Developing a proteomics pipeline for characterisation of a synthetic microbial co-culture

Inspired by microbial communities can undertake more complex tasks than pure cell cultures and can achieve continuous production of valuable products, an increasingly number of attempts have been made to grow artificial or synthetic microbial communities composed of designated species. However, detailed understanding of their communications and stability is often insufficient. A detailed understanding of the composition and dynamics of microbial communities is necessary to gain fundamental insights into the interactions among species.
In this project, a synthetic microbial co-culture system was constructed using genetically engineered strains of photosynthetic cyanobacterium Synechococcus elongatus cscB/SPS and nitrogen-fixing bacterium Azotobacter vinelandii nifL, in which the sucrose produced from S. elongatus cscB/SPS would be the carbon source for A. vinelandii nifL and the ammonium produced from A. vinelandii nifL would be the nitrogen source for S. elongatus cscB/SPS. This synthetic microbial co-culture can convert sunlight, carbondioxide, and atmospheric nitrogen into desired bio-product polyhydroxybutyrate (PHB).
Quantitative proteomics can provide valuable insights into how microbial strains adapt to changing conditions. However, current workflows and methodologies are not suitable for simple artificial co-culture systems where strain ratios are dynamic. Therefore, a mass spectrometry-based label-free shotgun proteomics workflow was established for the analysis of the composition and changes of the synthetic microbial co-culture proteome. Furthermore, a new normalization method was proposed, named “LFQRatio”, to reflect the relative contributions of the two distinct cell types emerging from the cell ratio changes during co-cultivation and minimise the impact of cell number changes for proteome quantification in microbial co-culture.
Lastly, comparative proteomics was performed to analyse the protein changes of A. vinelandii nifL and S. elongatus cscB/SPS in the synthetic co-culture, showing higher relative abundance of proteins in nitrogen fixation and photosynthesis pathways, which is evidence of the cross-feeding between the two members. In addition, co-culture proteomics changes over time were studied, demonstrating some interactions between co-cultures, including general carbon metabolism, two-component system, bacterial chemotaxis, etc. The proteomics results reveal potential targets for optimisation to keep the synthetic microbial co-culture healthier and longer-lived.