Week 12 HW: Building Genomes

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?

While E. coli naturally possesses the MEP pathway to produce the precursors IPP and DMAPP, it lacks the downstream enzymes required to synthesize lycopene. To enable lycopene production, the following three genes are required: crtE: Encodes geranylgeranyl pyrophosphate (GGPP) synthase, crtB: Encodes phytoene synthase, crtI: Encodes phytoene desaturase

The introduction of this gene set, collectively known as crtEBI allows the recombinant strain to accumulate lycopene.

The gene encoding lycopene cyclase is typically designated as crtY.

In addition to these primary synthesis genes, other sources highlight that production can be further enhanced by overexpressing genes like dxs (1-deoxy-D-xylulose-5-phosphate synthase) and idi (isopentenyl diphosphate isomerase) to increase metabolic flux toward the carotenoid pathway.

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

In the Du et al. (2016) paper, the recombinant strain E. coli K12f-pACLYC carries the plasmid pACLYC, which contains necessary genes for lycopene synthesis (crtE, crtB, and crtI). To ensure that the bacteria do not lose this plasmid as they divide, chloramphenicol (an antibiotic) is added to the culture medium. Because the plasmid provides resistance to chloramphenicol, only the cells that successfully retain the plasmid can survive and grow in the treated medium, so selective pressure means the bacteria retain the plasmid, thereby ensuring consistent lycopene production throughout the fermentation process.

(3) What outcomes might we expect to see when we vary the media, presence of fructose, and temperature conditions of the overnight cultures? Varying the growth conditions of the recombinant E. coli K12f-pACLYC strain demonstrates that fructose is the superior carbon source, outperforming glucose with a 3-fold increase in cell mass and a 7-fold increase in lycopene yield. This occurs because fructose uniquely reconfigures the bacteria’s metabolism: it up-regulates genes for its own transport while down-regulating pathways that produce waste (like acetate and lactate), leading to an accumulation of the essential precursors pyruvate and G-3-P. Additionally, fructose boosts the TCA cycle and oxidative phosphorylation to provide the abundant ATP and NADPH required for synthesis. To achieve these outcomes, cultures must be maintained at 37 °C with chloramphenicol to prevent plasmid loss, with harvesting typically occurring at 14 hours for fructose-grown cells to capture the peak mid-growth phase.

(4) Generally describe what “OD600” measures and how it can be interpreted in this experiment. OD600 determines relative cell concentration, and it can be interpreted as normalising each sample’s absorption peak measurement for the relevant pigment by the OD600 measurement from the corresponding bacterial culture. It can measure which culture conditions led to the highest production of either Lycopene or Beta-Carotene.

(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?

Based on the chemical properties of the compounds discussed, other experimental setups where acetone could be used to separate cellular matter from a target compound include: - Since lycopene is a tetraterpenoid carotenoid, acetone is a standard choice for extracting other members of this pigment family, such as beta-carotene, which the sources also identify as a target for metabolic engineering in E. coli

The sources mention that lycopene is naturally obtained from plants like tomatoes
Acetone can be used in experimental setups to separate these pigments from plant tissues, specifically tomato peels, often in conjunction with cell-wall degrading enzymes to improve yield
While the primary method described involves post-growth extraction, the sources reference “organic/aqueous culture systems” for in situ extraction
In such a setup, an organic solvent could potentially be used during the fermentation process itself to continuously separate the lipophilic product from the cellular biomass.

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

Recombinant E. coli K12f-pACLYC achieves a 7-fold increase in lycopene yield** (192 mg/g DCW) when grown on fructose compared to glucose. This high productivity is driven by transcriptional changes that redirect metabolic flux to increase precursor availability (pyruvate and G-3-P) while boosting the abundant energy and cofactors required for synthesis.

(1) Let’s get in touch with our metabolic pathway What are the enzymes of the carotene pathway?

The carotene pathway primarily utilizes crtE (GGPP synthase), crtB (phytoene synthase), and crtI (phytoene desaturase) to synthesise lycopene, which can then be converted into beta-carotene by lycopene cyclase. This synthesis is supported by upstream enzymes like ispA, dxs, and idi that manage precursor supply, as well as TCA cycle and oxidative phosphorylation enzymes that provide the high levels of ATP and NADPH required for production.

