Design

Design Overview

To design an eco-friendly fragrance production system, we engineered Synechocystis sp. PCC 6803 by introducing different terpene synthase genes via shuttle vectors to produce the fragrance molecules pinene, limonene, bisabolene, farnesene, santalene and santalol.

Figure 1: Summary of the production process of terpenes in cyanobacteria.

Terpenes and Fragrances ///

Terpenes are a large family of hydrocarbons that are responsible for the way most plants smell. They are categorized based on the number of five-carbon units. The fragrance molecules we aim to produce are in the categories of monoterpenes (C10) and sesquiterpenes (C15) [1].

Taking into consideration the effect of different fragrance molecules in aromatherapy and the challenges in their production, we decided to produce the fragrances pinene, limonene, farnesene, bisabolene, santalene and santalol.

Overview of terpenes we aim to produce:

Pathway ///

All terpenes are produced in nature from the same 5-carbon-atom building blocks: dimethylallyl-pyrophosphate (DMAPP) and isopentenyl-pyrophosphate (IPP). These are further modified to form different terpenes. Cyanobacteria are capable of producing terpenoids via the non-mevalonate (MEP) pathway. This process relies on G3P and pyruvate derived from photosynthesis. In the MEP pathway, which is present in our chassis cyanobacteria, IPP condenses with DMAPP to produce geranyl pyrophosphate (GPP), which are the precursors of all monoterpenes. GPP upon one additional condensation with IPP produces farnesyl pyrophosphate (FPP), which can be further modified into the sesquiterpenes we aim to produce with the addition of a specific terpene synthase [7].

Figure 2: Diagram of the MEP pathway.

Chassis selection ///

With recent achievements in synthetic biology and metabolic engineering, microbial production of terpenes has emerged as an attractive alternative. Cyanobacteria are a promising candidate for achieving this purpose. They are photosynthetic microorganisms that are emerging as attractive hosts for production of biochemicals. Their relatively simple physiology, fast photosynthetic growth and availability of model systems makes them ideal chassis. Unlike other chassis, such as yeast or E. coli, the photosynthetic nature of cyanobacteria allows them to harness solar energy directly and rely on CO2 as its sole carbon source, thereby reducing the need for external carbon sources contributing to a more environmentally friendly and cost-effective platform compared to other hosts and production methods. Moreover, taking into consideration that apart from the fragrance extracted, the engineered cyanobacteria itself will also be one of the final products that we would like to present to the public, cyanobacteria is a much better choice compared to other chassis due to its pleasant green sight and odorless characteristics. We’ve chosen Synechocystis sp. PCC 6803 specifically because it is a single cell organism and a model cyanobacteria. Additionally, its susceptibility to genetic manipulation since it is a natural competent cell. However, throughout our experiment, we also used E. coli DH5α before proceeding to cyanobacteria, as it was necessary to amplify the plasmids we constructed in E. coli first.

Plasmid Construction ///

To allow Synechosystis sp. PCC 6803 to produce our desired fragrance molecules, we introduced ten different terpene synthase genes into Synechosystis sp. PCC 6803 using a shuttle vector. We constructed ten plasmids using the broad host range replicative vector pPMQAK1, which is based on the replicon RSF1010. Our experimental process involves the use of both E. coli and cyanobacteria, therefore a shuttle vector that can replicate in both organisms is necessary. The spectinomycin immune sequence is present in our shuttle vector to facilitate the process of determining whether we successfully transformed our constructed plasmid into our host organism or not. We added spectinomycin into all culture mediums to ensure that the organisms that survive have our constructed plasmids in them.

Inspired by the work done by the Toulouse team in 2021, we decided to use an induction system composed of Ptrc promoter and theophylline dependent riboswitch theo E* to control the expression of the terpene synthases. The Ptrc promoter is a hybrid of lac and trp, making it stronger than the lac promoter (8). Transcription is regulated by IPTG and translation initiates only when there is theophylline present. This double regulation strictly regulates gene expression. We chose a theophylline inducible riboswitch specifically because it is not normally present in biological systems; therefore, it will not be affected by metabolic activities of organism. We used an inducible system because through it, we can control when our cyanobacteria will produce terpenes. This is very important because otherwise, the terpenes will be constantly produced, and it will excessively stress the cyanobacteria before it has grown to a high enough concentration to start producing terpenes efficiently. The genes coding for the different terpene synthases (αPS, βPS, PtPS, LIMS, MsLIMS, LIS, AgBS, PaFS, CYP736A167, SaSS) (1) are inserted between the riboswitch and the terminator (BBa_B0015).

Figure 3: Diagram of the plasmid we constructed for the terpene synthases αPS, βPS, PtPS, LIMS, MsLIMS, LIS, AgBS, PaFS, CYP736A167 and SaSS, which are inserted between the riboswitch and terminator.