Week 12 Lab: Bioproduction of Beta-Carotene and Lycopene

Post Lab Questions (Mandatory for All Students)

1) Which genes when transferred into E. coli will induce the production of lycopene and beta-carotene, respectively? According to the lab instructions, lycopene production in E. coli is induced by transferring the three genes from Erwinia herbicola: crtE, crtI, and crtB. These genes convert FPP into lycopene. Beta-carotene production uses the same pathway with the addition of crtY, which enables conversion toward beta-carotene.

2) Why do the plasmids that are transferred into the E. coli need to contain an antibiotic resistance gene? The antibiotic resistance gene allows selection for E. coli cells that successfully received the plasmid. Only transformed cells can grow on antibiotic-containing media.

3) What outcomes might we expect to see when we vary the media, presence of fructose, and temperature conditions of the overnight cultures? Different media composition and temperatures can affect both cell growth and pigment production. Richer media may increase biomass, fructose may improve lycopene production by changing carbon metabolism, and lower temperature may reduce stress or improve pathway performance, while 37°C may favor faster growth. Based on the lab framing, fructose is being tested because it may improve biomass yield and recombinant gene expression in E. coli. If it improves carbon flux or reduces metabolic stress, pigment production per culture may increase. However, the final result would need to be normalized by OD600 to distinguish higher pigment production from simply higher cell growth.

4) Generally describe what “OD600” measures and how it can be interpreted in this experiment. OD600 measures how much light at 600 nm is scattered by a bacterial culture. As the number of cells increases, the culture becomes more turbid, meaning it scatters more light and gives a higher OD600 value. The 600 nm wavelength is commonly used because it estimates cell density without strongly overlapping with many biological pigments or media components. In this experiment, OD600 helps estimate how much bacterial growth occurred under each condition. This is important because pigment absorbance alone could be misleading: a darker sample might have more pigment simply because it has more cells. By normalizing pigment absorbance by OD600, we can compare carotenoid production per amount of bacterial growth.

5) What are other experimental setups where we may be able to use acetone to separate cellular matter from a compound we intend to measure? Acetone can be used in experiments where we want to separate an organic-soluble compound from the rest of the cell material. In this lab, it helps extract carotenoid pigments from bacterial pellets while leaving much of the cellular debris behind. Similar setups could include extracting chlorophyll or carotenoids from algae and plant tissue, recovering hydrophobic metabolites from microbial cultures, or preparing pigment extracts before absorbance measurements. It could also be useful as a cleanup step, because acetone can precipitate proteins and help remove cell debris before analyzing small molecules by absorbance, fluorescence, or chromatography.

6) Why might we want to engineer E. coli to produce lycopene and beta-carotene pigments when Erwinia herbicola naturally produces them? Even though Erwinia herbicola naturally produces these pigments, E. coli is a better model organism and engineering chassis. It is easier to grow, transform, measure, and genetically manipulate, with well-characterized plasmids, promoters, selection markers, and growth conditions. This makes it more useful for rapid prototyping, pathway optimization, and controlled bioproduction experiments. Engineering E. coli also lets us isolate and test the carotenoid pathway in a standardized host, instead of working with the natural producer where regulation and metabolism may be more difficult to control.

Post Lab Questions (For Committed Listeners)

1.1) What are the enzymes of the carotene pathway?

1.2) Within this pathway, which is the rate determining step (the step that takes the longest)? Which enzyme is responsible for this step?

Within the carotenoid pathway, my hypothesis is that the likely rate-determining step is the conversion of phytoene into lycopene, catalyzed by CrtI, the phytoene desaturase.

The reason is that crtE and crtB first build the upstream carotenoid intermediate: CrtE helps produce GGPP, and CrtB converts GGPP into phytoene. Then CrtI carries out the desaturation steps that convert phytoene into lycopene. Since this step involves multiple oxidation/desaturation reactions, I would expect it to be slower and more limiting than the upstream condensation steps.

The literature support this hypothesis, but also show that CrtI is probably not the only bottleneck. Du et al. 2016 confirm that E. coli requires crtE, crtB, and crtI to produce lycopene, and they show that fructose strongly improves lycopene production by changing central metabolism, especially pathways linked to precursor, cofactor, and energy supply. So I would identify CrtI/crtI as the most likely pathway-level enzymatic bottleneck, while recognizing that whole-cell lycopene production also depends on upstream metabolic supply. This is also consistent with Aristidou, Sam and Bennett 2008, who show that fructose can reduce acetate overflow and improve biomass/recombinant expression in E. coli, suggesting that fructose supports a more favorable metabolic state for bioproduction than glucose under these conditions.

2) Notes for design of a DNA construct for bioproduction

2.1) The first thing to do is to decide what organism you are going to use for this (E. coli or S. cerevisiae) for production. Which would you choose and why (emphases on production differences)?

I would choose E. coli. S. cerevisiae could be useful for more complex eukaryotic engineering or when compartmentalization and eukaryotic metabolism are advantageous, but for fast carotenoid pathway testing, I think E. coli is the more practical chassis.

