Plastid Engineering for Photosynthesis‐Driven Synthesis of Hyaluronic Acid in Tobacco

Hyaluronic acid (HA) is a glycosaminoglycan composed of alternating units of N-acetylglucosamine and glucuronic acid. High moisture retention, viscoelasticity and biocompatibility are unique features that make HA polymers attractive compounds for medical applications and aesthetic purposes. Current synthesis of HA polymers relies on microorganisms and requires supply of glucose in bioreactors to produce glucose-6-phosphate and fructose-6-phosphate as precursors for HA biosynthesis. By contrast, photosynthetic organisms generate glucose-6-phosphate and fructose-6-phosphate as autotrophic products of CO₂ fixation via the Calvin-Benson-Bassham (CBB) cycle. Here we explored the possibility to harness chloroplast metabolism for the light-driven production of HA in the model organism tobacco (Nicotiana tabacum). An operon of five streptococcal genes were introduced into the plastid genome of tobacco to drive HA-synthesis by expression elements that confer low, medium or high expression levels. Photoautotrophic growth over the entire life cycle was only achieved in transplastomic lines with low transgene expression levels. Surprisingly, accumulation of HA polymers was observed only under heterotrophic growth conditions. Proteomic analysis revealed low accumulation levels of the first pathway enzyme in the transplastomic lines, and low contents of the final pathway enzyme (HA synthase) upon autotrophic growth. Altered abundances of proteins involved in photosynthesis and central metabolism were observed under autotrophic growth conditions, and metabolite profiling confirmed that photoautotrophic HA biosynthesis depleted CBB cycle derivatives and triggered plastid-associated stress responses. Our work demonstrated the feasibility of tapping the CBB cycle for HA synthesis and identified bottlenecks for plant-based production of carbohydrate polymers.