Design

Overview

The ultimate goal we aimed to achieve when designing our constructs was to enable the overproduction of protoporphyrin IX (PPIX) in an Escherichia coli chassis. Previous literature has demonstrated that different intermediates in the heme biosynthetic pathway, including protoporphyrin IX, could be overproduced by increasing expression of intermediate enzymes [1][2]. These studies determined that overproduction of protoporphyrin IX (PPIX), our target intermediate in the biosynthesis pathway, could be produced at high yields (~80 μM) by overexpressing the genes hemA, hemB, hemC, hemD¸ hemE, and hemF in combination. Thus, Team UAlberta sought to design a unified system which packages the requisite hemA through hemF genes for the express purpose of overproducing PPIX and aim to improve on previous approaches.

Heme Synthesis Pathway

Heme and other metalloporphyrins are ubiquitous in nature as they are integral parts of many enzymes including various cytochromes and hemoglobin. There are two major routes which heme is produced which and it is determined by how D-aminolevulinic acid (ALA) is obtained [3]:

• The Shemin-pathway in which ALA is produced in one reaction between glycine and succinyl-CoA.
• The C5-pathway in which ALA is produced by two reactions involving a glutamyl-tRNA precursor.

In most bacteria, including E. coli, the prevalent pathway is the C5-pathway, though, the intermediates and steps following the production of ALA are the same in both pathways. A schematic of the heme synthesis pathway is outlined in Figure 1.