Engineered cyanobacterium Synechococcus elongatus can use light and CO2 to produce sucrose, making it a promising candidate for use in co-cultures with heterotrophic workhorses. However, this process is challenged by the mutual stresses generated from the multispecies microbial culture. Here we demonstrate an ecosystem where S. elongatus is freely grown in a photo-bioreactor (PBR) containing an engineered heterotrophic workhorse (either β-carotene-producing Yarrowia lipolytica or indigoidine-producing Pseudomonas putida) encapsulated in calcium-alginate hydrogel beads. The encapsulation prevents growth interference, allowing the cyanobacterial culture to produce high sucrose concentrations enabling the production of indigoidine and β-carotene in the heterotroph. Our experimental PBRs yielded an indigoidine titer of 7.5 g/L hydrogel and a β-carotene titer of 1.3 g/L hydrogel, amounts 15–22-fold higher than in a comparable co-culture without encapsulation. Moreover, 13C-metabolite analysis and protein overexpression tests indicated that the hydrogel beads provided a favorable microenvironment where the cell metabolism inside the hydrogel was comparable to that in a free culture. Finally, the heterotroph-containing hydrogels were easily harvested and dissolved by EDTA for product recovery, while the cyanobacterial culture itself could be reused for the next batch of immobilized heterotrophs. This co-cultivation and hydrogel encapsulation system is a successful demonstration of bioprocess optimization under photobioreactor conditions.

Many carbon-fixing organisms have evolved CO 2 concentrating mechanisms (CCMs) to enhance the delivery of CO 2 to RuBisCO, while minimizing reactions with the competitive inhibitor, molecular O 2 . These distinct types of CCMs have been extensively studied using genetics, biochemistry, cell imaging, mass spectrometry, and metabolic flux analysis. Highlighted in this paper, the cyanobacterial CCM features a bacterial microcompartment (BMC) called 'carboxysome' in which RuBisCO is co-encapsulated with the enzyme carbonic anhydrase (CA) within a semi-permeable protein shell. The cyanobacterial CCM is capable of increasing CO 2 around RuBisCO, leading to one of the most efficient processes known for fixing ambient CO 2 . The carboxysome life cycle is dynamic and creates a unique subcellular environment that promotes activity of the Calvin-Benson (CB) cycle. The carboxysome may function within a larger cellular metabolon, physical association of functionally coupled proteins, to enhance metabolite channelling and carbon flux. In light of CCMs, synthetic biology approaches have been used to improve enzyme complex for CO 2 fixations. Research on CCMassociated metabolons has also inspired biologists to engineer multi-step pathways by providing anchoring points for enzyme cascades to channel intermediate metabolites towards valuable products.

Synechococcus elongatus UTEX 2973 can use light and CO2 to produce sucrose, making them promising candidates to construct cocultures with heterotrophic workhorses. This envisioned process is, however, challenging to implement because of photosynthetic oxidative stress, light shading effect by heterotrophic cells, degradation of light sensitive metabolites, and high cost to separate intracellular products. Here, we demonstrated an effective ecosystem, where the sucrose producing cyanobacterium was freely grown in photo-bioreactors (PBRs), while an engineered heterotrophic workhorse (β-carotene producing Yarrowia lipolytica or indigoidine producing Pseudomonas putida) was encapsulated in calcium-alginate hydrogel beads and then placed inside the PBRs. The compartmentalization by hydrogels prevented growth interference so that the cyanobacterial culture could reach high sucrose concentrations, resulting the production of indigoidine (7.5g/L hydrogel) and β-carotene (1.3g/L hydrogel), respectively (i.e., the titers were 15 ~ 22 folds higher than that in the free cell coculture). Moreover, 13C-metabolic analysis indicated that hydrogels provided a favorable microenvironment so that the flux network of cells inside hydrogel was similar to the free culture. Finally, this novel system allowed the heterotroph- containing hydrogel beads to be easily harvested and dissolved by an EDTA solution for product and cell recovery, while the cyanobacterial culture could be continuously used for growing the next batch of immobilized workhorse heterotrophs.