Biology subjects

Syren, P.-O.

Publications and source records attributed to Syren, P.-O..

3 recordsLinked to original sources

bioRxiv2025

The fitness landscape of a Form II Rubisco in a photosynthetic bacterium guides engineering of oxygen tolerance

Rubisco is an important but challenging protein engineering target. Fast and selective rubiscos could enhance photosynthesis in plants and accelerate biobased production processes. To facilitate engineering of rubisco, we applied an in vivo screen that couples rubisco activity to growth rate of the photoautotrophic cyanobacterium Synechocystis sp. PCC 6803. We screened a barcoded mutagenesis library of the form II rubisco from Gallionella sp. containing 15,000 single-site and multi-site variants. Exchanges in loop 6 near the active site, at the dimer interface, and in potential gas tunnels improved rubisco fitness. The dataset also informed protein engineering, using recombination and a trained transformer model. In vitro characterisation of two high-fitness variants showed reduced catalytic efficiency for oxygenation (kcat/Ko) in both and an increased carboxylation turnover (kcatC) in one. This large labeled fitness dataset, containing examples of epistasis, can be useful for benchmarking computational models of rubisco. TeaserEvolving a foreign rubisco to photosynthesis leads to reduced oxygen sensitivity.

biochemistry↗

bioRxiv2025

A cyanobacterial screening platform for Rubisco mutant variants

Rubisco is the main entry point of inorganic carbon into the biosphere and a central player in the global carbon system. Its relatively low catalytic constant as well as its tendency to also accept O2 as a substrate have made it a common target of enzyme engineering. We have developed an enzyme engineering and screening platform for Rubisco using the model cyanobacterium Synechocystis sp. PCC 6803. Starting with the Form II Rubisco from Gallionella, we first show that the enzyme can replace the native Form I Rubisco in Synechocystis and that growth rates become sensitive to CO2 and O2 levels. We address the challenge of designing a zero-shot input library, without prior experimental knowledge, by coupling the phylogenetically-guided model EVmutation with "in silico evolution". Starting with this targeted mutagenesis library, we used competitive growth coupled to deep sequencing to compare the properties of Rubisco protein variants under different cultivation conditions. We identified an amino acid exchange which increased the thermostability of Gallionella Rubisco and conveyed resilience to detrimental exchanges. The establishment of this platform is a first step towards high-throughput screening of Rubisco variants in Synechocystis and creating optimized enzyme variants to accelerate the Calvin-Benson-Bassham cycle in cyanobacteria and possibly chloroplasts. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/633911v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@b0e549org.highwire.dtl.DTLVardef@19d2a56org.highwire.dtl.DTLVardef@1468557org.highwire.dtl.DTLVardef@dc3e7e_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Metabolite interactions in the bacterial Calvin cycle and implications for flux regulation

Metabolite-level regulation of enzyme activity is important for microbes to cope with environmental shifts. Knowledge of such regulations can also guide strain engineering to improve industrial phenotypes. Recently developed chemoproteomics workflows allow for genome-wide detection of metabolite-protein interactions that may regulate pathway activity. We applied limited proteolysis small molecule mapping (LiP-SMap) to identify and compare metabolite-protein interactions in the proteomes of two cyanobacteria and two lithoautotrophic bacteria that fix CO2 using the Calvin cycle. Clustering analysis of the hundreds of detected interactions showed that some metabolites interacted in a species-specific manner, such as interactions of glucose-6-phosphate in Cupriavidus necator and of glyoxylate in Synechocystis sp PCC 6803. These are interpreted in light of the different central carbon conversion pathways present. Metabolites interacting with the Calvin cycle enzymes fructose-1,6/sedoheptulose-1,7-bisphosphatase (F/SBPase) and transketolase were tested for effects on catalytic activity in vitro. The Calvin cycle intermediate glyceraldehyde-3-phosphate activated both Synechocystis and Cupriavidus F/SBPase, which suggests a feed-forward activation of the cycle in both photoautotrophs and chemolithoautotrophs. In contrast to the stimulating effect in reduced conditions, glyceraldehyde-3-phosphate inactivated the Synechocystis F/SBPase in oxidized conditions by accelerating protein aggregation. Thus, metabolite-level regulation of the Calvin cycle is more prevalent than previously appreciated and may act in addition to redox regulation.

systems biology↗