(523d) Photobiological Production of High-Value Compounds Via Compartmentalized Co-Cultures Using Ca-Alginate Hydrogels

In contrast to current bioproduction techniques using plant-based carbohydrates as the primary input for microbial culture, increasing interest pivots towards the investigation of alternative feedstocks with lower environmental footprint. Fast growing cyanobacterial species (e.g., Synechococcus 2973) using light and CO₂ as primary inputs, is amenable for genetic modifications. Due to efficient sugar phosphate pathways in cyanobacteria, its metabolic network can be effectively rewired to produce sucrose at high concentrations, offering an efficient CO₂ capture route and sustainable sugar sources (Lin et al., 2020). Moreover, the resulting sucrose can be consumed by heterotrophs to make high-value products. However, co-culturing of cyanobacteria and heterotrophs faces several challenges including: 1) oxidative radicals from photosynthesis may be inhibitory to heterotroph growth, 2) growth competition between subpopulations, and 3) incompatible medium compositions. To resolve this problem, hydrogel-based technology can be applied for the co-culture. Specifically, we hypothesize that alginate hydrogels, formed after crosslinking by divalent cations to encapsulate heterotrophs, can provide a spatial barrier that protects them from the oxidative stressed microenvironment close to cyanobacteria (Tan et al., 2021). In this work, we developed a method for synthetic microbial consortium of a sucrose producing cyanobacterium (Synechococcus 2973 cscB) with heterotrophic workhorses (e.g., engineered Yarrowia lipolytica for β-carotene production and Pseudomonas putida for indigoidine production) through compartmentalization using calcium-alginate hydrogels (Czajka et al., 2018, 2021).

Results

Firstly, from axenic microbial fermentation experiments with capsulated heterotrophic strains, the engineered Y. lipolytica and P. putida cells produced well inside the alginate hydrogels. The yeast and bacteria had ~104% and ~87% per-cell production yield respectively inside the hydrogels compared to that in free culture. Cell leakage from the hydrogel beads was observed from both capsulated Y. lipolytica and P. putida, and the amount of leakage correlated with cell size. Incubated on a shaker, the larger-sized yeast had more than 95% cells staying inside the hydrogel without leakage while the smaller-sized bacteria had ~69% of initial cells remaining inside the beads. Then, in co-cultures set up in multi-cultivators (Fig. 1), the alginate hydrogels were able to protect the engineered heterotrophs from environmentally stressful conditions. At a temperature 5 ⁰C higher than the optimal fermentation temperature and 5 µM of antibiotics in the co-culture media (the workhorse strains had no antibiotic resistance), the living population of Y. lipolytica and P. putida inside the hydrogel increased to one thousand and ten thousand times of their initial population, respectively, while the heterotrophs in free co-culture died within 24 hours. Both hosts could consume the sugar produced by cyanobacteria in the medium (invertase was added in the medium) - yeast cell density inside hydrogels increased by ~240% in 5 days, and bacteria by ~172%. Production wise, the peak mass of β-carotene produced from hydrogel co-culture was 1.99 times that from free co-culture, and indigoidine 1.58 times. Inside the heterotroph-embedded hydrogels in consortia with cyanobacteria, the yield of β-carotene reached 0.11 g/L/OD₆₀₀ and that of indigoidine 0.33 g/L/OD₆₀₀.In coculture conditions, workhorse strains could survive inside the hydrogels for more than 8 days according to colony forming unit measurements, while free heterotrophic cells died within 48 hours.

Conclusions

We integrated engineered phototrophic and heterotrophic strains through hydrogel compartmentation for sustainable and economical production of high-value compounds. This phototrophic/heterotrophic consortium method with hydrogel compartmentation exhibited promising performance for microbial synthesis at high titer using a phototrophic feedstock.

Acknowledgement

The study was supported by funding from DOE Office of Fossil Energy and Carbon Management as well as National Science Foundation (2037887).

References

Lin, P. C., Zhang, F., & Pakrasi, H. B. (2020). Enhanced production of sucrose in the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973. Scientific Reports, 10(1). https://doi.org/10.1038/s41598-019-57319-5

Tan, A. X., Michalski, E., Ilavsky, J., & Jun, Y.-S. (2021). Engineering Calcium-Bearing Mineral/Hydrogel Composites for Effective Phosphate Recovery. ACS ES&T Engineering, 1(11), 1553–1564. https://doi.org/10.1021/acsestengg.1c00204

Czajka, J. J., Nathenson, J. A., Benites, V. T., Baidoo, E. E. K., Cheng, Q., Wang, Y., & Tang, Y. J. (2018). Engineering the oleaginous yeast Yarrowia lipolytica to produce the aroma compound β-ionone. Microbial Cell Factories, 17(1). https://doi.org/10.1186/s12934-018-0984-x

Czajka, J. J., Banerjee, D., Eng, T., Menasalvas, J., Yan, C., Munoz Munoz, N., Poirier, B. C., Kim, Y.-M., Baker, S. E., Tang, Y. J., & Mukhopadhyay, A. (2021). Optimizing a High Performing Multiplex-CRISPRi P. putida Strain with Integrated Omics Analyses [Manuscript submitted for publication]