Engineering

Experiments for Picrocrocin Production

We have produced Parts (BBa_K4955001) and succeeded in biosynthesizing Picrocrocin, a secondary metabolite of Saffron, in E. coli for the first time in the world. This was achieved by following the DBTL cycle, which is a fundamental principle of iGEM.

Overview

Design 1:

From the beginning, our goal was to biosynthesize picrocrocin in E. coli under culture conditions that did not include any special substrates. Therefore, we designed a new metabolic pathway (enzyme group A) that was known to biosynthesize 4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde (HTCC) (precursor of picrocrocin) in E. coli under the conditions described above and a new metabolic pathway (enzyme group B) that was known to biosynthesize picrocrocin in vitro from HTCC. We decided to biosynthesize picrocrocin by introducing the metabolic pathway (enzyme group A), which was known to biosynthesize HTCC (precursor of picrocrocin) in E. coli, and the enzyme group B, which was known to convert HTCC to picrocrocin in vitro, into BL21(DE3) strain.

Build 1:

We designed Parts (BBa_K4955000) as enzyme group A and received a plasmid from RIKEN BRC (https://web.brc.riken.jp/en/) cloned into the Duet-1 vector, pRK404. Next, Parts (BBa_K4955001) was designed as enzyme B, synthesized by Teist Bioscience, and cloned into the pET21a vector. The two plasmids were then introduced into the BL21(DE3) strain, which was constructed to have both enzyme group A and enzyme B.

Test 1:

Lycopene, β-Carotene, and Zeaxanthin, which are intermediates in the biosynthetic pathway to HTCC, are carotenoids. If the colonies of the BL21(DE3) strain produced in Build 1 are yellow, the production of the intermediates has been successful, but if the colonies are white, even the production of the intermediates has not been successful.

Learn 1:

When we checked the colonies of the strain produced in Build 1, we found that they were clearly white in color. The color did not change even after several days, indicating that not even carotenoids, which are intermediates in the biosynthetic pathway of picrocrocin, were being produced.

Design 2:

In Round 1, enzyme group A was designed downstream of the tac promoter, whereas enzyme group B was designed downstream of the T7 promoter; since the T7 promoter is a very strong promoter, only enzyme B was expressed in the cells, possibly suppressing the expression of enzyme group A. The T7 promoter is a very strong promoter.

Build 2:

The plasmid was redesigned so that enzyme group A and enzyme group B were downstream of the same promoter. Then, transformation of BL21(DE3) strain was performed.

Test 2:

As in Round 1, we first checked the production of intermediates in color.

Learn 2:

The color of the colony was confirmed to be yellow. Therefore, we conducted a culture for substance production and analyzed by HPLC-MS, which confirmed the production of picrocrocin.