Team:SUIS Shanghai/Design
Design
Once our team decided upon expression of vibrioferrin, we began to face into the challenge of obtaining the associated biosynthetic genes. A gene cluster consisting of five open reading frames for the biosynthesis and transport of vibrioferrin was previously identified through cloning of chromosomal loci around siderophore receptor regions in the halophilic gram-negative pathogenic marine bacterium Vibrio parahaemolyticus (Tanabe et al., 2003). This bacteria has been known to be capable of acquiring iron through the action of the native siderophore vibrioferrin (Yanamoto et al., 1994). The five genes are named pvsABCDE with pvs standing for “V. parahaemolyticus vibrioferrin synthesis”.
Fig 1 - Gene Cluster for the biosynthesis and secretion of Vibrioferrin in the organism Vibrio parahaemolyticus. (Source: NCBI)
The transcription of these genes is regulated by the Fur (ferric uptake regulator) protein in response to exogenous iron concentrations through the Fur box promoter region (Tanabe et al., 2003). Vibrio parahaemolyticus synthesizes and releases vibrioferrin through the action of these genes, of which it is strongly believed that pvsA encodes an enzyme catalyzing the ester bond formation between citrate and ethanolamine, both PvsB and PvsD, are annotated as enzymes for amide bond formation between 2-ketoglutarate and L-alanine with PvsE thought to encode and enzyme which catalyzes decarboxylation from an amino acid (probably serine) to an ethanolamine moiety, while PvsC being the transport protein involved in secretion of the siderophore (Tanabe et al., 2003; and Fujita et al., 2011).
Fig 2 - Theoretical pathway for the biosynthesis of Vibrioferrin by enzymes encoded by genes pvsABCE. (source: Fujita et al., 2011)
Our design aimed for the expression of this gene cluster, was achieved by placing the genes under regulation from an inducible promoter region on an expression plasmid, so that it would not be under regulation of the Fur box. When the Fur box is lost, vibrioferrin isolation yields by heterologous expression in e.coli were at 92.6 mg/L compared to measured values of 2.5 mg/L, for wild type V. parahaemolyticus, which requires strictly iron- regulated media (Fujita et al. 2011).
With this information, we decided to overexpress the synthesized gene cluster under a T7 promoter region using the expression vector pET30a(+). DNA sequences were obtained from NCBI and synthesis of the genetic sequences for the four biosynthetic enzymes and one transport protein within the cluster was completed by genscript China. This resulted in a large total construct of 12kbp which would need to be transformed into expression cells. To ensure such a large vector could be successfully transformed, we obtained high efficiency BL21 (DE3) cells which would also ensure our controlled expression of the gene cluster via the T7 expression system. We decided to culture our cells in normal LB broth to ensure any native siderophores produced by our e.coli would not be turned on by the Fur box, ensuring any detection of siderophore activity in our culture broth would be because of heterologous expression of our T7 induced gene cluster.
Fig 3 - Map of expression vectoir pET-30a(+). (Source: Genscript)
Fig 4 - Plasmid map of pET-30a(+) vector containing the biosynthetic gene cluster pvsA-E.
References:
Fujita, M., Kimura, N., Sakai, A., Ichikawa, Y., Hanyu, T., & Otsuka, M. (2011). Cloning and heterologous expression of the vibrioferrin biosynthetic gene cluster from a marine metagenomic library. Bioscience, Biotechnology, and Biochemistry, 75(12), 2283-7.
Tanabe, Tomotaka, Funahashi, Tatsuya, Nakao, Hiroshi, Miyoshi, Shin-Ichi, Shinoda, Sumio, & Yamamoto, Shigeo. (2003). Identification and Characterization of Genes Required for Biosynthesis and Transport of the Siderophore Vibrioferrin in Vibrio parahaemolyticus. The Journal of Bacteriology, 185(23), 6938-6949.
Yamamoto, Okujo, Yoshida, Matsuura, & Shinoda. (1994). Structure and iron transport activity of vibrioferrin, a new siderophore of Vibrio parahaemolyticus. Journal of Biochemistry, 115(5), 868-74.
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