Proteomics assisted synthetic biology applied to autotrophic bacteria

QC 2026-02-17

Abstract

Civilization and Earth’s natural environments are under threat by rapid climate change induced by human activity. A substantial driver for this change is greenhouse gas emissions, which motivates the development of sustainable alternatives for producing chemicals, fuels and materials. One such alternative is using engineered microbial cell factories that fix CO2 directly from the atmosphere into desirable products while utilizing sunlight for energy. This approach solves one of the inherent problems with other forms of bio-based production: the need for arable land. However, engineering these bacteria for human gain is not a trivial challenge: they have evolved over billions of years to be expert survivors with little interest in maximizing productivity of foreign compounds. Achieving sustainable and economically viable chemical production with such organisms requires a deep understanding of how protein activity, resource allocation and stress responses are regulated, and how such regulation can be manipulated.

In this thesis, proteomics has been a central toolkit, used both for discovery and diagnosis to investigate regulatory mechanisms in autotrophic bacteria. Special emphasis has been placed on carbon fixation and growth-arrest strategies in cyanobacteria, primarily Synechocystis sp. PCC 6803, with the aim of identifying metabolic control points and leveraging them to achieve high and stable productivity.

The first study applied interaction proteomics to identify metabolite-protein interactions across the bacterial Calvin-Benson-Bassham cycle. This revealed multiple instances of potential allosteric regulation of central enzymes, some of which were successfully validated biochemically. Among them was a redoxdependent activation of fructose-/sedoheptulose-bisphosphatase by glyceraldehyde-3-phosphate, implying a feed-forward activation mechanism that is coordinated with cellular energy supply.

The second developed a screening platform for Rubisco mutants in Synechocystis, enabling evaluation of large variant libraries through in vivo fitness. Quantitative proteomics was used to characterize the platform and thereby reveal that changing the gas composition of the culture headspace could tune fitness selection toward different enzyme properties. Subsequent work demonstrated this platform's capacity to use high-throughput libraries to identify Rubisco variants with enhanced kinetics. 

The third study assayed protein thermal stability in a proteome-wide manner to identify binding targets of the alarmone ppGpp in cyanobacteria and plant chloroplasts. The screens revealed potential interactions that were conserved across species, as well as interactions unique to specific species. Validation experiments confirmed a major post-translational regulatory mechanism on pyrimidine metabolism in chloroplasts: ppGpp inhibits aspartate carbamoyltransferase (PyrB), implying a mechanism for throttling nucleotide supply under stress. In addition, significant effects on carboxysome organization and structure was discovered in cyanobacteria.

Finally, the fourth study utilized a global labeling strategy for quantitative proteomics that enabled proteome-wide estimation of protein turnover rates. In combination with proteome allocation analysis, lactate production measurements and YFP expression levels, two different growth-arrest strategies were characterized. This revealed a (p)ppGpp-accumulating mutant capable of sustained and elevated lactate productivity that reallocated resources from growth-associated machinery toward product while maintaining photosynthetic capacity and cell viability. This work provides valuable insights into the dynamic resource allocation of cyanobacteria and chloroplasts that can be used to inform future engineering efforts.

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