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?

Introducing the Erwinia herbicola genes CrtE, CrtI, and CrtB into E. coli directs the metabolic conversion of endogenous farnesyl diphosphate into lycopene. To achieve beta-carotene production, the pathway is extended by transferring those same three core genes along with a fourth gene, CrtY, which encodes lycopene β-cyclase to cyclize the terminal groups.

2. Why do the plasmids that are transferred into the E. coli need to contain an antibiotic resistance gene?

Plasmids must carry an antibiotic resistance gene (such as chloramphenicol resistance) to serve as a selective marker during cultivation. Growing the bacteria in media containing the antibiotic ensures that only cells that actively retain the plasmid can survive, preventing plasmid loss over multiple generations and suppressing the growth of contaminating organisms.

3. What outcomes might we expect to see when we vary the media, presence of fructose, and temperature conditions of the overnight cultures?

Shifting incubation temperatures between 30°C and 37°C alters metabolic rates, where 30°C often optimizes heterologous enzyme folding while 37°C maximizes biomass growth rates. Varying the basal medium from LB to richer 2YT supplies a denser nutrient pool for higher biomass accumulation, while supplementing with fructose bypasses standard catabolite repression to boost recombinant pathway fluxes and overall pigment yields.

4. Generally describe what “OD600” measures and how it can be interpreted in this experiment.

OD600 measures optical density by quantifying the amount of light scattered at a wavelength of 600 nm by a liquid bacterial culture. In this experiment, it provides a rapid estimate of bacterial cell density, which serves as a baseline value to normalize total pigment extraction absorbance data against the concentration of biomass.

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’s ability to lyse cell membranes and precipitate proteins while solubilizing lipophilic small molecules makes it ideal for extracting hydrophobic compounds across biology. It is frequently used to isolate chlorophyll from plant tissue, extract lipids from yeast cells, or recover secondary hydrophobic metabolites and small-molecule drugs from microbial pellets.

6. Why might we want to engineer E. coli to produce lycopene and beta-carotene pigments when Erwinia herbicola naturally produces them?

E. coli is a highly optimized industrial host with fast replication kinetics, thoroughly mapped genetics, and scalable fermentation protocols. Engineering it allows for significantly higher metabolic yields, simpler downstream purification processes, and avoids the challenges of optimizing growth parameters for less characterized wild-type organisms.

Post Lab Questions - For Committed Listeners Only

1. Let’s get in touch with our metabolic pathway. What are the enzymes of the carotene pathway? Within this pathway, which is the rate determining step (the step that takes the longest)? Which enzyme is responsible for this step?

The biosynthetic pathway includes geranylgeranyl pyrophosphate synthase (CrtE), phytoene synthase (CrtB), phytoene desaturase (CrtI), and lycopene β-cyclase (CrtY). The rate-limiting bottleneck of the network is the multiple desaturation steps that convert colorless phytoene into pink-red lycopene, a process catalyzed entirely by the CrtI enzyme.

2. 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)?

E. coli is the ideal choice here because it supports incredibly rapid doubling times, features highly efficient transcription-translation coupling, and accommodates straightforward plasmid-based expression without requiring genomic integration. While S. cerevisiae excels at processing complex eukaryotic post-translational modifications or membrane-bound enzymes, E. coli provides a faster, higher-yield expression platform for these specific bacterial carotenoid enzymes.

3. What is the function of a promoter? What types of promoters do we have?

A promoter is a regulatory DNA sequence located upstream of a gene that provides a specific recruitment site for RNA polymerase to initiate transcription. Promoters generally fall into two categories: constitutive promoters, which drive continuous gene transcription at a fixed rate, and regulated promoters, which can be dynamically activated or suppressed by transcription factors.

4. 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 shut down gene transcription in response to an accumulating metabolite, a repressible promoter is required. Conversely, to activate or increase gene transcription upon the addition or detection of a target metabolite, an inducible promoter must be integrated into the construct design.

5. 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 pair the rate-limiting CrtI gene with an IPTG-inducible lac promoter (or T7 promoter). This choice allows the culture to build up substantial cellular biomass during an uninduced growth phase before adding the chemical trigger, preventing early pathway enzyme accumulation from overstressing the cell or stunting growth.

6. What is the origin of replication? What types of origin of replication do we have?

The origin of replication (ori) is the specific DNA sequence on a plasmid where host replication machinery binds to initiate plasmid duplication, effectively dictating its copy number within the cell. Origins are categorized by copy number into low-copy (e.g., pSC101), medium-copy (e.g., p15A), and high-copy types (e.g., pUC).

7. (Extra) What are compatibility groups?

Compatibility groups classify plasmids based on their specific replication and partitioning mechanisms. Two plasmids belonging to the same compatibility group cannot stably coexist inside the same bacterial cell because they compete for the exact same replication machinery, eventually causing one to be lost during cell division.

8. Now for the previously chosen promoter and gene what will be the best origin or replication?

A medium-copy origin like p15A is the best fit for the inducible CrtI construct. It maintains a stable, moderate plasmid presence inside the cell that generates high levels of transcript upon induction without overwhelming the host’s metabolic machinery or causing structural plasmid instability.

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

The construct requires a strong Ribosome Binding Site (RBS), such as an optimized Shine-Dalgarno sequence, positioned upstream of the start codon to ensure efficient translation initiation. Downstream of the stop codon, a rho-independent terminator loop is essential to form a hairpin structure that releases RNA polymerase cleanly, ensuring transcript stability and protecting downstream plasmid components.

10. What are aptamers and riboswitches and how can they be used for metabolic tuning or engineering in prokaryotes?

Aptamers are structured RNA domains that fold to bind specific small molecules, and riboswitches are cis-regulatory elements containing these aptamer domains embedded within an mRNA transcript. In prokaryotes, they can be engineered into 5’ untranslated regions to dynamically modulate translation or transcription in direct response to intracellular metabolite concentrations, enabling real-time feedback loops.

11. Now what approach can be used to join all these parts together? Make a quick analysis of their sequence in search of possibilities (search for restriction sites, etc)

Golden Gate Assembly using Type IIS restriction enzymes (like BsaI or BsmBI) is the most efficient choice. Type IIS enzymes cleave outside of their recognition sites to generate unique, non-palindromic sticky overhanging sequences, allowing multiple sequence parts to be assembled seamlessly in a single, scarless reaction.

12. Try to elaborate further on a biosynthetic pathway you would want to engineer in E. coli for production of a metabolite or product. What use could this bio-product have? Imagine dream applications!!!

A dream application would be engineering the metabolic pathway for crocetindialdehyde and associated glycosyltransferases into E. coli to bioproduce crocin, the highly valuable antioxidant and water-soluble carotenoid pigment typically harvested from saffron. Producing crocin via scalable bacterial fermentation would lower production costs for natural food colorants and therapeutic antioxidants, offering an eco-friendly alternative to traditional agricultural harvesting.