Week 12 HW: Bioproduction of Beta-Carotene and Lycopene

Bioproduction lab

Post-Lab questions

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

Three genes are responsible crtE, crtI, and crtB for the activation of the lycopene pathway and four genes (same as lycopene) plus one more gene crtY for the production of beta-caroteno from Erwinia herbicola in E.coli.

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

The antibiotic resistance gene allows the plasmid to replicate and produce the enzymes in E.coli through multiple cell divisions. Without it, E.coli will not produce the plasmid because it is not necessary for its survival.

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

Differences in the cell concentration and lycopene and beta carotene.

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

OD600 measurements reflect the proportion of light absorbed by the concentration of cells that the light of 600 nm interacts with.

  1. 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 to extract hydrophobic pigments or small molecules from cells, such as chlorophylls, carotenoids, or other nonpolar metabolites. In this lab, acetone disrupts cellular material and allows the carotenoid pigment to go into solution.

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

Because E.coli is a well-know characterized microorganism for bioproduction and genetic engineering and also has a faster growth compared to Erwinia herbicola.

You may need the following papers to answer these questions:

Gene expression pattern analysis of a recombinant Escherichia coli strain possessing high growth and lycopene production capability when using fructose as carbon source

Improvement of Biomass Yield and Recombinant Gene Expression in Escherichia coli by Using Fructose as the Primary Carbon Source

Let’s get in touch with our metabolic pathway

What are the enzymes of the carotene pathway?

The key enzymes involved in lycopene/carotene synthesis in E.coli through the MEP pathway are: crtE - Geranylgeranyl pyrophosphate (GGPP) synthase, crtB - Phytoene synthase and crtI - Phytoene desaturase. Other enzymes important in precursor supply include: DXS (1-deoxy-D-xylulose-5-phosphate synthase) and IDI (isopentenyl diphosphate isomerase).

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

The papers suggest that precursor supply into the MEP pathway is one of the major bottlenecks. In particular, the DXS enzyme (1-deoxy-D-xylulose-5-phosphate synthase) is commonly considered a rate-limiting step because it controls carbon flux toward IPP and DMAPP production.

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)?

I would choose Escherichia coli for carotene/lycopene production because it grows rapidly, is easy to genetically engineer, and can achieve very high lycopene yields compared to other organisms. The articles showed that engineered E. coli grown on fructose reached high biomass and lycopene production while reducing acetate accumulation, which improves recombinant expression and overall productivity. In addition, E. coli has well-characterized metabolic pathways and many available molecular tools, making it an efficient platform for industrial bioproduction.

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 With the links below we are going to answer a few questions and think about the correct use of promoter: (https://blog.addgene.org/plasmids-101-the-promoter-region, https://www.addgene.org/mol-bio-reference/promoters/, https://blog.addgene.org/plasmids-101-repressible-promoters, https://blog.addgene.org/plasmids-101-inducible-promoters)

What is the function of a promoter?

The function of a promoter is to initiate the transcription of the gene of interest. It establishes the rate and amount of transcripts produced in the cell.

What types of promoters do we have?

Constitutive, and inducible (mediated by transcription factors as repressor or activators, temperature and light).

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?

If you wanted to turn OFF transcription in response to a metabolite, the best choice would be a repressible promoter and if we wanted to increase the transcription of a gene you will use an inducible promoter.

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 will use an inducible promoter to produce DXS enzyme that is going to activate according to cell concentration (quorum sensing system). Origin of replication of plasmid

With the links below we are going to answer a few questions and think about the correct use of origin of rep: (https://blog.addgene.org/plasmid-101-origin-of-replication, https://blog.addgene.org/plasmids-101-plasmid-incompatibility, https://blog.addgene.org/plasmids-101-ebook-4th-edition)

What is the origin of replication?

Also, call it ORI, is the place where DNA replication begins and it is usually a sequence rich of ATs for

What types of origin of replication do we have?

High-copy origins: produce many plasmid copies per cell. Example: pUC (~500-700 copies/cell). Medium-copy origins: around 15-40 copies/cell. Example: pBR322, pET, pGEX. Low-copy origins: around 5-10 copies/cell. Example: pSC101, p15A

(Extra) What are compatibility groups? Compatibility groups refer to groups of plasmids that cannot stably coexist in the same bacterial cell because they use similar replication machinery. Plasmids with the same ori are usually incompatible and compete with each other, causing instability. Therefore, if two plasmids are used together, they should contain different compatible origins of replication.

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

A medium- or low-copy-number origin, such as p15A or pSC101. Since DXS is involved in a metabolic pathway, excessive expression from a very high-copy plasmid could create metabolic burden and stress for the cells. A medium/low-copy origin provides more stable growth while still allowing controlled induction of expression through the quorum sensing promoter.

(Mandatory for Global listeners, Optional MIT/Harvard) Elaborate further on other bioparts like RBS, terminators, operators you would use for a correct design and further bioproduction?