Team:UAlberta/Design

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.

Figure 1:The two main heme biosynthesis pathways where the first step in (1.i) the C5-pathway and (1.ii) the Shemin-pathway are explicitly denoted. The remaining steps (2-8) depict the steps found in the E. coli endogenous pathway and its associated genes. The first compound produced in this schematic is ALA and the last step depicts heme. Our molecule of interest is PPIX is the last intermediate produced before heme.

Producing PPIX

Studies have shown that a combination of hemA, B, C, D, E, F can push flux through the heme synthesis pathway beyond natural levels, and high titres of up to 80 uM of PPIX can be obtained from E. coli. [1][2]. However, in these studies, the initial two steps of the C5-pathway, mediated by two enzymes encoded by hemA and hemL of E. coli are bypassed by the introduction of the hemA gene from Rhodobacter capsulatus.

hemA from R. capsulatus, encodes for the aminolevulinic acid synthase of the Shemin-pathway, which E. coli was was able to express in these previous studies. The reasons cited for the motivation behind using this alternate route in E. coli include reducing the number of steps needed to generate ALA, from two to one, and that high utilization of the glutamyl-tRNA precursor in the C5-pathway may but a load on metabolism as a component used in protein synthesis is being directly competed for.

With these previous results as a motivation, Team UAlberta also sought to overexpress these six genes in E. coli to fulfill our first objective for APIS: the engineering of a PPIX-producing strain. Thus, we present our design for APIS, which is a six-gene system optimized for inducible expression in E. coli!