Systematic Identification of Target Genes for Cellular Morphology Engineering in Synechococcus elongatus PCC7942

Abstract

Cyanobacteria are serving as promising microbial platforms for development of photosynthetic cell factories. For enhancing the economic competitiveness of the photosynthetic biomanufacturing technology, comprehensive improvements on industrial properties of the cyanobacteria chassis cells and engineered strains are required. Cellular morphology engineering is an up-and-coming strategy for development of microbial cell factories fitting the requirements of industrial application. In this work, we performed systematic evaluation of potential genes for cyanobacterial cellular morphology engineering. Twelve candidate genes participating in cell morphogenesis of an important model cyanobacteria strain, Synechococcus elongatus PCC7942, were knocked out/down and overexpressed, respectively, and the influences on cell sizes and cell shapes were imaged and calculated. Targeting the selected genes with potentials for cellular morphology engineering, the controllable cell lengthening machinery was also explored based on the application of sRNA approaches. The findings in this work not only provided many new targets for cellular morphology engineering in cyanobacteria, but also helped to further understand the cell division process and cell elongation process of Synechococcus elongatus PCC7942.

Orthogonal Degron System for Controlled Protein Degradation in Cyanobacteria

“…Zhang et al used RNA-based gene repression to study proteins involved in cell morphology (e.g., FtsZ, MreB, and ZipN), but significant changes in cell length were not present until 2−5 days after induction. 94 Similarly, Yao et al observed maximal CRISPRi repression of 94% in a fluorescent reporter after 4 days of induction. 28 Taton and co-workers developed a NOT gate, a type of genetic logic gate that represses expression upon activation of the gate, 95 which was able to achieve over 90% downregulation in 2−4 days in S. elongatus, but this system cannot be used to target genes in their native context, due to how the circuit was constructed.…”

“…Zhang et al used RNA-based gene repression to study proteins involved in cell morphology ( e.g. , FtsZ, MreB, and ZipN), but significant changes in cell length were not present until 2–5 days after induction . Similarly, Yao et al observed maximal CRISPRi repression of 94% in a fluorescent reporter after 4 days of induction .…”

Reconstitution and Expression of mcy Gene Cluster in The Model Cyanobacterium Synechococcus 7942 Reveals a Roll of MC-LR in Cell Division

“…The FtsZ protein is a tubulin-like GTPase protein which would assemble Z-ring structure as the skeleton and scaffold to the cellular divisome (Dong et al 2010, Zhang et al 2020). The localization of FtsZ were examined by immunofluorescence microscopy, as shown in Fig.…”

Engineering a Controllable Targeted Protein Degradation System and a Derived OR-GATE-Type Inducible Gene Expression System in Synechococcus elongatus PCC 7942

“…We performed a direct comparison between the protein degradation system and a previously developed microRNA system, which was designed to down-regulate the GFP expressions in the translation step (Figure A). In the microRNA system, the MicC asRNA (targeting the gfp gene) and the Hfq chaperone will be induced (by theophylline) to be transcribed and expressed by the heterologous T7 system, which has been successfully used to regulate several morphogenesis genes in PCC 7942 . However, as shown in Figure B, for the GFP expressed by the strong promoter PcpcB , the Hfq-micC system could only reduce the intensities of GFP signals by 11% after 2 h of induction by 1 mM theophylline, whereas the protein degradation system caused a reduction of 87%, indicating a significantly better response rate and degradation activity.…”