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?