Cloning, Expression and Structural Modeling of the MlrA Protein from Novosphingobium sp. KKU25s for Microcystin Degradation

Abstract

MlrA is a gene involved in the degradation of toxic cyanobacterial microcystins. This gene encodes microcystinase, mlrA, the 1st enzyme in the pathway that breaks down toxic cyanobacterial microcystins. In this study, primers were designed, and polymerase chain reaction (PCR) was performed to amplify the mlrA gene in Novosphincgobium sp. KKU25s. A PCR product of 752 base pairs was obtained. The nucleotide sequence of the mlrA gene of Novosphingobium sp. KKU25s was similar to that of Sphingomonas sp. ACM-3962 (98 % similarity). The mlrA gene of Novosphingobium sp. KKU25s was further cloned into the pGEM T-Easy plasmid to obtain the nucleotide sequence of the mlrA gene. The gene was also ligated into the pET32a plasmid for gene expression. Expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and verified using SDS-PAGE. The expressed protein was approximately 22 kilodaltons. The cell-free extract (CE) containing the crude protein from confirmed recombinant cells showed high activity in the biodegradation of [Dha7] MC-LR. [Dha7] MC-LR at an initial concentration of 30 mg L-1 and was completely biodegraded within 30 h. A distinct product derived from [Dha7] MC-LR appeared with a decrease in the [Dha7] MC-LR peak in the HPLC profile. The product (m/z 999.51) showed a molecular weight of 18, which is higher than that of native [Dha7] MC-LR (m/z 981.50), and was determined to be a linearized peptide fragment of [Dha7] MC-LR using LC-MS analysis. The 3-dimensional structure of microcystinase was predicted from the amino acid sequence deduced from the mlrA gene by the Swiss Model and Phyre2 programs. The structure contained a predicted alpha helix. The predicted 3-dimensional structure was also similar to that of a protein in the CAAX protease group. HIGHLIGHTS Research focused on characterization of microcystinase (MlrA) protein First research worked on the degradation of [Dha7] MC-LR by MlrA This work is useful for the applications aimed at the removal of MCs in freshwater environments GRAPHICAL ABSTRACT

Microcystin-Detoxifying Recombinant Saccharomyces cerevisiae Expressing the mlrA Gene from Sphingosinicella microcystinivorans B9

“…On the other hand, Moolwang et al (2021) worked with purified mlrA recombinant enzyme expressed in E. coli BL21 strains. A complete degradation of 30 μg.mL −1 of MC-LR was reported within 30 h of incubation [ 36 ]. In addition, Liu et al (2020) overexpressed the mlrA gene in K12 B1 strain reducing intracellular and extracellular MC content.…”

“…Furthermore, Microcystis aeruginosa growth in BG11 medium was inhibited, which represents a useful biological tool in the control of toxigenic cyanobacteria [ 37 ]. In all the studies mentioned above, expression of the mlrA gene was induced with IPTG (isopropyl-β-D-thiogalactoside), and experiments reported by Moolwang et al (2021) and Liu et al (2020) were performed with the purified enzyme [ 35 , 36 , 37 ]. In contrast, here, a constitutive promoter was used, which does not require adding a reagent for induction and MC degradation was assessed during yeast growth.…”

Effects of Pond Water Depth and Method of Aeration on Phytoplankton Communities in Macrobrachium rosenbergii Farming Ponds

“…Te outbreak of cyanobacteria produces a variety of toxins and decreases dissolved oxygen in the water, which could reduce productivity as a result of frequent disease outbreaks of M. rosenbergii [3,15,17,18]. Also, cyanobacterial blooms could cause taste and odor adverse efects in M. rosenbergii [19][20][21], which further reduces the price of M. rosenbergii and the income of aquaculture.…”