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bioRxiv · 10.1101/2022.02.17.480855

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

Yanli, Z.·Chunling, X.·Hui, C.·Anqi, J.·Liang, Z.·Junli, Z.·Lixin, Z.·Qiang, W.
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Abstract

Cyanobacterial blooms pose a serious threat to public health due to the presence of cyanotoxins. The most common cyanotoxins, microcystins (MCs), can cause acute poisoning at high concentrations and hepatocellular carcinoma following chronic exposure. Among all MC variants, MC-LR produced by Microcystis aeruginosa PCC 7806 is the most common toxic MC. Although the biosynthetic pathway for MC-LR has been proposed, experimental support of this pathway is lacking. In an effort to experimentally validate this pathway, we expressed the 55 kb microcystin biosynthetic gene cluster (mcy cluster) (mcyA-J) and produced MC-LR in the model cyanobacterium Synechococcus 7942. We designed and constructed the strong bidirectional promoter biPpsbA2 between mcyA and mcyD, reassembled the mcy cluster in yeast by transformation-associated recombination (TAR cloning), transformed the gene cluster into the NSII site of Synechococcus 7942, and successfully expressed MC-LR at a level of 0.006-0.018 fg cell-1 day-1. The expression of MC-LR led to abnormal cell division and the filamentation of Synechococcus 7942 cells, further analysis proved a role of MC-LR in functional assembly of the cell division protein FtsZ, by competing its GTP binding site. These results represent the first synthetic biological expression of the mcy cluster and the autotrophic production of MC-LR in a photosynthetic model organism, which lays the foundation for resolving the MC biosynthesis pathway. The suggested role of MC-LR in cell division reveals a mechanism of how blooming cyanobacteria gain a competitive edge over their non-blooming counterparts. HighlightsO_LIWe expressed the 55 kb mcy cluster and produced MC-LR in the model cyanobacterium Synechococcus 7942. C_LIO_LIThis is the first realized production of MC in the model non-toxin-production cyanobacteria from CO2 by photosynthesis. C_LIO_LICompared with the self-replicating plasmid, the recombination of the mcy cluster into the genome of Synechococcus 7942 is more suitable for the heterologous production of microcystin. C_LIO_LIMC-LR inhibits cell division by irreversibly competing the GTP binding domain of the cell division protein FtsZ. C_LIO_LIThe newly discovered effect of MC-LR on cell division reveals a mechanism of how blooming cyanobacteria gain competitive edge over their non-blooming counterparts. C_LI

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Yanli, Z., Chunling, X., Hui, C., Anqi, J., Liang, Z., Junli, Z., Lixin, Z., Qiang, W.. 2022-02-17. Reconstitution and Expression of mcy Gene Cluster in The Model Cyanobacterium Synechococcus 7942 Reveals a Roll of MC-LR in Cell Division. https://doi.org/10.1101/2022.02.17.480855

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