Plant Synthetic Biology

Instead of using a model organism like Eschericiha coli, our team opted for building our pathways in a single-celled algal plant. Chlamydomonas reinhardtii, or as we call it, “chlamy”, is a unique candidate for the uptake of excess fertilizers due to features this algae has that other model organisms are lacking. Plants offer many benefits that heterotrophic organisms cannot provide. For example, due to its unique phototrophic abilities; we do not need to feed chlamy with additional carbon sources other than carbon dioxide. This allows for easy application of a treatment to a water treatment facility, for example. Plants offer a powerful platform for producing a diverse set of synthetic biology products that can be applied to a large number of application-specific outcomes.

Nitrogen and phosphorus are readily utilized by chlamy. This makes chlamy an ideal candidate for excess neutrient clean up, especially in aqueous environments. For C. reinhardtii, nitrates are a viable energy source which are then converted into nitrous oxide. Chlamy also readily uptakes phosphates as nutrients. The usage of chlamy as a chassis for phosphorus and nitrogen pathways is a natural choice as it already completes many nutrient fixation stages on its own. Our engineering for this alga is unique in that it accentuates chlamy’s natural tendencies and allows for it to keep its normal functions overall. Without the use of a plant, these cycles may be less efficient; inserting an abundance of foreign parts into a chassis less suited for performing these functions could lead to an overall decreased function of the organism.

On our “Experiments” and “Results page!” pages, you can see much more detail about the steps of the Engineering Cycle we went through to optimize this photosynthetic chassis as a wastewater-cleaning machine. Below, we show some of the main proteins that are expressed by our chassis by utilizing synthetic biology principles.

Overview of our engineered plant chassis

Figure 1: This image shows the theoretical uptake system that our team designed for nitrogen and phosphorus uptake in C. reinhardtii. Using Psr1, phosphates will accumulate within the cell and a miRNA will prevent the phosphate transporters from releasing it. For nitrates, chlamy naturally converts nitrates to nitrites, then to nitric oxide and nitrous oxide. Our engineered part, nosZ from denitrifying bacteria works to convert nitrous oxide, a potent greenhouse gas, to nitrogen gas.

Phosphate

To engineer chlamy to uptake more phosphate from wastewater, we used the Chlamydomonas MoClo toolkit to clone it’s Psr1 gene with a constitutively-active Psad promoter. This gene, normally only expressed under phosphate-starvation conditions, induces increased uptake of phosphorous from the environment.

Figure 2: Protein model of the PSR1 transcription factor. This model was generated using AlphaFold2 as a predicted structure for PSR1. The dark blue coloring is used to distinguish regions of high confidence from regions of low confidence, in red.

As a result of over-expressing Psr1, we found that chlamy was more efficient at removing phosphate from wastewater, as shown below.

Figure 3. Phosphate concentration of TAP media inoculated with untransformed (orange) chlamy or chlamy transformed to express Psr1 with a constitutive promoter (blue). The engineered chlamy removes phosphate more quickly from the wastewater.

Nitrogen

To engineer chlamy to convert nitrous oxide into dinitrogen gas, we cloned codon-optimized versions of the nosZ gene; one taken from the bacterium Dechloromoas denitrificans, and the other from Pseudomonas stutzerii. The structures of the encoded proteins are shown below.

Figure 4: This predicted protein structure of nosZ that comes from D. denitrificans. This is the highest ranking predicted structure from AlphaFold2. D. denitrificans was chosen for its capability as a denitrifying bacterium.

Figure 5: This predicted protein structure of nosZ that comes from P. stutzeri. This is the highest ranking predicted structure from AlphaFold2. P. stutzeri was chosen for its ability to be implemented into plants based on previous research.

Future Directions and Conclusion

In future endeavors, algae that has been used to collect nitrates and phosphates could be used as a regenerative fertilizer that would prevent the need to collect new materials. As algae decompose, their nutrients could be redistributed into fields used by farmers. Instead of farmers purchasing fertilizers from outside sources and the runoff from their farms carrying the excess fertilizer to a body of water, nutrient supplements could be used in a closed system. This would mean that no new fertilizers would be added to a farm as it would be recycled back to a source that normally needs nutrient boosts. By containing a system of nutrient replenishment, future algal blooms are prevented as there is a decrease of nutrients added to an environment plagued by cyanobacteria. Our engineered algae could be used as a replacement for commercial fertilizers which could further help prevent blooms from growing in the first place.

Successful builds of our parts could potentially be moved to another plant which would allow for further development of algal bloom reduction mechanisms. Starting our plasmid builds in a simple, single-celled organism allows for testing and design which can grow into more complex systems. Knowing how a simple system works allows us to search for a future, more sophisticated system that may be better suited for needs in other domains of algal bloom prevention. One such future chassis we have considered is seaweed, which can take on a larger load of nutrient uptake and provide a secondary function as a potential food source for cattle due to the reduction of methane emissions seen in bovines that consume it. Such implmentation would help in both cleanup of our waterways and further prevention of future pollution.