21EngBio: Organelle engineering for the production of useful bioproducts

Nicotiana benthamiana is a plant similar to tobacco. Certain strains of Agrobacterium (a soil bacteria) can be taken up or infect N. benthamiana and express genes transiently to form protein or metabolite based products. The efficiency or properties of the host N. benthamiana line play a major role in yield and quality of the products produced. To date the main products from the N. benthamiana cell factory that have benefited humanity have been proteins, including vaccine components and antibodies.
Chloroplasts are key structures within photosynthetic plant cells that are essential for the functioning of the plant. Specific biochemical pathways exist in the chloroplasts and thus certain valuable chemicals are only generated in the plastid. The metabolism performed in the chloroplast also drives the metabolism of the plant. The chloroplast also represents a cellular structure that can be used to store or deposit chemicals (metabolites).
The potential exits to also produce small molecules (metabolites /chemicals) in N. benthamiana. This would in many cases improve the environmental credentials of production as presently chemical synthesis, using precursors derived from petrochemical refining industry are used predominantly. Numerous valuable plant derived metabolites and precursors that we use daily are synthesised or stored in the chloroplast. The chloroplast also provides vital energy for the cell through its unique metabolism. In the present project our ambitions goal is to create cells with more chloroplasts. Thus, lines of N. benthamiana will be generated that contain more chloroplasts per cell. The increase in the chloroplast complement of the cell will be achieved by modulating regulators that perceive the quantity of light. Effectively the plant will be switched on to experience light incidence all the time. Creating stable lines will generate more chloroplasts which will enable them to act as hosts to produce other valuable small molecules.
In the project we will demonstrate that the plant created can act as a more efficient host to produce Astaxanthin and Squalene. These are valuable biosynthetically related molecules that are used across multiple industrial sectors. Astaxanthin is a valuable feed additive that is incorporated into aquaculture and poultry feeds. It is responsible for conferring the colour to the flesh of salmon/trout and the egg yolks of chickens. Without this colour the produce generated cannot be sold. Squalene is used in personal care products and is the leading adjuvant for vaccines. Presently, it is a commodity in great demand as vaccine production is paramount with the rapid development and production of vaccines against sever acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The present major source of squalene is shark's liver. Demand is outstripping supply and in both cases and production methods have poor environmental credentials. Thus, delivery of new plant cell factories represents a new sustainable production method.

Nicotiana benthamiana can be used as a plant cell factory. Agroinfiltration of N. benthamiana to produce valuable proteins and organic compounds is classified as a New Plant Breeding Technique (NPBT). To date the approach has been used to produce pharmaceutical proteins such as antibodies and vaccine components. In fact it is likely that the next SARS-CoV-2 adjuvanted vaccine will be produced in N. benthamiana. The potential also exists to produce small molecules in N. benthamiana in either an independent manner or combined as a multipurpose feedstock. In order to advance N. benthamiana as an amenable chassis for small molecule production, our goal is to create new N. benthamiana lines that contain a higher chloroplast complement per cell. To achieve this goal a genome editing approach will be used to impair and knock out the function of the regulators DAMAGED DNA BINDING PROTEIN-1 (DDB-1), and DE-ETIOLATED-1 (DET-1). These gene products are components of the light signal transduction pathway. Loss or impairment of these regulators results in the plant effectively perceiving light irradiation constantly. At a cellular level a greater complement of chloroplasts occurs in the cell. Concurrently, the metabolome is affected, with changes in intermediary and specialised metabolism. Our hypothesis is based on foundational work performed in tomato with the HIGH PIGMENT 1 and 2 mutant alleles of DDB-1 and DET-1. The utility of stable N. benthamiana lines with altered DDB-1 and DET-1 will be tested using a construct encoding the genes for astaxanthin formation and squalene. The squalene synthesising vector facilitates the orthogonal engineering of squalene in the plastid. Both astaxanthin and squalene forming vectors have been shown to work efficiency in N. benthaminana. With these tools available we will determine if high chloroplast containing chassis can be used as a resource to increase the production of valuable bioactive compounds.