New tools for effective production and long-term stabilization of microcystinase (MlrA) - A biotechnological perspective towards hepatotoxic microcystins remediation

Abstract

Microcystinase (MlrA) is the most specific catalyst and the most efficient enzyme of known microcystins (MCs) detoxification pathways. Very recently, direct MlrA application has effectively degraded MCs within industrial processes, demonstrating MlrA application for sustainable in situ MCs remediation. Heterologous MlrA expression in cyanobacteria offers a unique opportunity, linking harmful MCs remediation with emerging cyanobacterial biotechnologies. Here, we first generate a novel Synechocystis sp. PCC 6803 (6803) using non-native trc promoter for MlrA expression. Whole-cell MlrA activity was comparable to previously described expression via native 6803 PcpcB₅₆₀ promoter, while cellular extracts of the new strain showed significantly higher MlrA yields (2–15 times, depending on the age of the cultures). Furthermore, efficiency of MlrA production under multiple photoautotrophic cultivation conditions varied, but was not improved by Na₂CO₃ supplementation nor under increased light, indicating the need to explore new photoautotrophic chassis for higher MlrA productivity.

Methods for MlrA stabilization are critical for industrial development, thus lyophilization of MlrA-enriched cellular extracts was explored. Recovered MlrA activity was not statistically different from initial MlrA activity following storage of lyophilized extracts at −20 °C for 20 weeks. In contrast, storage at 20 °C and storage of aqueous lysates at 4 °C resulted in progressive MlrA activity loss. Such stabilized lyophilizate may offer a checkpoint for further optimization of upstream processes (production), while expanding potential for downstream investigations (application), contributing simultaneously to novel MlrA-based MCs remediation approaches and to cyanobacterial biotechnology.