Team:Macquarie Australia/Composite Part
Magnesium chelatase plasmid
BBa_K1998000
The chlorophyll biosynthesis pathway shares similar precursors and enzymes with the heme biosynthetic pathway. The main common precursor between these two pathways is Protoporphyrin IX (PPIX). PPIX is a tetrapyrrole that can be synthesised by many organisms from simple precursors such as glycine, succinyl CoA or glutamate.
The fate of the PPIX is dependent upon which enzyme is present to convert PPIX to the next product. For instance, ferrochelatase converts PPIX to FePPIX – a precursor for heme. Our Magnesium chelatase plasmid is an assembly of six biobricks previously registered by a past Macquarie iGEM team.
Each individual biobrick was assembled in the following order using 3A assembly: pLac-chlI1-chlD-gun4-chlI2-cTH1-pLac-chlH [BBa_R0010], [BBa_K1326008], [BBa_K1080011], [BBa_K1080005] and [BBa_K1640019]. Five of these genes code for different subunits of the enzyme Magnesium chelatase, with the cTH1 gene encoding a component of a later enzyme in the pathway, the oxidative cyclase.
Magnesium chelatase is responsible for catalysing the insertion of Mg2+ into Protoporphyrin IX which is the precursor for chlorophyll biosynthesis. All genes within this plasmid are sequences obtained from Chlamydomonas reinhardtii and codon optimised to be expressed in E. coli. Lac promoters are also present at the front of the chlI1 and chlH gene with an RBS sequence at the beginning of all genes.
The magnesium chelatase is a complex enzyme consisting of a chlI1:chlD:chlI2 hexameric enzymatic motor complex which forms in the presence of ATP. The chlH and gUN4 proteins bind Protoporphyrin IX and form an activated substrate complex which behaves as a substrate for the motor complex to insert magnesium into the bound Protoporphyrin IX upon ATP hydrolysis.
The assembly of this complex requires milliMolar concentrations of Mg2+ and ATP to assemble. While the substrate complex also requires microMolar concentrations of Protoporphyrin IX and chlH for optimal activity.
This part has the potential to be used with other plasmids and genes to complete the chlorophyll biosynthesis pathway.
1. The 150kDa chlH protein is highly expressed and visible on SDS-PAGE and confirmed by proteomic analysis. This should sufficient to produce Mg-Protoporphyrin IX.
2. Mg-protoporphyrin is not produced in DH5- E. coli containing this plasmid when induced with IPTG. But Mg-protoporphyrin IX is produced in a hemH mutant background of E. coli containing this plasmid when induced with IPTG.
3. The Mg-protoporphyrin IX produced in this strain demonstrates that metabolic engineering of E. coli is required to generate sufficient Protoporphyrin IX substrate for the assembled magnesium chelatase complex to successfully make Mg-protoporphyrin IX.
