Synthetic Biology of N2-Fixing Cyanobacteria for Photosynthetic Production of Perfumed Linalool from Air and Water

Linalool (C₁₀H₁₈O) is a naturally-occurring terpene alcohol, emitted as a volatile from many flowers and over 200 aromatic plants. In plants, linalool is produced from the universal isoprenoid intermediate geranyl diphosphate (GPP) through the MEP pathway. Linalool has many commercial applications including perfumed hygiene products, flavor and fragrances, pharmaceuticals, and drop-in biofuels. Commercial production of linalool from plant extracts is limited by availability of raw feedstocks and high extraction costs. Engineering N₂-fixing cyanobacteria to photosynthetically produce and secrete linalool may have potential to simultaneously solve these two problems.

Unlike plants, N₂-fixing cyanobacteria are capable of performing both solar-powered N₂-fixation and CO₂-fixation. Their simple input requirements for rapid growth and ease of genetic manipulation, together with the highest photosynthetic efficiency and suitability for industrialized production, N₂-fixing cyanobacteria are attractive organisms for engineering production of linalool. Cyanobacteria, like plants, have native metabolic pathways (Calvin cycle and MEP pathway) to photosynthetically convert CO₂ and water into a variety of reduced carbon compounds, including GPP, the precursor for linalool. However, cyanobacteria lack the linalool synthase that plants use to convert GPP into linalool. In this research, we have created three generations of linalool-producing cyanobacteria. First, a linalool synthase gene from Norway Spruce was fused to a synthetic, dual cyanobacterial P_nir–P_psbA1 promoter and subcloned into a shuttle vector for transformation of an N₂-fixing cyanobacterium Anabaena sp. PCC7120. The first generation of transgenic Anabaena (with a single plant linalool synthase gene) was confirmed to continuously synthesize and secrete linalool using only air (CO₂ and N₂ gas), mineralized water and light energy. To increase linalool production, a synthetic operon coding for three key enzymes (DXS-IDI-GPPS) from the MEP pathway were over-expressed in Anabaena. This second generation strain produced 2.8 fold more linalool than the first generation strain. Under 50 µE·m^-2·s^-1 illumination, the maximum linalool productivity was 35µg/L/day/OD₇₂₀ when nitrate was used as the sole nitrogen source, and 36.1µg/L/day/OD₇₂₀ when atmospheric N₂ gas was the sole nitrogen source. To further increase linalool production, we created a third generation of the linalool-producing Anabaena by introducing a synthetic CO₂-fixing photorespiratory bypass that is based on the 3-hydroxypropionate bi-cycle (3-HP-bicycle). This synthetic photorespiratory bypass route containing six enzymes is expected to avoid the loss of fixed carbon during photorespiration, and also functions as an additional O₂-tolerant, CO₂-fixing pathway, alongside the Calvin cycle. The third generation strain showed a maximum productivity of 133.7µg/L/day/OD₇₂₀ when the culture was supplemented with 1.0% CO₂,producing about10.7-fold more linalool than the first generation strain.

In summary, we used synthetic biology to have developed N₂-fixing cyanobacteria that can directly convert solar energy, CO₂ and water into a fragrant linalool. The linalool produced by the engineered cyanobacterium was secreted into the media and volatilized into the flask headspace, allowing for easy separation of linalool from the culture biomass. Thus, the engineered N₂-fixing cyanobacterium can serve as a living cellular factory to continuously produce and emit a wide range of commodity chemicals/fuels using only atmospheric gases (CO₂ & N₂), mineralized H₂O, and sunlight.