Week 1 HW: Final Project Ideas

1. What would you like to biomanufacture, and why?

Target product: A beta-carotene-rich oil derived from the mesocarp (pulp) of aguaje (Mauritia flexuosa), produced through plant cell suspension culture and intended for use as an active ingredient in an antioxidant and photoprotective serum or cream.

Motivation:

• Environmental sustainability: Aguaje is traditionally harvested by cutting down the entire palm because the fruit-producing female trees can only be harvested by climbing or felling them. This practice has led to the overexploitation of aguaje palm stands in the Loreto and Ucayali regions of the Peruvian Amazon, damaging key ecosystems known as aguajales—wetlands that serve as carbon sinks. Producing the active compound through cell culture would decouple commercial demand from tree felling, providing a scalable production method that does not place additional pressure on wild populations.
• Genuine nutraceutical and cosmetic value: Aguaje mesocarp has one of the highest reported beta-carotene concentrations among fruits and has been reported to contain more than carrots. This makes it an attractive candidate for use as a natural topical antioxidant and photoprotective ingredient because its beta-carotene absorbs part of the UV radiation spectrum and neutralizes sun-induced free radicals.

Who is it for? It is intended for the cosmetics industry, particularly formulators of antioxidant and anti-aging serums and creams who seek natural active ingredients with traceable and sustainable production.

Why is it important? This approach addresses the overexploitation of aguajales by providing a biotechnological alternative for producing high-value beta-carotene for the cosmetics industry without relying on the continuous extraction of wild fruits. In addition, it applies plant tissue culture to an undomesticated and understudied species, presenting a technical challenge with the potential to generate a sustainable, traceable, and scalable solution.

2. What living system would you use?

Proposed chassis: A plant cell suspension culture derived from callus induced from aguaje mesocarp tissue.

What goes into the system?

• Initial explant: Mesocarp (pulp) tissue from aguaje fruit, obtained from a wild source only once—or from pulp already harvested commercially for food—to avoid additional tree felling for research purposes.
• MS (Murashige and Skoog) or B5 culture medium supplemented with plant growth regulators, such as the auxin 2,4-D and the cytokinin BAP, to induce callus formation and subsequently maintain the cell line in liquid suspension.
• A carbon source (sucrose) and, during the carotenoid-induction phase, possibly controlled light exposure, as carotenoid production in culture is often induced or enhanced by light.

What biological process occurs?

1. Callus induction: The mesocarp explant is cultured on solid medium containing plant growth regulators until it dedifferentiates into a callus, an unorganized mass of cells.
2. Establishment of a cell suspension line: The callus is transferred to agitated liquid medium in a bioreactor or an orbital shaker flask to generate a homogeneous, rapidly growing cell suspension.
3. Induction of carotenoid biosynthesis: Culture conditions—such as light exposure, precursors like mevalonic acid, or elicitors—are adjusted to activate the carotenoid biosynthetic pathway (the MEP/mevalonate pathway) and maximize beta-carotene accumulation in the cells.
4. Oil extraction: The cells are harvested and processed by solvent extraction or pressing, depending on whether the beta-carotene accumulates in intracellular lipid bodies, to obtain a beta-carotene-rich oil.

Assumptions:

• It is assumed that callus can be induced from aguaje mesocarp using standard protocols developed for palms, as this process is not as well documented as it is for model species.
• It is assumed that the carotenoid biosynthetic pathway remains active—or can be induced—in dedifferentiated cultured cells. However, because beta-carotene accumulates in chromoplasts within differentiated fruit tissue, an additional differentiation step or the use of elicitors may be required.
• Access to a standard MS culture medium and a simple stirred bioreactor is assumed, without requiring a highly specialized bioprocess design at this stage.

3. How would you use automation?

A desktop liquid-handling robot such as an Opentrons OT-2 could be used, combined with a small automated shaking and bioreactor system, to maintain and screen the suspension cultures.

Which part of the workflow would you automate, and why?

• Screening and selection of cell lines: After inducing the callus, it is necessary to test dozens or hundreds of individual cell lines to measure beta-carotene yield, since carotenoid accumulation varies greatly among clonal lines. I would automate media dispensing and sampling in 96-well plates, allowing many induction conditions to be tested in parallel instead of manually pipetting each variation. This makes it possible to identify the cell lines and induction conditions that truly produce significant beta-carotene levels.
• Sampling and measurement: The same platform would automate periodic sampling of culture aliquots for beta-carotene quantification by spectrophotometry or HPLC, replacing manual sampling and reducing contamination risk from repeated handling of the cultures.
• Small-scale culture maintenance: Automated control of agitation/aeration would standardize subculture intervals and inoculum density.

Final Project