Parts

Nomination: Best New Composite Parts
BBa_25SXFI1M (More details: please refer to https://registry.igem.org/parts/bba-25sxfi1m)

This year, we have designed 14 basic parts (8 new basic parts) and 11 new composite parts in total. We nominated ourselves for the award of the Best New Composite Part (BBa_25SXFI1M) for: Successfully developing and optimizing a spatial metabolic engineering toolkit for enhanced subcellular targeting to lipid droplets. This composite part enables an effective compartmentalization strategy that significantly improves the biosynthesis of hydrophobic compounds, including carotenoids, triterpenoids, and terpenes.

Best Composite Part BBa_25SXFI1M: POX1-PTDH3-CarRP-HD2-TCYC1 + PTEF1-CarB-linker(G4S)2-HD2-TADH1-POX1 (https://registry.igem.org/parts/bba-25sxfi1m)

Properties

Our project aims to use the saccharomyces cerevisiae to produce β-carotene as a preventative supplement for eye issues like dry eye syndrome and nyctalopia. To enhance β-carotene yield, we designed and optimized a genetic construct that employs a compartmentalization strategy, specifically targeting lipid droplets for improved accumulation. This composite construction markedly increases β-carotene yield, and this strategy extends beyond β-carotene biosynthesis which means it can also be applied to the biosynthesis of other hydrophobic compounds, including carotenoids, triterpenoids, and terpenes. The core elements are shown in Figure 1.

We modified the following part of mevalonate (MVA) pathway of S. cerevisiae (Figure 2).

This modification introduces two key enzymes:

• CarB (from Mucor lusitanicus): phytoene desaturase
• CarRP (from Mucor circinelloides): bifunctional phytoene synthase/lycopene cyclase

Both enzymes were codon-optimized for yeast expression and fused with the HD2 signal peptide, ensuring LD targeting. Structural modeling guided linker selection to avoid disrupting enzyme activity:

• CarB-linker-HD2 configuration
• CarRP-HD2 configuration

CarRP-HD2 configuration

The engineered pathway catalyzes:

• Condensation of GGPP → phytoene (carRP)
• Desaturation of phytoene → lycopene (carB)
• Cyclization of lycopene → β-carotene (carRP)

By targeting β-carotene biosynthetic enzymes to lipid droplets—hydrophobic organelles with a neutral lipid core—we provided a dedicated compartment for the synthesis and storage of hydrophobic intermediates (GGPP, phytoene, lycopene) and the final product β-carotene. This strategy minimizes cytotoxic aggregation in the cytosol and prevents disruptive accumulation in membrane systems, thereby enhancing overall productivity. The entire pathway was integrated into the S. cerevisiae genome at the POX1 locus via CRISPR-Cas9-mediated homologous recombination using plasmid-borne flanking sequences.

However, it is worth explaining how the lipid droplet targeting toolkit (this composite part) was developed and optimized (Figure 3). There are 4 steps: (1) screening of targeting signal peptides; (2) optimization of linker positioning; (3) evaluation of linker configurations; (4) structure-guided fusion with HD2.

Our Solid Basis: BBa_25JCA3OI: POX1-PTDH3-CarRP-TCYC1+ PTEF1-CarB-TADH1-POX1 (https://registry.igem.org/parts/bba-25jca3oi)

Properties

Our project employs Saccharomyces cerevisiae as a microbial chassis for β-carotene production. To establish this system, we introduced two key genes, carRP and carB, into S. cerevisiae. The design of the expression cassettes is shown in Figure 4. The CarRP gene encodes a bifunctional enzyme with phytoene synthase and lycopene cyclase activities, while the CarB gene encodes phytoene desaturase. Both genes were codon-optimized for S. cerevisiae, enabling efficient β-carotene biosynthesis in the host. The entire part was integrated into the S. cerevisiae genome at the POX1 locus via CRISPR-Cas9-mediated homologous recombination using plasmid-borne flanking sequences.

As described above, this part introduces two key enzymes:

• CarB (from Mucor lusitanicus): phytoene desaturase
• CarRP (from Mucor circinelloides): a bifunctional enzyme with phytoene synthase and lycopene cyclase activities

Figure 5 shows how these enzymes catalyze the following steps in the β-carotene biosynthetic pathway:

• Condensation of GGPP → phytoene (CarRP)
• Desaturation of phytoene → lycopene (CarB)
• Cyclization of lycopene → β-carotene (CarRP)

Both genes were codon-optimized for expression in yeast, ensuring efficient β-carotene production.

Parts Collection

Note: Orange means the new parts.

References

1. Guo, Q., Peng, Q. Q., Li, Y. W., Yan, F., Wang, Y. T., Ye, C., & Shi, T. Q. (2024). Advances in the metabolic engineering of Saccharomyces cerevisiae and Yarrowia lipolytica for the production of β-carotene. Critical Reviews in Biotechnology, 44(3), 337-351.
2. Dasso, M. E., Centola, C. L., Galardo, M. N., Riera, M. F., & Meroni, S. B. (2025). FSH increases lipid droplet content by regulating the expression of genes related to lipid storage in Rat Sertoli cells. Molecular and Cellular Endocrinology, 595, 112403.
3. Zhao, Y., Zhang, Y., Nielsen, J., & Liu, Z. (2021). Production of β-carotene in Saccharomyces cerevisiae through altering yeast lipid metabolism. Biotechnology and Bioengineering, 118(5), 2043-2052.
4. Greenspan, P., Mayer, E. P., & Fowler, S. D. (1985). Nile red: a selective fluorescent stain for intracellular lipid droplets. The Journal of cell biology, 100(3), 965-973.