Overview：

(S)-equol is a highly bioactive metabolite with antioxidant capacity that prevents and alleviates cardiovascular diseases, skin aging, hair loss, obesity, and other hormone-related conditions^[1]. However, only 25-50% of the global population harbors the specialized gut microflora required to naturally produce (S)-equol, and merely a small fraction of these “equol producers” generate therapeutically effective doses during menopause^[2]. To enable low-cost (S)-equol production through synthetic biology, our team aimed to regulate biosynthesis by modulating (S)-equol enzyme expression at the translational level via RBS optimization. We executed two engineering design (DBTL) cycles:

1) Designing and constructing the pETM6-pnar-mCherry plasmid and evaluating its expression.

2) Designing and constructing a pETM6-pnar-RBS (1-8)-mCherry mutant library and assessing the regulatory effects of different RBS mutants on protein expression.

Figure 1 Design diagram of this project.

Cycle 1: Construction of the plasmid pETM6-pnar-mCherry

Design 1:

We chose the pETM6-pnar plasmid as a vector, in which the nar promoter is anaerobically inducible, enabling induction at any cell growth stage. For the model protein, we chose mCherry, which is the most commonly used red fluorescent protein with great photostability and is ideal for tracking expression levels. We designed the expression frame of pETM6-pnar-mCherry (Figure 2), and after obtaining this plasmid, we tested whether the nar promoter could successfully initiate the transcription of target proteins under anaerobic conditions.

Figure 2 Expression frame of the pETM6-pnar-mCherry.

Build 1:

To construct plasmid pETM6-pnar-mCherry, we first double-digested pET28a-mCherry and pETM6-pnar with XhoI and AvrII restriction enzymes, respectively (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 them by colony PCR to identify the transformant with the correct band and inoculated it for subsequent plasmid extraction.

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

Test 1:

We inoculated positive transformants, followed by fluorescence intensity testing to measure mCherry expression after induction of expression under hypoxic conditions. As shown in Figure 4, 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.

Figure 4 Fluorescence intensity assay results of pETM6-pnar-mCherry.

Learn 1:

The anaerobically induced nar promoter successfully induced mCherry expression, and its expression could be evaluated by fluorescence intensity. We demonstrated that mCherry fluorescence intensity (i.e., expression level) exhibited a good linear relationship with induction time, and pETM6-pnar without mCherry did not produce fluorescence. This reflects the reliability of this method for subsequent testing of the regulatory effects of different RBS sequences on protein expression levels.

Cycle 2: Construction of the pETM6-pnar-RBS (1-8)-mCherry mutant library

Design 2:

The role of RBS is to help the ribosome properly bind with messenger RNA (mRNA) and initiate protein synthesis. The sequence of RBS can be adjusted to control the binding efficiency between the ribosome and mRNA, thereby affecting the protein expression level. To test the regulation of protein expression by the series of RBS sequences downstream of the nar promoter, we mutated the RBS sequence on the pETM6-pnar-mCherry plasmid. We designed eight RBS mutants using the RBS Calculator (https://salislab.net/software/) and tested the regulation of protein expression by different RBS mutants.

Figure 5 Design diagram of DBTL cycle 2.

Build 2:

We performed whole plasmid PCR on pETM6-pnar-mCherry using eight synthesized primers with mutation sites, and the results showed that we successfully amplified plasmids with different RBS sequences to obtain the pETM6-pnar-RBS (1-8)-mCherry mutant library. We then transformed the pETM6-pnar-RBS (1-8)-mCherry plasmids into E. coli BL21(DE3) cells, respectively. Colony PCR identification showed that we obtained the pETM6-pnar-RBS (1-5)-mCherry mutant library (Figure 6).

Figure 6 Construction results of pETM6-pnar-RBS (1-8)-mCherry mutant library.

Test 2:

We inoculated the positive transformants of the pETM6-pnar-RBS (1-8)-mCherry mutant library, and then performed fluorescence intensity tests to measure the results of the regulation of mCherry expression by different RBS sequences. As shown in Figure 7, in pETM6-pnar-RBS (1-8)-mCherry cells, there were three RBS sequences (RBS 4, RBS 6, and RBS 1) that exhibited the strongest fluorescence. These three RBS sequences are promising for regulating the expression of (S)-equol pathway enzymes.

Figure 7 Fluorescence intensity assay results of RBS mutant library.

Learn 2:

By optimizing the RBS sequence, we achieved regulation of target protein expression at the translational level. The three best-performing RBS sequences have potential for application in producing (S)-equol biosynthetic pathway enzymes. This method provides another useful tool for regulating protein expression, facilitating the optimization of biological processes in synthetic biology and metabolic engineering.

Reference

[1] Legette L L, Prasain J, King J, et al. Pharmacokinetics of equol, a soy isoflavone metabolite, changes with the form of equol (dietary versus intestinal production) in ovariectomized rats [J]. Journal of agricultural and food chemistry, 2014, 62(6): 1294-1300.

[2] Mayo B, Vázquez L, Flórez A B. Equol: A Bacterial Metabolite from The Daidzein Isoflavone and Its Presumed Beneficial Health Effects [J]. Nutrients, 2019, 11(9): 2231.