Within this pathway, which is the rate determining step (the step that takes the longest). Which enzyme is responsible for this step? Rate determining step is the condensation of two molecules of geranylgeranyl diphosphate to form 15-cis-phytoene. Phytoene synthase (PSY) is considered the enzyme used for this step, it is a major rate-limiting enzyme in the biosynthesis of carotenoids in plants. Because PSY drives the entire pathway, it is the primary target for genetic engineering to increase carotenoid content in crops.

(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 as S. cerevisiae is often used for similar purposes because it is a Generally Recognized As Safe organism and is highly robust in industrial fermenters. However, unlike E. coli, yeast typically uses the Mevalonate pathway rather than the MEP pathway for precursor synthesis. E. coli will be chose if our priority is high speed and maximum titer. S. cerevisiae lacks the endotoxins found in E. coli. It is also superior for producing more complex downstream xanthophylls like astaxanthin because it has the internal membranes and redox systems needed for Cytochrome P450 enzymes to function.

(2.2) 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

(3i) 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.

To design an expression vector for the overproduction of carotenoids in E. coli, I will focus on Phytoene desaturase, the enzyme encoded by the crtI gene. This enzyme is critical because it catalyses the conversion of phytoene into lycopene.

Plasmid outline:

Promoter: Initiates transcription. Operator: A segment of DNA to which a repressor or activator binds, allowing for inducible control (e.g., triggering production only when a specific substrate like fructose is present) Ribosome binding site (RBS): A sequence that recruits the ribosome to the mRNA to begin the translation of the crtI enzyme. Gene of interest (crtI): The DNA sequence that encodes the phytoene desaturase enzyme Transcription terminator: A sequence that signals the end of transcription, preventing the RNA polymerase from continuing into other parts of the plasmid. Antibiotic resistance gene: (e.g. chloramphenicol resistance) To ensure only bacteria carrying our plasmid survive in the culture

(3) 1a(i): the promoter initiates transcription, a start codon denotated as ATG. It acts as the binding site for RNA polymerase and transcription factors, initiating and regulating the transcription of a gene into mRNA.

(3) 1a(ii) What types of promoters do we have? Primary promoter types include constitutive, which are always active, as well as inducible and repressible systems

(3) 1a(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?

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?

To regulate transcription via a metabolite, repressible promoters stop gene expression, while inducible promoters increase it.

An inducible T7 promoter is suitable for controlling the CrtI enzyme in carotenoid biosynthesis, allowing for biomass accumulation prior to product synthesis.

(3) 1a(iv) To create an expression construct for the metabolic pathway, I will select the Lycopene beta-cyclase (LCYb) enzyme, which is the key driver for converting red lycopene beta-carotene.LCYb (Lycopene beta-cyclase), the CaMV 35S promoter (Cauliflower Mosaic Virus 35S), is a strong constitutive promoter, meaning it will drive high and continuous levels of the enzyme across almost all tissues of the host organism. Using a strong constitutive promoter like 35S will ensure that the bottleneck step (lycopene cyclisation) is consistently bypassed to maximise the yield of beta-carotene.

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? What types of origin of replication do we have? (Extra) What are compatibility groups? Now for the previously chosen promoter and gene what will be the best origin or replication?

The origin of replication is a specific DNA sequence that initiates plasmid replication within a host cell, determining copy number and incompatibility. Types include high-copy (e.g. pUC) and low-copy (e.g. pBR322) origins, while incompatibility groups define plasmids that cannot coexist due to shared replication machinery. For maximum expression of an LCYb enzyme, a high-copy origin such as pUC is typically recommended.

For the CaMV 35S promoter and LCYb gene construct, the best choice is a high-copy pUC origin of replication (ori). pUC origin (derived from pMB1/ColE1). A high-copy origin like pUC produces 500–700 copies per cell, which maximizes the “gene dosage” available for the CaMV 35S promoter to drive massive enzyme production. For metabolic engineering of pathways like beta-carotene synthesis, maximizing the amount of the bottleneck enzyme (LCYb) is usually the primary goal to ensure high final yields.

(4) (Mandatory for Global listeners, Optional MIT/Harvard) Elaborate further on other bioparts like RBS, terminators, operators you would use (5) for a correct design and further bioproduction? (Hot! Extra points) What are aptamers and riboswitches and how can they be used for metabolic tuning or engineering in prokaryotes?

