Abstract Title

Application of CyanoGate, a Cyanobacterial Synthetic Biology Molecular Cloning Suite, for Exploring Optimal Heterologous Microcystinase (MlrA) Production for Freshwater Bioremediation from Cyanotoxins

Start Date

27-5-2022 11:00 AM

End Date

27-5-2022 11:15 AM

Abstract

Microcystinase (MlrA) is currently the most specific catalyst and the most efficient enzyme of all known microcystins (MCs) detoxification pathways. Direct MlrA application has been demonstrated to degrade MCs under modeled environmental conditions, potentially informing sustainable methodologies for in situ MCs remediation. Heterologous MlrA expression in cyanobacteria offers a unique perspective, linking harmful cyanobacterial MCs remediation with the state of the art in cyanobacterial biotechnology. Here, we report application of the most recent cyanobacterial synthetic biology molecular cloning suite, CyanoGate, for peptide engineering of the MlrA N-terminus. Utilizing CyanoGate allowed for generation of broad-host range plasmid vectors for application of the following N-terminal sequences to MlrA in various combinations: (1) 23 amino acid (AA) secretion peptide from Synechocystis sp. PCC 6803 PilA (sll1694), (2) 38 AA secretion peptide from Thermobifida fusca lytic polysaccharide monooxygenase (TfAA10A), (3) full length TfAA10A, and (4) USTB-05 MlrA native 26 AA signal peptide. Resultant plasmids were transformed into Synechocystis sp. PCC 6803 and the industrially-relevant Synechococcus elongatus UTEX 2973. Successful transformants were verified via PCR assay, followed by analysis of both cytosolic and extracellular MlrA activity. Application of (1) and (2) (and combination) demonstrated cytosolic MlrA activity, offering novel, synthetic MlrA towards industrial applications.

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May 27th, 11:00 AM May 27th, 11:15 AM

Application of CyanoGate, a Cyanobacterial Synthetic Biology Molecular Cloning Suite, for Exploring Optimal Heterologous Microcystinase (MlrA) Production for Freshwater Bioremediation from Cyanotoxins

Microcystinase (MlrA) is currently the most specific catalyst and the most efficient enzyme of all known microcystins (MCs) detoxification pathways. Direct MlrA application has been demonstrated to degrade MCs under modeled environmental conditions, potentially informing sustainable methodologies for in situ MCs remediation. Heterologous MlrA expression in cyanobacteria offers a unique perspective, linking harmful cyanobacterial MCs remediation with the state of the art in cyanobacterial biotechnology. Here, we report application of the most recent cyanobacterial synthetic biology molecular cloning suite, CyanoGate, for peptide engineering of the MlrA N-terminus. Utilizing CyanoGate allowed for generation of broad-host range plasmid vectors for application of the following N-terminal sequences to MlrA in various combinations: (1) 23 amino acid (AA) secretion peptide from Synechocystis sp. PCC 6803 PilA (sll1694), (2) 38 AA secretion peptide from Thermobifida fusca lytic polysaccharide monooxygenase (TfAA10A), (3) full length TfAA10A, and (4) USTB-05 MlrA native 26 AA signal peptide. Resultant plasmids were transformed into Synechocystis sp. PCC 6803 and the industrially-relevant Synechococcus elongatus UTEX 2973. Successful transformants were verified via PCR assay, followed by analysis of both cytosolic and extracellular MlrA activity. Application of (1) and (2) (and combination) demonstrated cytosolic MlrA activity, offering novel, synthetic MlrA towards industrial applications.