2.2) Now choose one of the enzymes and lets outline the parts of the construct for expression

I would choose the phytoene desaturase, encoded by crtI, because it catalyzes the conversion of phytoene into lycopene and may be one of the key pathway-level bottlenecks in lycopene production.

A minimal plasmid design would be: Origin of replication, antibiotic resistance marker, promoter, operator, RBS, crtI, terminator.

If the goal were only to test crtI expression, this construct would be enough. But if the goal is full lycopene production, crtI would need to be expressed together with the upstream pathway genes crtE and crtB, because E. coli requires crtE, crtB, and crtI to synthesize lycopene. For beta-carotene production, crtY would also be included.

2.3.i.1.a.i) What is the function of a promoter? The promoter is the DNA region that initiates transcription of the gene of interest. It controls RNA polymerase binding and therefore strongly affects when, where, and how much of the target enzyme is produced. In bacteria, promoter recognition depends on bacterial RNA polymerase and sigma factors, so the promoter must be compatible with a prokaryotic host like E. coli. Source: Educational Resources > Molecular Biology Reference > Promoters.

2.3.i.1.a.ii) What types of promoters do we have? Promoters can be grouped by their expression behavior. Constitutive promoters are active continuously, inducible promoters are turned on or increased by a signal such as IPTG, lactose, arabinose, heat, or light, and repressible promoters are turned off or reduced in response to a signal or metabolite. Common bacteria promoter examples are included in the table below.

2.3.i.1.a.iii) If we wanted to turn off the transcription of a gene in response to a metabolite, what type of promoter would be most useful? What if we wanted this to increase in the presence of the metabolite? To turn transcription off in response to a metabolite, I would use a repressible promoter, such as the trp promoter, where high tryptophan represses transcription. To increase transcription in response to a metabolite, I would use an inducible promoter, such as lac/IPTG or araBAD/arabinose, where the inducer activates expression or removes repression.

2.3.i.1.a.iv) Now choose one of the genes of the metabolic pathway previously described (Carotene/lycopene )and choose one enzyme to make an expression construct. What promoter could you use for this? Why did you choose it? I would choose crtI, which encodes phytoene desaturase, the enzyme that converts phytoene into lycopene. I chose this gene because this step is a good candidate for pathway-level control: if CrtI expression is too low, phytoene may accumulate and lycopene output may remain limited.

For the promoter, I would use a tunable inducible bacterial promoter, such as pBAD/araBAD or lac/IPTG. I would prefer pBAD/araBAD for an initial design because arabinose-inducible expression allows controlled activation of the gene. The reason I would not immediately use a strong constitutive promoter is that carotenoid production can create metabolic burden. The goal is not simply to express crtI as strongly as possible, but to tune expression and find the level that improves lycopene production without compromising cell growth.

Therefore, a minimal expression cassette would be: pBAD promoter, RBS, crtI, terminator.

In the full plasmid context: Origin of replication, antibiotic resistance marker, pBAD promoter, RBS, crtI, terminator.

3.1.i What is the origin of replication? The origin of replication, or ori, is the DNA sequence where plasmid replication begins. It allows the plasmid to copy itself inside the host cell and be maintained over generations. Together with its control elements, the ori is part of the plasmid replicon. Source: Adgene’s Article “Plasmids 101: Origin of Replication” available here.

3.1.ii What types of origin of replication do we have? Origins of replication differ by copy number, replication control, compatibility group, and host requirements. Copy number affects gene dosage and burden; replication control affects how tightly plasmid replication is regulated; compatibility group matters when using more than one plasmid; and host requirements determine whether the plasmid can replicate in a given strain. Here goes some examples from Adgene’s Article “Plasmids 101: Origin of Replication” available here.

3.1.iii (Extra) What are compatibility groups? Compatibility groups describe whether two plasmids can be stably maintained in the same bacterial cell. Plasmids with the same or very similar replication/partitioning systems are usually incompatible because they compete for the same replication control machinery. Over time, one plasmid may be lost. This matters if we want to use more than one plasmid in the same E. coli strain: they should have compatible origins, meaning different incompatibility groups. For example, pMB1/ColE1-derived plasmids such as pUC, pBR322, pET, and pGEX are all in compatibility group A, so they should generally not be combined in the same cell. A p15A/pACYC plasmid, group B, could be combined with a ColE1/pMB1 plasmid more safely.

3.1.iv Now for the previously chosen promoter and gene what will be the best origin or replication? For a crtI expression plasmid in E. coli, I would choose a medium-copy origin rather than a very high-copy pUC-type origin. This should provide enough CrtI expression while reducing metabolic burden. If I combine this plasmid with another carotenoid-pathway plasmid, I would choose compatible origins, for example p15A with ColE1/pMB1-derived origins.

4. Elaborate further on other bioparts like RBS, terminators, operators you would use for a correct design and further bioproduction?

I did not complete questions 5, 6, 7, and 8, as they were marked as extra-point questions. For this submission, I prioritized the mandatory All Students and Committed Listener sections.