Week 12 HW: Building Genomes

Lab Homework: Bioproduction of Beta-Carotene and Lycopene

Post-Lab questions

Which genes when transferred into E. coli will induce the production of lycopene and beta-carotene, respectively?

Lycopene production in E. coli typically requires the carotenoid biosynthesis genes crtE, crtB, and crtI from organisms such as Erwinia herbicola (now Pantoea ananatis). Beta-carotene production additionally requires the crtY gene, which converts lycopene into beta-carotene through cyclization.

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

The antibiotic resistance gene acts as a selectable marker so only bacteria containing the plasmid survive when grown on antibiotic-containing media.

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

Different media compositions can affect bacterial growth rate and nutrient availability, influencing pigment production levels. For instance, the presence of fructose may increase carbon availability and metabolic flux toward carotenoid synthesis, while temperature changes can alter enzyme activity, protein folding, and plasmid stability, leading to differences in pigment accumulation.

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

OD600 measures optical density at 600 nm and is used as an estimate of bacterial cell density in liquid culture. In this experiment, higher OD600 values generally indicate greater bacterial growth, which can correlate with increased carotenoid production if the engineered pathway is functioning efficiently.

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 is commonly used to extract hydrophobic pigments or metabolites such as chlorophyll, carotenoids, and lipids from cells. It can also precipitate proteins while leaving smaller metabolites in solution for downstream analysis.

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

E. coli is easier to genetically manipulate, grows rapidly, and is widely used for industrial-scale recombinant protein and metabolite production. Studies have shown that engineered E. coli strains can achieve enhanced growth and lycopene production, especially when optimized carbon sources such as fructose are used.

Metabolic Pathway

What are the enzymes of the carotene pathway?

The main enzymes are:

  • CrtE: geranylgeranyl pyrophosphate synthase
  • CrtB: phytoene synthase
  • CrtI: phytoene desaturase
  • CrtY: lycopene cyclase

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

The phytoene desaturation step catalyzed by CrtI is often considered rate-limiting because it involves multiple sequential desaturation reactions required to form lycopene.

Notes for design of a DNA construct for bioproduction

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)?

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

For E. coli lets create a expression vector that works as a plasmid you choose E. coli let’s create a expression vector that works as a plasmids Now, for making a functional construct there are a variety of biological parts needed for this, like ribosome binding sites, terminators, operators and promoters. The last ones are the most important in terms of enzyme or protein production. Let’s elaborate further on this biopart

Promoter

What is the function of a promoter?

A promoter is a DNA sequence where RNA polymerase binds to initiate transcription of a gene.

What types of promoters do we have?

There is a varity of promoters, which includes:

  • Constitutive promoters
  • Inducible promoters
  • Repressible promoters
  • Tissue-specific promoters
  • Synthetic promoters

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?

For turning off the transcription in response to a metabolite, a repressible promoter is useful. On the other hand, an inducible promoter would be useful if transcription should increase in the presence of the metabolite.

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?

The lac promoter or T7 promoter could be used for crtI expression in E. coli. These promoters allow strong inducible expression, giving researchers control over carotenoid production timing and reducing metabolic burden before induction.

Origin of replication of plasmid

What is the origin of replication?

The origin of replication (ori) is the DNA sequence where plasmid replication begins inside the host cell.

What types of origin of replication do we have?

Origins can be:

  • High-copy origins
  • Low-copy origins
  • Broad-host-range origins
  • Narrow-host-range origins

(Extra) What are compatibility groups?

Compatibility groups describe whether two plasmids can coexist in the same cell without interfering with each other’s replication systems.

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

A high-copy origin such as ColE1/pUC would be useful for strong crtI expression and increased carotenoid production in E. coli.

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

  • Strong RBS for efficient translation
  • Terminators to stop transcription properly
  • Operators for transcriptional regulation
  • Selection markers for plasmid maintenance

These components improve stability, expression efficiency, and control of metabolic production.

(Hot! Extra points) What are aptamers and riboswitches and how can they be used for metabolic tuning or engineering in prokaryotes?

Aptamers are nucleic acid structures that bind specific molecules, while riboswitches are RNA regulatory elements that alter gene expression in response to metabolite binding. They can be used for metabolic tuning by dynamically regulating pathway genes depending on intracellular metabolite concentrations.

(Extra points) 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)

Methods include:

Gibson Assembly Golden Gate Assembly Restriction enzyme cloning Yeast homologous recombination

Restriction site analysis helps identify compatible cloning strategies and avoid unwanted cutting inside coding sequences.

(Extra Hot!!! Extra Points) 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!!!

I would engineer E. coli to produce biodegradable bioplastics such as polyhydroxyalkanoates (PHAs). These materials could replace petroleum-based plastics in packaging and medical applications, reducing environmental pollution and dependence on fossil fuels.

(Extra points) For S. cerevisiae create an integration cassette for homologous recombination.

As well as for prokaryotes, eukaryotic DNA designs need bioparts used for construction of a function design and further expresion. Now search for a biosynthetic pathway if interested and describe one of the genes of the pathway.

One interesting eukaryotic biosynthetic pathway is the resveratrol biosynthesis pathway engineered in Saccharomyces cerevisiae (Meng et. al, 2023). One important gene in this pathway is 4CL (4-coumarate:CoA ligase), which converts p-coumaric acid into p-coumaroyl-CoA, an essential precursor for resveratrol production. This step is important because it activates the substrate that will later be converted into resveratrol by stilbene synthase.

Now, remember that for making a functional construct there are a variety of biological parts needed for this, like ribosome binding sites or Kozak sequences, terminators, and promoters. List the ones you could use for DNA design.

Possible parts include:

  • GAL1 promoter
  • TEF1 promoter
  • Kozak sequence
  • CYC1 terminator
  • Selectable markers such as URA3 or LEU2

In yeast engineering we use DNA construction designs for making genome integration. What chromosome site could you use for integration of these and why?

Safe harbor loci such as the HO locus are commonly used because integration there minimizes disruption of essential genes while maintaining stable expression.

(Hot! Extra points) Following the next chart of how a DNA integration cassette should be designed and with the previously chosen parts elaborate the DNA sequence you could use to synthesize with Twist.