We faced a similar dilemma recently... We are using a hybrid approach of using two promoters, each expressing two proteins using a 2A cleavage sequence. So we get 4 proteins total.

2A sequence general info: https://www.intechopen.com/chapters/47960

Example in plants: Article Non-viral 2A-like sequences for protein coexpression

2A sequences for polycistronic expression: https://doi.org/10.1038/s41598-017-02460-2

Coordinated expression of multiple transgenes in dicotyledonous plants requires careful attention to promoter choice, transcriptional termination, gene arrangement, and insulation to avoid silencing, interference, and positional effects. The following guidelines synthesize best practices from pathway engineering studies.

1. Promoter and Terminator Diversity

Using distinct promoters for each gene minimizes homology-based transcriptional silencing and promoter competition. Ideally, choose a panel of 4–6 promoters with varying strengths and tissue or developmental specificity (e.g., CaMV 35S, Arabidopsis Ubiquitin10, parsley ubiquitin, tomato E8, potato Lhca3, peanut LBP). Pair each with a unique strong terminator—such as the nopaline synthase (NOS), octopine synthase (OCS), pea rbcS E9, or Arabidopsis heat-shock protein terminators—to prevent read-through and avoid repeated sequence blocks.

2. Promoter Compatibility and Strength Balancing

For pathway flux balance, assign promoters according to enzyme kinetics and metabolic control points. Use high-strength constitutive promoters for rate-limiting enzymes, moderate promoters for branch-point enzymes, and weaker or inducible promoters for enzymes whose overexpression risks metabolite toxicity. Publicly available promoter-strength databases (e.g., the NCBI Plant Promoter Database) can guide relative activity selection.

3. Gene Orientation and Order

Arrange genes as tandem, unidirectional cassettes to minimize convergent transcription and cryptic read-through. Place the first gene cassette closest to the T-DNA left border to ensure early integration and expression. Empirical studies show that expression can decline for cassettes positioned farthest from border sequences; intersperse MAR (matrix attachment region) elements between distal cassettes to buffer positional effects if necessary.

4. Transcriptional Terminators and Read-Through Prevention

Strong terminators with dual RNA-processing signals (polyadenylation site and downstream stability elements) ensure efficient transcript cleavage. The E9 terminator from pea rbcS and the rbcS-3A terminator from Arabidopsis consistently yield >95% termination efficiency. Incorporate double terminator modules—two different terminators in series—to further reduce read-through into downstream cassettes.

5. Insulators, Linkers, and MARs

Incorporating insulator sequences (e.g., gypsy insulator from Drosophila or plant-derived STAR elements) flanking your entire multi-gene assembly can shield against chromosomal position effects. MARs/WARs placed between cassettes break up heterochromatin spread and reduce promoter crosstalk. Short spacer linkers (~500 bp of non-coding stuffer DNA) can also minimize direct promoter–promoter interactions.

6. Vector Backbone and T-DNA Configuration

Use a binary vector with a minimal backbone and single T-DNA region. Place a single selectable marker (e.g., hygromycin phosphotransferase) under its own promoter–terminator cassette at one border. Avoid multiple antibiotic markers to reduce T-DNA size and complexity.

7. Modularity and Assembly Methods

Employ modular cloning systems (Golden Gate, MoClo, or Gibson) to assemble individual promoter–gene–terminator modules in a standardized manner. This approach eases reuse, troubleshooting, and future pathway optimization.

8. Empirical Validation and Further Optimization

After stable transformation, quantify transcript levels by qRT-PCR and measure enzyme activities or metabolite accumulation. If imbalances appear, swap promoters or reorder cassettes. Consider split-intron strategies to reduce homologous recombination between similar terminators or promoters.

By combining promoter diversity, strong terminators, unidirectional gene order, and insulation elements, you can construct a stable six-gene T-DNA that minimizes silencing and interference while enabling balanced pathway expression in dicot plants.

My colleague developed the system with dTALE, and it seems to work well for multiple gene expressions in rice: Article A single promoter‐TALE system for tissue‐specific and tuneab...

The bes strategies for designing a multiple genes construct are very well explained by Shwet Dwivendil and Quentin Herzig.

Then I fully recommend then and I hope you can obtained very good results.

Perea Dallos Margarita Thank you for your recommendation! I appreciate your suggestion regarding Shweta Dwivedi and Herzig explanations .