Characterising complex metabolic responses in an engineered, cross-feeding microbial co-culture using quantitative...

Microbial communities play a key role in biogeochemical transformations in a wide range of ecosystems, but they also hold significant potential to enhance the bioproduction of desired chemicals. Although designing synthetic microbial consortia has generated a lot of interest, a more in-depth understanding of the interactions between strains is required, particularly when strains are engineered to cross-feed, but are not isolated from related environments. Challenges include enhancing stability, productivity and controllability. Here, we used a synthetic microbial co-culture consisting of engineered strains of the photosynthetic cyanobacterium Synechococcus elongatus PCC 7942 cscB/SPS and nitrogen-fixing bacterium Azotobacter vinelandii AV3. Each relies on the other for conversion of atmospheric carbon (CO₂) and nitrogen (N₂) into organic forms, i.e. sucrose and ammonia, respectively, resources which can be shared. As both strains have such contrasting growth dynamics in co-culture compared to monoculture, we applied a label-free quantitative proteomics approach to characterise metabolism in both strains. The proteomes of both shifted when in co-culture to reflect adaptive restructuring of carbon and nitrogen metabolism, although A. vinelandii appeared to transition to a more stressed state, inducing proteins linked to polymer biosynthesis. An analysis of the co-culture over 16 days led to phenotypic changes, including cell structure alterations in A. vinelandii AV3 over time, with the proteome suggesting cell envelope remodelling and potentially encystment. These findings suggest that physiological control of parameters, such as oxygen and nutrient availability, may enable cultivation of more stable co-cultures.