Biology subjects

Klaehn, S.

Publications and source records attributed to Klaehn, S..

4 recordsLinked to original sources

bioRxiv2026

Conversion of CO2 into valuable products: Engineering the PirC-PGAM switch in cyanobacteria to direct carbon flux into desired products

BackgroundIn response to rising CO2 emissions driving global warming, there is an urgent need for a transition toward a sustainable bioeconomy. Photo-biotechnological processes based on oxygenic photosynthesis hold high potential for achieving CO2 neutrality and in this regard, cyanobacteria have emerged as promising biocatalysts. Rational metabolic engineering of cyanobacteria depends on a thorough understanding of native regulatory mechanisms governing primary metabolism, which can limit metabolic flux through specific pathways and, consequently, the formation of target products. Recent insights have identified a key regulatory node at the 2,3-bisphosphogylcerate-independent phosphoglycerate mutase (PGAM) reaction, where the metabolic flux from newly fixed carbon is redirected from the Calvin-Benson-Bassham (CBB) cycle towards lower glycolysis. This metabolic valve is controlled by the small inhibitor protein PirC, whose binding to PGAM is determined by the central signal transduction protein PII. ResultsIn this study, we exploit the PirC-PGAM interaction as a novel target for regulatory metabolic engineering in the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). Chassis strains with engineered control of PGAM, defined as PGAM-ON or PGAM-OFF states, were generated using two complementary approaches: tuning pgam gene expression and modulating PirC abundance to regulate PGAM activity. The effectiveness of this regulatory engineering strategy was demonstrated by redirecting carbon flux toward two representative, naturally occurring products: sucrose, produced via gluconeogenesis fueled by the Calvin-Benson-Bassham (CBB) cycle, and succinate, an intermediate of the tricarboxylic acid (TCA) cycle. Narrowing the PGAM valve resulted in a threefold increase in sucrose accumulation. In contrast, opening the PGAM valve by relieving PGAM inhibition through pirC deletion or separate pgam overexpression resulted in up to an 18-fold increase in succinate excretion. Furthermore, similar genetic configurations were applied to enhance production of a heterologous compound, isoprene, derived from pyruvate. ConclusionsThis study establishes the PGAM valve as a tunable control point for the rational re-direction of carbon flux in Synechocystis and highlights small regulatory proteins as powerful targets for metabolic engineering. Together, these findings provide proof of concept for an advanced level of molecular engineering in cyanobacteria and to fully harness their biocatalytic potential in future photosynthesis-driven biotechnological applications.

bioengineering↗

bioRxiv2025

The small protein SbtC is a functional component of the CO2 concentrating mechanism of cyanobacteria

Oxygenic phototrophs fix CO2 via the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) that shows relatively low CO2 affinity and specificity. To circumvent low and fluctuating CO2 concentrations in aquatic systems, cyanobacteria and algae have evolved sophisticated inorganic carbon (Ci) concentrating mechanisms (CCMs). Bicarbonate transporters such as SbtA play a crucial role in the cyanobacterial CCM and hence are tightly regulated at multiple layers. The control of sbtA gene expression and corresponding transporter activity involves the PII-like protein SbtB, whose gene is frequently co-transcribed with sbtA. Here we report on the discovery of a so far non-annotated gene in the model Synechocystis sp. PCC 6803, which is located upstream of the sbtAB operon and encodes the small protein SbtC, composed of 80 amino acids. Presence of SbtC was confirmed by immunoblotting after fusing the sbtC-coding sequence to a Flag-tag. Similar to sbtAB, transcription of the sbtC locus is induced by low CO2 availability but controlled independently. Mutation of the sbtC locus in a wild-type background showed only a mild phenotype even under low CO2, but the diurnal growth was impaired as found before in the mutant {Delta}sbtB. Biochemical analysis provided evidence for a trimeric SbtABC complex in the membrane. Recombinant Synechocystis strains harboring only SbtA as single Ci uptake system with either deleted sbtB or sbtC genes showed that bicarbonate leakage from the cell was strongly elevated in both mutants. Our results provide evidence that SbtC contributes to the formation of the SbtAB complex, thereby regulating bicarbonate exchange at the cytoplasmic membrane. Well-conserved SbtC-like proteins encoded in the neighborhood of sbtAB exist in many cyanobacterial genomes pointing towards an important role in the cyanobacterial CCM.

