Overview

In this study, we controlled the expression of (S)-equol biosynthetic enzymes in engineered E. coli using the nar promoter induced by anaerobic conditions. This avoided the use of expensive inducer IPTG during fermentation and provided a microaerobic environment favorable for (S)-equol biosynthesis. To regulate the expression levels of exogenous genes, we adjusted the strength of the ribosome-binding site (RBS) by modifying its sequence, thereby influencing the initiation and rate of protein translation without constructing multiple variants. Using mCherry as a model protein, we constructed a nar promoter library with different RBS sequences and characterized the mutant library by fluorescence intensity detection. We aimed to obtain optimal RBS sequences to achieve regulated expression of (S)-equol pathway enzymes.

Our experimental results mainly include:

(1) Construction of plasmid pETM6-pnar,

(2) Construction of plasmid pETM6-pnar-mCherry,

(3) Mutation library construction for the plasmid pETM6-pnar-RBSx-mCherry,

(4) Fluorescence intensity detection of RBS mutant library.

Figure 1 Schematic flow diagram of this experiment.

(*The creation of engineering diagrams received guidance and assistance from Associate Researcher Xia Xiudong, the Jiangsu Academy of Agricultural Sciences.)

1. Construction of plasmid pETM6-pnar

To construct the plasmid pETM6-pnar, we first double-digested the synthetic pCDM4-pnar and pETM6 with NdeI and AvrII restriction enzymes, respectively, as shown in Figure 2A. After recycling the target fragments, we ligated the nar fragment with the pETM6 vector using T4 DNA ligase to obtain the complete recombinant plasmid (Figure 2B).

We then transformed the recombinant plasmid into E. coli DH5α competent cells. After overnight culture, positive transformants grew on LB plates. We identified transformants with the correct bands by colony PCR, and inoculated them for subsequent plasmid extraction. The next day, we extracted plasmid from the transformant for later construction of plasmid pETM6-pnar-mCherry.

Figure 2 Construction result of the plasmid pETM6-pnar.

2. Construction of plasmid pETM6-pnar-mCherry

To construct plasmid pETM6-pnar-mCherry, we first double-digested pET28a-mCherry and pETM6-pnar with XhoI and AvrII restriction enzymes, respectively, as shown in Figure 3A. After recycling the target fragments, we ligated the mCherry fragment with the pETM6-pnar vector using T4 DNA ligase to obtain the complete recombinant plasmid (Figure 3B).

We then transformed the recombinant plasmid into E. coli DH5α competent cells. After overnight culture, positive transformants grew on LB plates. We identified transformants with the correct bands by colony PCR, and inoculated them for subsequent plasmid extraction. The following day, we extracted the plasmid of the transformant for the construction of pETM6-pnar-RBSx-mCherry mutant library containing different RBS sequences.

Figure 3 Construction result of the plasmid pETM6-pnar-mCherry.

3. Mutation library construction for the plasmids pETM6-pnar-RBSx-mCherry

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 1 RBS sequences designed by RBS Calculator

Name	Sequence (5’-3’)
RBS 1	AGGTAGCCG
RBS 2	AGGCCCGAC
RBS 3	AGAAATGCA
RBS 4	AGAAACAAC
RBS 5	AGGACCAAA
RBS 6	AGAGAACAC
RBS 7	AGGCGGGGA
RBS 8	AGAAACAGA

We then introduced the RBS sequence mutations by whole plasmid PCR. As shown in Figure 4, we successfully amplified pETM6-pnar-RBS(1-8)-mCherry plasmids containing different RBS sequences.

Figure 4 Electrophoresis results of whole plasmid PCR.

We transformed pETM6-pnar-RBS (1-8)-mCherry plasmids into competent E. coli BL21(DE3) cells, respectively. After overnight culture, positive transformants grew on LB plates. Colony PCR identification showed the correct bands for pETM6-pnar-RBS (1-5)-mCherry transformants, while no bands were observed for pETM6-pnar-RBS (6-8)-mCherry. This may be due to mistakes in picking colonies or omissions in adding templates. Nevertheless, we inoculated all eight transformants for subsequent fluorescence intensity testing.

Figure 5 Transformation and colony PCR identification results of plasmids pETM6-pnar-RBSx-mCherry

4. Fluorescence intensity detection of RBS mutant library.

We measured mCherry expression every half hour after induction using a fluorometer. Cell density (OD₆₀₀) was measured on a UV/VIS spectrophotometer. Total red fluorescence of whole cells was determined by the fluorometer with excitation at 580 nm and emission at 610 nm. Background from transformants with mutant promoter plasmids was subtracted under the same conditions to obtain actual red fluorescence intensity.

As shown in Figure 6A, as culture time increased, the fluorescence intensity of pETM6-pnar-mCherry cells became stronger, while no fluorescence was detected in pETM6-pnar cells. This indicates successful mCherry expression induction by the nar promoter. As shown in Figure 6B, there were three RBS sequences (RBS 4, RBS 6 and RBS 1) exhibited the strongest fluorescence in pETM6-pnar-RBS (1-8)-mCherry cells. These three RBS sequences are promising for regulating the expression of (S)-equol pathway enzymes.

Figure 6 Fluorescence intensity assay results of RBS mutant library.

In summary, using mCherry as a model protein, we successfully constructed the pETM6-pnar-mCherry plasmid. Based on this, we obtained a nar promoter library with different RBS sequences. The mutant library was characterized by fluorescence intensity detection, and the three best-performing RBS sequences were identified. These RBS sequences have the potential to achieve high-level regulated expression of (S)-equol pathway enzymes.