Overview

Currently, heterologous expression of (S)-equol synthesis pathway enzymes by engineered E. coli is an effective means of producing (S)-equol^[1]. In order to achieve low-cost and efficient production of (S)-equol, our team engineered a series of RBS sequences to realize the regulation of (S)-equol expression at the translational level. We designed and obtained a mutant library of RBS using mCherry as a model protein, aiming to achieve regulated expression of (S)-equol biosynthetic enzymes and potentially increase (S)-equol yield while decreasing production costs.

We provide useful contribution for future iGEM teams, as these parts could be used in various contexts, as follows:

1. Add new information to the existing part：promoter nar(fnr regulated) (BBa K3408000 )

2. Add new information to the existing part: mCherry (BBa_J04450)

Table 1. Parts Contribution Collection

Design and Build

1. Add new information to the existing part：promoter nar(fnr regulated) (BBa K3408000 )

We designed eight RBS sequences downstream of the nar promoter using the RBS Calculator (https://salislab.net/software/), as shown in the table below.

Table 2. RBS sequences designed by RBS Calculator

We then introduced the RBS sequence mutations by whole plasmid PCR. As shown in Figure 1, we successfully amplified pETM6-pnar-RBS(1-8)-mCherry plasmids containing different RBS sequences.

Figure 1. Electrophoresis results of whole plasmid PCR.

2. Add new information to the existing part: mCherry (BBa_J04450), obtained a new part BBa_K4842003

To test the regulation of protein expression by the series of RBS sequences downstream of the nar promoter, we used mCherry as a model protein. mCherry has superior photostability and is the most commonly used red fluorescent protein, making it an ideal choice for tracking expression levels. Therefore, we constructed the pETM6-pnar-mCherry plasmid. We inserted mCherry into the pETM6-pnar plasmid and obtained this plasmid by transformation. Then, we designed eight RBS mutants using the RBS Calculator (https://salislab.net/software/). Using the primers with mutation sites for whole plasmid PCR of pETM6-pnar-mCherry, we obtained a pETM6-pnar-RBS (1-8)-mCherry mutant library.

Finally, we inoculated these transformants and tested their fluorescence intensities. The results showed that as culture time increased, the fluorescence intensity of pETM6-pnar-mCherry cells became stronger, while pETM6-pnar cells had no fluorescence (Figure 2A). This indicates successful induction of mCherry expression by the nar promoter. Three RBS sequences (RBS 4, RBS 6, and RBS 1) exhibited the strongest fluorescence in pETM6-pnar-RBS (1-8)-mCherry cells (Figure 2B). These test results demonstrate that mutating the RBS can achieve regulated protein expression, and these three RBS sequences have the potential to regulate the expression of (S)-equol pathway enzymes in the future.

Figure 2 Fluorescence intensity assay results of RBS mutant library

Reference

[1] Wang Z, Li X, Azi F, et al. Biosynthesis of (S)-Equol from Soy Whey by Metabolically Engineered Escherichia coli [J]. Journal of agricultural and food chemistry, 2023, 71(15): 6110-6119.