Bio-based production of hydrocarbons using a new photoenzyme – PHOTOALKANE

In vitro experiments were carried out on recombinant FAP to better characterize the enzyme photocatalytic process. Time-resolved spectroscopic measurements, CO2 production measurements by membrane inlet mass spectrometry, and measurements of volatile hydrocarbons by thermodesorption coupled to gas chromatography were carried out to monitor the operation of the photoenzyme under variable illumination conditions and in the presence of fatty acids of varying chain lengths. All these approaches converged to show that FAP was capable of converting octanoic acid to heptane via an autocatalytic process. The engineering of bacterial strains expressing FAP was undertaken to create and optimize cellular factories producing medium- and short-chain hydrocarbons. Optimization of volatile hydrocarbon production was carried out on bacterial cultures in batch or continuous mode using instrumented photobioreactors.

The existence of an autocatalytic mechanism enabling the enzyme to function efficiently using fatty acids with medium chain lengths (C8 to C12) has been demonstrated. In cyanobacteria, the inactivation of the alkane biosynthesis pathway and its replacement by FAP has shown that hydrocarbons are involved in the stabilization of photosynthetic membrane systems. When FAP is co-expressed in bacteria with short-chain-specific thioesterases, continuous production of volatile hydrocarbons is observed. Depending on the thioesterase employed, it is possible to vary the chain length of the hydrocarbons produced, broadening the spectrum of possible applications. We have thus established a proof of concept for the production of volatile hydrocarbons, either medium-chain (C11 to C15) or shorter-chain (C7-C11), depending on the enzyme chosen.
Dedicated photobioreactors (1L) were designed to evaluate the performance of FAP-expressing strains and develop initial production protocols. The production of volatile hydrocarbons was confirmed at process scale, but the light activation phase proved critical, as expression of the FAP protein led to a drop in microbial growth. This led us to propose a continuous protocol based on alternating light and dark phases in order to find a compromise between growth and hydrocarbon production. The stability of the genetic constructs proved to be a key factor in the eventual development of a continuous, stable production process.

The Photoalkane project has had a major impact on both fundamental and applied research levels. The demonstration of FAP activity on short-chain fatty acids through an autocatalytic process was finely described (Samire et al. 2023) thus opening up important biotechnological prospects. In cyanobacteria, the role of hydrocarbons in photosynthetic membrane dynamics has been demonstrated (Miao et al. in preparation). The engineering of bacterial strains allowed demonstrating the production of volatile short-chain hydrocarbons in the gas phase of cultures. However, experiments carried out in continuous culture encountered certain limitations due in part to the instability of FAP in the light and in part to the instability of genetic constructs carried by a plasmid.

Research results have been published in international peer-reviewed journals, including a publication in Science Advances deciphering the autocatalytic mechanism involved in the production of short-chain hydrocarbons, a publication in Scientific Report on the bacterial production of volatile hydrocarbons, and a publication in Plant Physiology, describing, among other things, the substrate specificity of FAP from biodiversity-derived microalgae.

Growing concerns over fossil reserve limitations and greenhouse gas effects have focused attention on the need to develop sustainable biofuels. While most industrial developments have focused on the production of ethanol (bioethanol) or oil (converted to biodiesel), microbial production of medium-chain hydrocarbons (C8-C14) appears as a promising alternative. Indeed, hydrocarbons (alkanes or alkenes) have similar properties as petrol-derived fuels, have a higher energy density than other biofuels due to the absence of oxygen, and can ‘drop in’ to the existing transportation infrastructure. Progress in metabolic engineering and synthetic biology has made possible the engineering of microbes to produce advanced biofuel molecules with similar properties to petroleum-based fuels. While most enzymatic systems developed so far for the microbial production of hydrocarbons rely on two enzymes to convert fatty acids into hydrocarbon, the recent discovery in microalgae of a photoenzyme (FAP) converting fatty acids into hydrocarbons in a single enzymatic step and solely depending on blue light as a cofactor (Sorigué et al. 2017 Science 357, 903-907) opens new perspectives for the biological production of alkanes. The PHOTOALKANE project aims at exploring the biotechnological potential of FAP by engineering the fatty acid metabolism of two cellular platforms, the bacteria E. coli and the cyanobacteria Synechocystis. A major challenge of the PHOTOALKANE project will be to optimize hydrocarbon production by the FAP photoenzyme based on a coordinated management of both substrate availability through metabolic engineering and light input (intensity, frequency, and spectral quality) in order to optimize light energy conversion while limiting enzyme inactivation due to an excess of absorbed photons. A predictive modeling approach based on radiative transfer properties will be developed in order to manage the light supply for optimized hydrocarbon productivity in both heterotrophic (blue-light FAP activation) and photoautotrophic (red and blue light to monitor independently photosynthesis and FAP activity) microbial models. By developing specific illumination devices and advanced models of light transfer, experiments will be carried out using instrumented photobioreactors to characterize FAP efficiency in recombinant organisms subjected to highly controlled environments and contrasted illumination conditions (in frequency and intensity). This will allow assessing microbial hydrocarbon production at lab-scale photobioreactor level, and further defining strategies for future large-scale production.