Week 12 Lab: Bioproduction of Beta-Carotene and Lycopene

Special Note: This lab had both a section mandatory for all students and one especially for committed listeners.

Post Lab Questions | Mandatory for All Students

(References supplied from lab:

A) Du W, Song Y, Liu M, Yang H, Zhang Y, Fan Y, Luo X, Li Z, Wang N, He H, Zhou H, Ma W, Zhang T. Gene expression pattern analysis of a recombinant Escherichia coli strain possessing high growth and lycopene production capability when using fructose as carbon source. Biotechnol Lett. 2016 Sep;38(9):1571-7. doi: 10.1007/s10529-016-2133-0. Epub 2016 Jul 5. PMID: 27379652.

B) Aristidou, A.A., San, K.-Y. and Bennett, G.N. (1999), Improvement of Biomass Yield and Recombinant Gene Expression in Escherichia coli by Using Fructose as the Primary Carbon Source. Biotechnol Progress, 15: 140-145. https://doi.org/10.1021/bp980115v

Additional used for below:

C) Shumskaya, M., & Wurtzel, E. T. (2013). The carotenoid biosynthetic pathway: thinking in all dimensions. Plant science : an international journal of experimental plant biology, 208, 58–63. https://doi.org/10.1016/j.plantsci.2013.03.012

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

The genes that when transferred into E.Coli will induce the production of lycopene and beta-carotene are crtB, crtE, crtl, and crtY.

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

The antibiotic resistance genes allow for selection pressue among E.Coli who have the plasmid vs those who do not.

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

I’d imagine that we would see changes in bacterial growth and protein expressions.

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

OD600 works to measure cell density via turbidity. You can use this to estimate population growth and overall production 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?

Applications towards pigment isolation, lipid extraction, and generally any that lean on solvent mixture optimizations may suffice.

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

We can scale up production and farm E.coli in far more diverse environments. Further, we can edit E.Coli more easily for this process.

Post Lab Questions | For Committed Listeners Only

1. Let’s get in touch with our metabolic pathway

• 1. What are the enzymes of the carotene pathway?

The enzymes of the carotene pathway are Phytoene synthase, GGPP synthase, Phytoene desaturase, and Lycopene ε-cyclase.

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

phytoene synthase (PSY) synthesis

Zhou, X., Rao, S., Wrightstone, E., Sun, T., Lui, A. C. W., Welsch, R., & Li, L. (2022). Phytoene Synthase: The Key Rate-Limiting Enzyme of Carotenoid Biosynthesis in Plants. Frontiers in plant science, 13, 884720. https://doi.org/10.3389/fpls.2022.884720

2. Notes for design of a DNA construct for bioproduction

• 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 lean on E. Coli for the purpose of rapid prototyping and famailiairity (including easier genetic engineering)

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

Enzyme chosen: Phytoene synthase

• 3. 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
• • 1. 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.
• • • 1. Promoter
• • • • 1. 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)
• • • • • 1. What is the function of a promoter?

Promoters function to recruit RNA polymerase to kickstart transicription.

• • • • • 2. What types of promoters do we have?

Eukaryotic, Prokaryotic

• • • • • 3. 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?

A) For turning off transcroption, we would need a promoter that can be repressed (Repressible) B) To increase transcription, we need an inducible promoter.

• • • • • 4. 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?

3. Origin of replication of plasmid

• 1. 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)

Ptac might be the best for the ability to induce production tightly.

• • 1. What is the origin of replication?

The origin of replication refers the a DNA sequence that determines plasmid replicability within a host.

• • 2. What types of origin of replication do we have?

We have many types such as ColE1, pMB1, pSC101, R6K, 15A, and more. Some have relaxed and stringent controls of replication. There are numerous incompatibility groups and varied copy numbers among them. Host compatibility/incompatibility and copy number are common means to categorize them.

• • 3. (Extra) What are compatibility groups?

Compatibility groups are categories of plasmids that can coexist within the same bacterial cells. Competition for replication material components within bacteria are a source of incompatibility.

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

15A might be a reasonable candidate.

4. (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?

For these, I would hunt parts found on IGEM given prior reserch, standardization, and testing. I would like an inducible promoter. For the RBS, terminators, and operators, I might choose the following noting initial steps for protoyping:

For the RBS: BBa_B0034 Terminator: BBa_B0015 (Double terminator) OPerator: BBa_R0010 (lac regulated and inducible)

These would be emphasized for the prior reasons plus tigter regulation.

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

A) Aptamers refer to short genomic sequences that bind to specific molecules. B) Riboswitches are regulatory genomic elements that have a sensing region that acts like an aptamer and an expression-control region that can be used to modify either transcription or translation in the presence of a metabolite.

C) These can be used to tune protein expression in protkaryotic systems.

6. (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)

Gibson and Golden Gate could be used. For a quick analysis, the availibility of compatible restriction sites may lean one towards Gibson whereas a desire to use multiple RNA regulatory parts could lean someone moreso towards Golden Gate Assembly.

7. (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!!!

In a very high-level way, a biosynethic pathway that is responsive to changing macro-host conditions could be neat to engineer. This bio-product could have medications.

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

I’m not sure of the level that is desired here. At a high level: a cassette can include a left homology armm, promoter, Kozak sequence, our gene, terminator, a marker like KanMX (dominantn antibiotic selection), and a right homology arm.

• 1. First let’s check some concepts of yeast engineering and homologous recombination this in this notes
• 2. 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.

I’m happy to use Carotoid biosynthesis via S. cerevisiae and use crtYB which encodes for phytoene synthase.

“Phytoene synthase (PSY) catalyzes the first committed step in the carotenoid biosynthesis pathway and is a major rate-limiting enzyme of carotenogenesis”

See: Ledetzky N, Osawa A, Iki K, Pollmann H, Gassel S, Breitenbach J, Shindo K, Sandmann G. Multiple transformation with the crtYB gene of the limiting enzyme increased carotenoid synthesis and generated novel derivatives in Xanthophyllomyces dendrorhous. Arch Biochem Biophys. 2014 Mar 1;545:141-7. doi: 10.1016/j.abb.2014.01.014. Epub 2014 Jan 30. PMID: 24486200.

Phytoene synthase has been described above.

• 3. 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.

Again, using IGEM parts, some of my hypothethical parts might be:

Koazak sequence BBa_K165002 See: https://registry.igem.org/parts/bba-k165002 Terminator BBa_K2637017 See: https://registry.igem.org/parts/bba-k2637017 Promoter BBa_K2637023 (Constitutive yeast promoter) Marker KanMX See: https://registry.igem.org/parts/bba-k300989

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

I’d consider the HO locus given research pointing at it being a relatively safe site.

See: Voth WP, Richards JD, Shaw JM, Stillman DJ. Yeast vectors for integration at the HO locus. Nucleic Acids Res. 2001 Jun 15;29(12):E59-9. doi: 10.1093/nar/29.12.e59. PMID: 11410682; PMCID: PMC55758.

Voth, W. P., Richards, J. D., Shaw, J. M., & Stillman, D. J. (2001). Yeast vectors for integration at the HO locus. Nucleic acids research, 29(12), E59–e59. https://doi.org/10.1093/nar/29.12.e59

• 5. (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.

At a high-level the following parts would be helpful to design: Homology arms (flanking the casette), Promoter, Kozak Sequence, our gene of interest (crtYB), terminator, and our selectible marker, to be eventually inserted into the HO locus. I’d be minfdul that the sequence does not contain incompatible restriction sites and minimize undesirable repeats and other sub-optimal sequence features.