Biology subjects

Muth-Pawlak, D.

Publications and source records attributed to Muth-Pawlak, D..

4 recordsLinked to original sources

bioRxiv2026

Overexpression of flavodiiron protein Flv3 in engineered Synechocystis stimulates sucrose production and growth by altering cellular redox balance through enhanced sulfur metabolism

Biotechnological applications of oxygenic photosynthetic organisms depend on conversion of light energy into chemical energy through photosystems (PS). This energy can then be used to drive engineered metabolic pathways that are designed as strong electron sinks. For optimal performance, the engineered host metabolism must also be balanced with the native photoprotective electron transfer network. This includes the energy-consuming function of flavodiiron (Flv) proteins, which are universal to cyanobacteria and all other oxygenic photosynthetic organisms except angiosperms. In the cyanobacterium Synechocystis sp. PCC 6803, four different Flv proteins have been shown to function in a Mehler-like reaction within two heterodimeric forms (Flv1/Flv3 and Flv2/Flv4), donating electrons to O2 without generating oxidative stress. Previously, deleting Flv3 in the Synechocystis sucrose-producing (S02) strain was shown to cause drastic metabolic changes in S02{Delta}flv3, shifting it from photoautotrophic to mixotrophic growth (Muth-Pawlak, et al., 2024). In this study, we took an opposite approach by complementing S02 with Flv3 overexpression at different levels using RBS tuning. Interestingly, this resulted in S02oeFlv3 strains with significantly increased overall photosynthetic activity and sucrose production, enhanced cell growth, and storage compound accumulation. However, these outcomes are shown not to be due to conventional O2 photoreduction activity catalysed by Flv1/Flv3. Instead, we postulate that the observed changes are linked to the previously unidentified function of homomeric Flv3/Flv3 and the strongly increased sulphate redox metabolism. Based on extensive proteomic and metabolite analyses, we hypothesise that the Flv3 homooligomer uses sulfate metabolites directly or indirectly as the final electron acceptor instead of O2. This would also explain the upregulation of sulfate-related enzymes, as well as SQR, which passes the electrons back to the PQ pool in the Flv3 overexpression strain.

biochemistry↗

bioRxiv2026

Optimizing photoautotrophic production of soluble 3-hydroxybutyrate in Synechocystis sp. PCC 6803 through combinatorial translational tuning

While photosynthetic cyanobacteria are potential biotechnological hosts for light-driven production of sustainable chemicals from CO2, engineering more efficient strains is critical for the development of competitive industrial processes. This study demonstrates significantly enhanced production of the soluble bioplastic precursor (R)-3-hydroxybutyrate (3HB) that has been engineered based on the polyhydroxybutyrate (PHB) pathway in photoautotrophic cyanobacterium Synechocystis sp PCC 6803. As the key novelty, we generated a library of engineered 3HB pathway variants that express the three key heterologous pathway enzymes PhaA, PhaB and TesB at varying efficiencies, followed by the screening of most efficient 3HB producers. This was achieved by placing each of the pathway enzymes under the translational regulation of three alternative RBSs in different combinations, resulting in strains with wide dynamic range of 3HB productivities. The best strains accumulated over 5 gl-1 under 200 mol photons m-2s-1 and 3% CO2 in a 14-day flask batch culture, with the highest titer reaching 12 gl-1, corresponding to nearly 3 gl-1 d-1 during the peak production phase. These are the highest 3HB production levels reported so far in cyanobacteria, and comparable to those previously established in heterotrophic production systems. Proteomic comparison of selected strains revealed that the different RBS combinations result in varying expression patterns of the pathway proteins, and that the strain-specific enzyme levels remained relatively constant over the monitored six-day period. The results show that altering the levels of the target pathway enzymes can dramatically improve product yield in Synechocystis, while even very small quantitative differences in the strain-specific expression profiles can have marked effects on the production efficiency. This could be used as a general tool for optimizing engineered pathways in cyanobacteria, provided that the flux to the end-product is not critically restricted by substrate availability but rather determined by the balance between the consecutive pathway steps.

synthetic biology↗

bioRxiv2024

Sustained non-photochemical quenching and regulation of PSII repair cycle during combined low temperature and high light stress in lettuce

In nature, light and other environmental conditions are constantly changing, requiring plants to have several overlapping regulatory mechanisms to keep light reactions and metabolism in balance. Here, we show that high light (HL) induces a much stronger down-regulation of light reactions when lettuce plants are exposed to 1500 {micro}mol photons m-2 s-1 for 4 h at 13{degrees}C (low temperature, LT) compared to 23{degrees}C (growth temperature, GT). GT/HL treatment induced non-photochemical quenching (NPQ), which relaxed during 1 h recovery in darkness. In contrast, LT/HL treatment induced an exceptionally high NPQ that only partially relaxed during 1 h in darkness at GT. Such a high sustained NPQ (sNPQ) cannot be explained by canonical NPQ mechanism(s). Instead, sNPQ was associated with partial disassembly of PSII-LHCII complexes and a transient increase in phosphorylation of the minor antenna proteins LHCB4.1/LHCB4.2. This coincided with increased expression of the light-harvesting-like proteins SEP2 and ELIP1.2, the PSII assembly proteins HCF173 and LPA3, and accumulation of the pre-D1 protein. These results lead us to propose that LHCB4.1/LHCB4.2 phosphorylation- dependent disassembly of PSII-LHCII supercomplexes allows SEP2 to bind to CP47 and hypothetically quenches the inner PSII core antenna, while free CP43 released during PSII repair is proposed to be protected by LPA3.

plant biology↗

bioRxiv2019

Cytochrome cM downscales photosynthesis under photomixotrophy in Synechocystis sp. PCC 6803

Photomixotrophy is a metabolic state, which enables photosynthetic microorganisms to simultaneously perform photosynthesis and metabolism of imported organic carbon substrates. This process is complicated in cyanobacteria, since many, including Synechocystis sp. PCC 6803, conduct photosynthesis and respiration in an interlinked thylakoid membrane electron transport chain. Under photomixotrophy, the cell must therefore tightly regulate electron fluxes from photosynthetic and respiratory complexes. In this study, we show via characterization of photosynthetic apparatus and the proteome, that photomixotrophic growth results in a gradual reduction of the plastoquinone pool in wild-type Synechocystis, which fully downscales photosynthesis over three days of growth. This process is circumvented by deleting the gene encoding cytochrome cM (CytM), a cryptic c-type heme protein widespread in cyanobacteria. {Delta}CytM maintained active photosynthesis over the three day period, demonstrated by high photosynthetic O2 and CO2 fluxes and effective yields of Photosystem II and Photosystem I. Overall, this resulted in a higher growth rate than wild-type, which was maintained by accumulation of proteins involved in phosphate and metal uptake, and cofactor biosynthetic enzymes. While the exact role of CytM has not been determined, a mutant deficient in the thylakoid-localised respiratory terminal oxidases and CytM ({Delta}Cox/Cyd/CytM) displayed a similar phenotype under photomixotrophy to {Delta}CytM, demonstrating that CytM is not transferring electrons to these complexes, which has previously been suggested. In summary, the obtained data suggests that CytM may have a regulatory role in photomixotrophy by reducing the photosynthetic capacity of cells. One sentence summaryThe cryptic, highly conserved cytochrome cM completely blocks photosynthesis in Synechocystis under three days of photomixotrophy, possibly by suppressing CO2 assimilation.

plant biology↗