for the aerobactin biosynthesis within a single codon, optimized for high-level expression in E. coli. It is built with three composite parts (gBlock), containing parts of each iuc gene involved in the biosynthesis of aerobactin. This part has undergone an assembly with a Gibson Assembly kit from NEB with the pBluescript (SK+) plasmid.

The four iuc genes are split across the three gBlock assemblies. iucA is completely found in gBlock 1. iucB is split across gBlock 1 and gBlock 2, and iucC is split across gBlock 2 and gBlock 3. Finally, the iucD gene is found in gBlock 3. The third gBlock ends with the terminator ECK1200296002 and a random sequence (to get a better PCR primer).

Ideas for future iGEM teams

These tools/parts will be practical for other iGEM teams wanting to work with the aerobactin gene or try to synthesize a siderophore. This section illustrates the general process to synthesize a siderophore as well as all the details about the four genes of the NIS pathways for K. pneumoniae.