microbiology↗

bioRxiv2025

When a chaotropic agent turns into a nutrient: Deciphering the assimilation of guanidine and its utilization to drive synthetic processes in cyanobacteria

Guanidine is a well-known chaotropic agent used to denature proteins and nucleic acids. However, recent studies have demonstrated both the widespread synthesis of guanidine, e.g. in plants and mammals, as well as the widespread occurrence of guanidine metabolism in bacteria, suggesting a broader biological role. Here, we provide insights into guanidine assimilation via guanidine hydrolases (GdmH) in cyanobacteria. The gdmH gene is widespread among cyanobacteria and enables growth on guanidine as sole nitrogen source. Strains lacking gdmH, naturally or by deletion, failed to grow on guanidine. Expression of gdmH increased under nitrogen limitation, regulated by the transcription factor NtcA. However, guanidine is toxic above 5 mM, necessitating GdmH activity and adaptive mutations activating the multidrug efflux system PrqA. The gdmH gene is frequently co-localized with ABC transporter genes, which are driven by an additional NtcA-regulated promoter. At low guanidine concentrations, their mutation disrupted guanidine-dependent growth of Synechocystis sp. PCC 6803, supporting that they encode a high affinity transport system. In presence of >1 mM guanidine, these mutants grew like wildtype, suggesting the existence of additional uptake mechanisms for guanidine. We next demonstrate the high-affinity binding of guanidine to a previously described, conserved RNA motif located within the gdmH 5 UTR, validating it as a guanidine I riboswitch. By combining it with various promoters, we achieved precise, titratable control of heterologous gene expression in cyanobacteria in vivo. Our findings establish guanidine assimilation as an integral element of cyanobacterial nitrogen metabolism and highlight guanidine riboswitches as valuable tools for synthetic biology. Significance statementCyanobacteria are promising whole-cell biocatalysts for the sustainable, CO2-neutral production of chemicals and fuels. Unlocking this potential requires deep understanding of their metabolism and advanced molecular tools for genetic engineering. We show that cyanobacteria can assimilate guanidine as sole nitrogen source. Because of its toxicity, guanidine metabolism is tightly controlled, involving the transcription factor NtcA and a riboswitch, an RNA element regulating gene expression by guanidine binding. By utilizing this riboswitch, we achieved precise regulation of heterologous genes. Guanidine is inexpensive and effective at low concentrations, making large-scale applications in cyanobacterial cell factories cost-efficient. This study advances our understanding of the metabolic capacities of environmentally important cyanobacteria and their metabolic engineering, highlighting riboswitches as valuable tools for controlling biotechnological processes.

microbiology↗

bioRxiv2021

Integrative analysis of the salt stress response in cyanobacteria

Microorganisms evolved specific acclimation strategies to thrive in environments of high or fluctuating salinities. Here, salt acclimation in the model cyanobacterium Synechocystis sp. PCC 6803 was analyzed by integrating transcriptomic, proteomic and metabolomic data. A dynamic reorganization of the transcriptome and proteome occurred during the first hours after salt shock, e.g. involving the upregulation of genes to activate compatible solute biochemistry balancing osmotic pressure. The massive accumulation of glucosylglycerol then had a measurable impact on the overall carbon and nitrogen metabolism. In addition, we observed the coordinated induction of putative regulatory RNAs and of several proteins known for their involvement in other stress responses. Overall, salt-induced changes in the proteome and transcriptome showed good correlations, especially among the stably up-regulated proteins and their transcripts. We define an extended salt stimulon comprising proteins directly or indirectly related to compatible solute metabolism, ion and water movements, and a distinct set of regulatory RNAs involved in post-transcriptional regulation. Our comprehensive data set provides the basis for engineering cyanobacterial salt tolerance and to further understand its regulation.

systems biology↗