Growth-decoupled terpene biosynthesis from CO2 by a photo-heterotrophic coculture

Abstract Microbial conversion of CO 2 to chemicals is essential for a sustainable bioeconomy. However, this process is often hampered by low carbon fixation rates. Photoautotroph-heterotroph cocultures can potentially overcome this bottleneck by leveraging a strong carbon sink in the phototroph to drive carbon fixation, which is then coupled to heterotrophic chemical production. Despite the potential, exploiting cocultures for chemical production, particularly of low-flux pathway products, remains challenging. In this study, we established a synthetic consortium comprising a sucrose-secreting Synechococcus elongatus strain and a limonene-producing Escherichia coli strain to produce limonene from CO 2 as the sole carbon input. Through multiplex promoter engineering of the csc regulon, we identified sucrose consumption dynamics favorable for limonene production in E. coli , showing that expression of the invertase ( cscA) alone is sufficient to support growth on sucrose via the moonlighting activity of the lactose permease ( lacY ). A detailed characterization of sucrose consumption and limonene production revealed a biphasic growth-production scheme in the engineered E. coli , consistent with carbon flux being preferentially directed toward biomass during exponential growth. Comparative proteomics supported this observation, with higher mevalonate-pathway enzyme levels in the post-exponential phase. These results suggest that chemical production from low-flux pathways in cocultures could be enhanced by strategies that restrain heterotrophic growth. Based on these insights, we achieved a proof-of-concept CO 2 -to-limonene conversion titer of 11 mg/L. This study provides a framework for overcoming metabolic bottlenecks in synthetic consortia and offers insights to guide the future development of CO 2 -to-chemical production systems.