Week 5: Bio production
This week we discussed about engineering a microbes to produce interesting small molecules and the advantage in yield over conventional chemical synthesis. We reviewed design, build and test strategies that are appropriate for engineering the metabolism of bacteria and yeast.
1. What chassis and pathway will you use?
The target molecule we chose is estrogen (estradiol) beginning with an endogenous cholesterol precursor. It is actually possible to use the cholesterol from egg yolks after some purification [source].
First, we researched metabolic pathways for estrogen biosynthesis and pinpointed the associated enzymes for the synthesis of our molecule (see red squares in Fig. 4).
You can also see the isolated pathway in the following image taken from KEGG. KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies. Our desired pathway is highlighted in yellow.
- cholesterol monooxygenase side-chain-cleaving
- 3beta-hydroxy-Delta5-steroid dehydrogenase
- steroid Delta-isomerase
- aromatase; CYP19A1
- estradiol dehydrogenase
In addition, with CYP1A1 we can go to ESTRIOL (Target estrogen, predominant during reproductive years). The creation of a synthetic protein through 8 enzymes is no small task, so we must next decide how we will test the efficacy of this hypothetical pathway design.
2. How will you measure product formation?
We can add a GFP reporter protein gene gated by an estrogen receptor. “Tightly Regulated, betaestradiol DoseDependent Expression System for Yeast” (2000)
Other methods are commercially available estrogen tests in the form of a Fertility Monitor (right).: