Week 12 Lab: Bioproduction of Beta-Carotene and Lycopene
Bioproduction of Beta-Carotene and Lycopene
1. Enzymes of the carotene pathway
CrtE, CrtB, CrtI, and CrtY are key enzymes involved in carotenoid biosynthesis. CrtE participates in geranylgeranyl pyrophosphate synthesis, CrtB catalyzes phytoene formation, CrtI mediates phytoene desaturation, and CrtY converts lycopene into beta-carotene. Although these enzymes are essential for carotenoid production, recent metabolic engineering studies suggest that carotenoid-specific enzymes do not solely limit lycopene yield. Instead, precursor availability, metabolic flux through the MEP pathway, cofactor balance, and overall cellular metabolism strongly influence production efficiency. (Huang et al., 2025; Jing et al., 2021; Sandmann & Misawa, 1992)
2. Rate-determining step
Although phytoene desaturation mediated by CrtI has historically been considered an important control point in carotenoid biosynthesis, recent studies suggest that carotenoid production is often limited by precursor supply and metabolic flux rather than by a single carotenoid-specific enzyme. In engineered E. coli systems, optimization of the MEP pathway, carbon metabolism, and cofactor availability can significantly influence lycopene and Ξ²-carotene yields (Wang et al., 2019; Liu et al., 2026).
DNA Construct Design
1. Choice of production organism
I would choose Escherichia coli as the production organism for this construct because the goal of this lab is to design a microbial system for carotenoid bioproduction, especially lycopene or Ξ²-carotene. E. coli is a convenient chassis because it grows quickly, is inexpensive to culture, and has well-established plasmid-based expression systems. In addition, several metabolic engineering strategies for carotenoid production in E. coli focus on improving precursor supply through the MEP pathway, optimizing central carbon metabolism, and balancing expression of heterologous carotenoid genes. For this reason, E. coli would be a practical host for a first plasmid-based design. (Liu et al., 2026; Wu et al., 2020)
2. Example enzyme for expression
For the expression construct, I would choose CrtY, the lycopene Ξ²-cyclase enzyme. This enzyme converts lycopene into Ξ²-carotene, so it is directly relevant if the desired final product is Ξ²-carotene. In this design, lycopene can be considered the precursor, and CrtY would redirect the pathway toward Ξ²-carotene accumulation. This choice also makes the construct easy to explain because the presence or absence of CrtY activity helps determine whether the engineered system accumulates lycopene or produces Ξ²-carotene. (Wang et al., 2020; Liu et al., 2026)
Promoters
| Based on Liu et al., 2026 | 
|
1. Function of a promoter
A promoter is a regulatory DNA sequence that controls the initiation of transcription by recruiting RNA polymerase and transcriptional machinery. In engineered microbial systems, promoters are essential for regulating the expression level of heterologous genes involved in biosynthetic pathways.
2. Types of promoters
Promoters can be classified as constitutive, inducible, or repressible promoters. Constitutive promoters continuously drive gene expression, whereas inducible promoters activate transcription in response to specific molecules such as IPTG or arabinose. Repressible promoters decrease transcription in response to regulatory signals or metabolites. In metabolic engineering, inducible promoters are commonly used to balance cell growth and product synthesis while minimizing metabolic burden.
3. Promoter response to metabolites
If the goal is to suppress transcription in response to a metabolite, repressible promoters would be useful because they decrease gene expression after sensing a specific molecule. Conversely, inducible promoters are useful when gene expression should increase in the presence of a metabolite or inducer. Dynamic promoter regulation is important in metabolic engineering because excessive expression of biosynthetic genes can impose a metabolic burden and reduce cell growth.
4. Promoter choice
For this construct, I would choose a strong inducible promoter such as the T5 promoter. Recent metabolic engineering studies demonstrated that replacing native promoters of MEP pathway genes with strong T5 promoters significantly improved Ξ²-carotene production in engineered E. coli strains. Strong promoters can enhance precursor flux and increase carotenoid biosynthesis, although excessive overexpression may also generate metabolic burden and reduce cellular fitness (Liu et al., 2026).
Origin of Replication
1. What is an origin of replication?
The origin of replication (Ori) is the DNA sequence that allows a plasmid to replicate independently inside the host cell. The selected origin strongly influences plasmid copy number, stability, and metabolic burden.
(Cooper, 2000)
2. Types of origins
Origins of replication can be classified as high-copy-number, medium-copy-number, or low-copy-number origins. High-copy plasmids typically produce larger amounts of recombinant proteins or metabolites, whereas low-copy plasmids reduce metabolic burden and improve cellular stability.
(Ding & Koren, 2020)
3. Compatibility groups
If the goal is to suppress transcription in response to a metabolite, repressible promoters would be useful because they decrease gene expression after sensing a specific molecule. Conversely, inducible promoters are useful when gene expression should increase in the presence of a metabolite or inducer. Dynamic promoter regulation is important in metabolic engineering because excessive expression of biosynthetic genes can impose a metabolic burden and reduce cell growth.
(Ding & Koren, 2020)
- Best origin for this construct
For this construct, a medium-copy-number origin such as p15A ori would be appropriate because it provides a balance between carotenoid production and cellular fitness. High-copy plasmids may increase carotenoid synthesis but can also generate significant metabolic burden and stress on the host cell.
(Liu et al., 2026)
Other bioparts
RBS
Ribosome binding sites (RBSs) are short regulatory sequences that recruit ribosomes and initiate translation. RBS strength strongly influences protein expression levels and can be tuned to balance metabolic flux in engineered biosynthetic pathways. (Wu et al., 2018)
Terminators
Terminators are DNA sequences that stop transcription and stabilize RNA transcripts. Proper terminator selection reduces transcriptional readthrough and improves the stability of engineered genetic circuits. (Torella et al., 2013; Deaner & Alper, 2016)
Operators
Operators are regulatory DNA elements recognized by transcription factors or repressors. They help regulate promoter activity and allow dynamic control of gene expression in response to environmental or metabolic signals. (Addgene, 2019)
Aptamers and riboswitches
Aptamers are nucleic acid structures capable of binding specific target molecules, whereas riboswitches are regulatory RNA elements that alter gene expression in response to metabolites. In metabolic engineering, riboswitches can dynamically regulate biosynthetic pathways to improve metabolic balance, reduce toxicity, and optimize carotenoid production.
(Zhang et al., 2019)
Joining DNA parts together
The plasmid construct could be assembled using restriction enzyme cloning through the Benchling assembly wizard. Restriction enzymes can generate compatible sticky ends between the plasmid backbone and the insert containing the promoter, RBS, crtY coding sequence, and terminator. This approach allows modular insertion of the carotenoid expression cassette into an existing plasmid backbone.
Dream biosynthetic pathway
One interesting dream biosynthetic pathway would involve engineering Escherichia coli to detect early biofilm-associated physiological states on food-contact surfaces and activate a fluorescent or antimicrobial response before mature biofilm formation occurs. This system could combine biosensing circuits with metabolic pathways capable of producing detectable pigments, antimicrobial peptides, or protective metabolites in response to quorum sensing or stress-associated signals. Such a platform could contribute to early contamination monitoring and improved food safety strategies while reducing the risk of persistent biofilm-associated infections.
1. Mockup Plasmid Creation
A conceptual crtY expression plasmid was designed in Benchling using a pAC-BETA-inspired backbone for simulated carotenoid bioproduction in Escherichia coli. The objective was to create a simplified synthetic biology construct containing a promoter, ribosome binding site, crtY coding sequence, and transcriptional terminator assembled into a plasmid backbone carrying an origin of replication and chloramphenicol resistance marker.
2. Benchling Folder
Folder Benchling: Week 12 Bioproduction lab Benchling
3. Design Overview
| Backbone | Insert |
|---|---|
| pAC-BETA | Promoter β RBS β crtY β Terminator |
Plasmid diagram:
4. Genetic Parts
All the genetic parts are clickable sections
4.1 crtY CDS:
Uniprot:
P21687Β Β·Β CRTY_PANAN (gen) Link: https://www.uniprot.org/uniprotkb/P21687/entry
Protein sequence:
Cusabio reverse translation (DNA sequence)
| Analyze DNA Sequence from Cusabio: | 
|
Link of the Reverse Translate Protein to DNA: Cusabio.online
5. Restriction Cloning Strategy
Restriction cloning was simulated using HindIII and Alw44I/ApaLI restriction enzymes. These enzymes were selected because they cut once within the pAC-BETA backbone while avoiding internal cuts within the crtY insert sequence, allowing directional insertion of the expression cassette into the plasmid backbone.
pAC-BETA crtY Insert Plasmid
| Enzyme | Position | Cut site sequence | Strand |
|---|---|---|---|
| HindIII | 1525-1525 | AAGCTT | Forward |
| Alw44I/ApaLI | 2499-2499 | GTGCAC | Forward |
Insert crtY
| Enzyme | Position | Cut site sequence | Strand |
|---|---|---|---|
| HindIII | 1-6 | AAGCTT | Forward |
| Alw44I/ApaLI | 976-981 | GTGCAC | Forward |
6. Assembly Workflow
Figure 1. Overview of the restriction cloning workflow performed in Benchling for the assembly of the crtY expression cassette into the pAC-BETA-inspired backbone.
6.1 The assembly workflow consisted of:
- Analyze restriction sites in the plasmid backbone.
- Select restriction enzymes compatible with the insert sequence.
- Add restriction sites to the crtY insert.
- Simulate backbone digestion in Benchling.
- Assemble the insert into the backbone using the Benchling Assembly Wizard.
- Validate the final circular plasmid construct.
6.2 Assembly Tutorial: (Digest Enzymes- Wizard Assembly)
| 1 | 2 | 3 |
|---|---|---|
| 
| 
|
6.3 Assembly Results:
A conceptual crtY expression plasmid was designed in Benchling using a pAC-BETA-inspired backbone containing the p15A origin of replication and chloramphenicol resistance marker. The crtY expression cassette included a T5 promoter, lac operator, B0034 ribosome binding site, crtY coding sequence, and B0015 terminator. Restriction cloning was simulated using HindIII and Alw44I restriction sites to assemble the insert into the plasmid backbone. Itβs summarized in Figures 2 & 3 for the insert and the final plasmid.
Figure 2. Conceptual crtY expression cassette containing the T5 promoter, lac operator, B0034 ribosome binding site, crtY coding sequence, and B0015 terminator flanked by HindIII and Alw44I/ApaLI restriction sites.
Link of the file inside the folder of the project: https://benchling.com/s/seq-sjjKvYhHZXTUR2aVGgvi?m=slm-4EMRU1KnzhjK8fZPacDy
Figure 3. Final circular pAC-BETA-inspired plasmid generated after simulated restriction cloning of the crtY expression cassette in Benchling.
Link of the file inside the folder of the project: https://benchling.com/s/seq-xhC0Hl0ZfXZ6G9BMwRGk?m=slm-MnPTn07FbXYKK8zFJ7Fp
7. Weekly reflection:
The weekly reflections can be read at Week 12: Bioproduction. Thanks for reading! Also, you can check the same info at my Notion webpage: Week12Notion
8. References & Sources
Addgene. (2019). AddGene: Promoters. https://www.addgene.org/mol-bio-reference/promoters/
Cooper, G. M. (2000). DNA replication. The Cell - NCBI Bookshelf. https://www.ncbi.nlm.nih.gov/books/NBK9940/
Deaner, M., & Alper, H. S. (2016). Promoter and terminator Discovery and Engineering. Advances in Biochemical Engineering, Biotechnology, 162, 21β44. https://doi.org/10.1007/10_2016_8
Ding, Q., & Koren, A. (2020). Positive and Negative Regulation of DNA Replication Initiation.Β Trends in genetics : TIG,Β 36(11), 868β879. https://doi.org/10.1016/j.tig.2020.06.020
Huang, G., Lan, Y., Duan, C., & Yan, G. (2025). Engineering microbial cell factories for the production of lycopene: Advances and perspectives. Food Research International, 227, 118270. https://doi.org/10.1016/j.foodres.2025.118270
Jing, Y., Guo, F., Zhang, S., Dong, W., Zhou, J., Xin, F., Zhang, W., & Jiang, M. (2021). Recent advances on biological synthesis of lycopene by using industrial yeast. Industrial & Engineering Chemistry Research, 60(9), 3485β3494. https://doi.org/10.1021/acs.iecr.0c05228
Liu, J., Shi, Y., Zhao, D., Lin, M., Wang, P., Zhou, Y., & Yan, X. (2026). Strategies for Metabolic Engineering ofΒ Escherichia coliΒ for Ξ²-Carotene Biosynthesis.Β Molecules (Basel, Switzerland),Β 31(4), 611. https://doi.org/10.3390/molecules31040611
Sandmann, G., & Misawa, N. (1992). New functional assignment of the carotenogenic genescrtBandcrtEwith constructs of these genes fromErwiniaspecies. FEMS Microbiology Letters, 90(3), 253β258. https://doi.org/10.1111/j.1574-6968.1992.tb05162.x
Torella, J. P., Boehm, C. R., Lienert, F., Chen, J., Way, J. C., & Silver, P. A. (2013). Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic Acids Research, 42(1), 681β689. https://doi.org/10.1093/nar/gkt860
Wang, C., Zhao, S., Shao, X., Park, J., Jeong, S., Park, H., Kwak, W., Wei, G., & Kim, S. (2019). Challenges and tackles in metabolic engineering for microbial production of carotenoids. Microbial Cell Factories, 18(1), 55. https://doi.org/10.1186/s12934-019-1105-1
Wang, Z., Sun, J., Yang, Q., & Yang, J. (2020). Metabolic Engineering Escherichia coli for the Production of Lycopene. Molecules, 25(14), 3136. https://doi.org/10.3390/molecules25143136
Wu, Y., Yan, P., Li, Y., Liu, X., Wang, Z., Chen, T., & Zhao, X. (2020). Enhancing Ξ²-Carotene Production in Escherichia coli by Perturbing Central Carbon Metabolism and Improving the NADPH Supply. Frontiers in Bioengineering and Biotechnology, 8, 585. https://doi.org/10.3389/fbioe.2020.00585
Wu, F., Zhang, Q., & Wang, X. (2018). Design of adjacent transcriptional regions to tune gene expression and facilitate circuit construction. Cell Systems, 6(2), 206-215.e6. https://doi.org/10.1016/j.cels.2018.01.010
Zhang, Y., Lai, B. S., & Juhas, M. (2019). Recent Advances in Aptamer Discovery and Applications.Β Molecules (Basel, Switzerland),Β 24(5), 941. https://doi.org/10.3390/molecules24050941