No abstract

Photosynthesis in plants, green algae, and cyanobacteria converts solar energy into chemical energy in the form of ATP and NADPH, both of which are used in primary metabolism. However, often more reducing power is generated by the photosystems than what is needed for primary metabolism. In this review, we discuss the development in the research field, focusing on how the photosystems can be used as synthetic biology building blocks to channel excess reducing power into light-driven production of alternative products. Plants synthesize a large number of high-value bioactive natural compounds. Some of the key enzymes catalyzing their biosynthesis are the cytochrome P450s situated in the endoplasmic reticulum. However, bioactive compounds are often synthesized in low quantities in the plants and are difficult to produce by chemical synthesis due to their often complex structures. Through a synthetic biology approach, enzymes with a requirement for reducing equivalents as cofactors, such as the cytochrome P450s, can be coupled directly to the photosynthetic energy output to obtain environmentally friendly production of complex chemical compounds. By relocating cytochrome P450s to the chloroplasts, reducing power can be diverted toward the reactions catalyzed by the cytochrome P450s. This provides a sustainable production method for high-value compounds that potentially can solve the problem of NADPH regeneration, which currently limits the biotechnological uses of cytochrome P450s. We describe the approaches that have been taken to couple enzymes to photosynthesis in vivo and to photosystem I in vitro and the challenges associated with this approach to develop new green production platforms.

HighlightA pathway containing two cytochrome P450s and a glucosyltransferase has been stably expressed in Nicotiana tabacum chloroplasts. The functional P450s are enriched in the thylakoids and receive electrons from photo-reduced ferredoxin.

SUMMARYChloroplasts in plants and algae and photosynthetic microorganisms such as cyanobacteria are emerging hosts for sustainable production of valuable biochemicals, using only inorganic nutrients, water, CO 2 and light as inputs. In the past decade, many bioengineering efforts have focused on metabolic engineering and synthetic biology in the chloroplast or in cyanobacteria for the production of fuels, chemicals and complex, high-value bioactive molecules. Biosynthesis of all these compounds can be performed in photosynthetic organelles/organisms by heterologous expression of the appropriate pathways, but this requires optimization of carbon flux and reducing power, and a thorough understanding of regulatory pathways. Secretion or storage of the compounds produced can be exploited for the isolation or confinement of the desired compounds. In this review, we explore the use of chloroplasts and cyanobacteria as biosynthetic compartments and hosts, and we estimate the levels of production to be expected from photosynthetic hosts in light of the fraction of electrons and carbon that can potentially be diverted from photosynthesis. The supply of reducing power, in the form of electrons derived from the photosynthetic light reactions, appears to be non-limiting, but redirection of the fixed carbon via precursor molecules presents a challenge. We also discuss the available synthetic biology tools and the need to expand the molecular toolbox to facilitate cellular reprogramming for increased production yields in both cyanobacteria and chloroplasts.

BackgroundPlant terpenoids are known for their diversity, stereochemical complexity, and their commercial interest as pharmaceuticals, food additives, and cosmetics. Developing biotechnology approaches for the production of these compounds in heterologous hosts can increase their market availability, reduce their cost, and provide sustainable production platforms. In this context, we aimed at producing the antimicrobial diterpenoid isopimaric acid from Sitka spruce. Isopimaric acid is synthesized using geranylgeranyl diphosphate as a precursor molecule that is cyclized by a diterpene synthase in the chloroplast and subsequently oxidized by a cytochrome P450, CYP720B4.ResultsWe transiently expressed the isopimaric acid pathway in Nicotiana benthamiana leaves and enhanced its productivity by the expression of two rate-limiting steps in the pathway (providing the general precursor of diterpenes). This co-expression resulted in 3-fold increase in the accumulation of both isopimaradiene and isopimaric acid detected using GC-MS and LC-MS methodology. We also showed that modifying or deleting the transmembrane helix of CYP720B4 does not alter the enzyme activity and led to successful accumulation of isopimaric acid in the infiltrated leaves. Furthermore, we demonstrated that a modified membrane anchor is a prerequisite for a functional CYP720B4 enzyme when the chloroplast targeting peptide is added. We report the accumulation of 45–55 μg/g plant dry weight of isopimaric acid four days after the infiltration with the modified enzymes.ConclusionsIt is possible to localize a diterpenoid pathway from spruce fully within the chloroplast of N. benthamiana and a few modifications of the N-terminal sequences of the CYP720B4 can facilitate the expression of plant P450s in the plastids. The coupling of terpene biosynthesis closer to photosynthesis paves the way for light-driven biosynthesis of valuable terpenoids.