To ensure an effective genetic design for bioproduction, you should incorporate several critical regulatory parts. A strong consensus RBS (like the Shine-Dalgarno sequence) is essential to recruit ribosomes efficiently and ensure high translation rates for the enzyme. To maintain genetic stability, a strong Rho-independent terminator should be placed at the end of the gene to prevent transcriptional read-through that could interfere with other plasmid elements or waste energy. For control, operators (such as the lac operator) can be integrated to pause production until the culture reaches an ideal density. Beyond these standard parts, aptamers—nucleic acids that bind specific molecules—can be used within riboswitches to provide metabolic tuning. This allows for dynamic control where the cell automatically senses metabolite levels and adjusts production, either boosting it when precursors are plenty or down-regulating the pathway to prevent the buildup of toxic intermediates. (6)

(7) I would engineer E. coli to produce L-DOPA, the primary precursor to dopamine, to create a living medicine for Parkinson’s disease. By inserting a highly active tyrosine hydroxylase gene and optimizing the pathway with a high-copy pUC origin and a strong RBS, I could turn the bacteria into a sustainable bio-factory for this critical neurological treatment.

(8.2) I chose the artemisinin biosynthetic pathway, typically engineered in S. cerevisiae (yeast) to produce the critical antimalarial drug. This pathway converts simple sugars into artemisinic acid, which is then chemically converted to artemisinin. It involves shifting the cell’s internal metabolic flux from the Mevalonate pathway toward complex sesquiterpenes. I chose CYP71AV1, who’s function is encoding Amorphadiene oxidase, a cytochrome P450 enzyme. It performs a vital three-step oxidation, converting the intermediate amorpha-4,11-diene into artemisinic alcohol, then artemisinic aldehyde, and finally artemisinic acid. Because this enzyme is membrane-bound and requires an electron donor to function, it must be co-expressed with a Cytochrome P450 Reductase. In eukaryotic hosts like yeast, these enzymes naturally localize to the Endoplasmic Reticulum , a compartment missing in E. coli, making yeast the superior factory for this specific gene.Bioparts for Eukaryotic To express CYP71AV1 correctly in yeast, you would use:Promoter: A strong, inducible promoter like GAL1, which allows you to grow the yeast on glucose first and then “switch on” drug production by adding galactose.Terminator is A eukaryotic-specific terminator like CYC1t to ensure proper mRNA polyadenylation and stability.Selection marke is URA3 gene, which allows to select only the yeast cells that have successfully taken up the DNA construct.

(8.3) To design a functional genetic construct, you must select biological parts tailored to your host organism’s cellular machinery. For translation initiation, prokaryotic systems require a Shine-Dalgarno ribosome binding site (RBS) to recruit ribosomes, while eukaryotic designs utilize a Kozak sequence to position the ribosome at the start codon. Transcription is driven by promoters, which can be constitutive for constant output (like BBa_J23100 in bacteria or CMV in mammals) or inducible for controlled timing (such as T7 or GAL1). To ensure the process ends cleanly, terminators like the Rho-independent T7 terminator in bacteria or the SV40 polyA signal in eukaryotes are used to stop RNA polymerase and stabilize the mRNA. Finally, these elements are housed in a plasmid backbone containing an origin of replication (like pUC for high copy numbers) and a selectable marker (such as Ampicillin resistance or URA3) to ensure only the engineered cells are maintained in culture.

(8.4,8.5) For yeast genome integration, I’ll target the HO locus or a site like the LPP1 or X-2 loci. These sites are chosen because they allow for stable, high-level gene expression without disrupting essential yeast functions or affecting the cell’s natural growth rate. For the Twist DNA Sequence Design, the DNA sequence should be ordered as a single continuous block arranged in this specific order: 5’ Integration Flank: A ~500bp sequence homologous to the upstream region of the HO locus to guide the DNA into the chromosome, Promoter: The GAL1 sequence to allow for inducible control of your pathway, RBS/Kozak: A strong Kozak sequence (AAAAAAATGG) for efficient translation in yeast, Gene: The codon-optimized sequence for CYP71AV1 (or your chosen metabolic gene), removing any internal restriction sites like BsaI, Terminator: The CYC1 terminator to ensure mRNA stability and proper polyadenylation, Selection Marker: A URA3 expression cassette to allow you to select for successful integration, 3’ Integration Flank: A ~500bp sequence homologous to the downstream region of the HO locus to complete the crossover during recombination.