Heterologous expression of mlrA in a photoautotrophic host – Engineering cyanobacteria to degrade microcystins

Dexter, Jason; Dziga, Dariusz; Lv, Jing; Zhu, Jianfei; Strzałka, Wojciech; Maksylewicz, Anna; Maroszek, Magdalena; Marek, Sylwia; Fu, Pengcheng

doi:10.1016/j.envpol.2018.01.071

Cited by 29 publications

(20 citation statements)

References 55 publications

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“…SDS/PAGE of the total cell lysates of E. coli C41 before and after induction with IPTG demonstrated the overexpression of the recombinant MlrA. The average activity of recombinant MlrA against MC-LR in lysate (Table 1) was significantly higher in comparison with the lysates from the previous expression systems, compared in a paper by Dexter et al [8]. Thus, the use of this system of MlrA production for field study and further applications is more justified and efficient.…”

Section: The Efficiency Of Mlra In Environmentally Relevant Conditionsmentioning

confidence: 75%

“…It is the only known enzyme that is capable of hydrolysing the stable MC cyclic structure. Heterologous expression and production systems for this enzyme have already been developed [7], including the recently proposed alternative production of this enzyme in autotrophic hosts [8]. Furthermore, recent research [9] provides an indication that the recombinantly produced MlrA should be considered a very effective agent which hydrolyses and detoxifies different MC variants.…”

Section: Introductionmentioning

confidence: 99%

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The Effect of a Combined Hydrogen Peroxide-MlrA Treatment on the Phytoplankton Community and Microcystin Concentrations in a Mesocosm Experiment in Lake Ludoš

Dziga

Tokodi

Drobac

et al. 2019

Toxins

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Harmful cyanobacteria and their toxic metabolites constitute a big challenge for the production of safe drinking water. Microcystins (MC), chemically stable hepatotoxic heptapeptides, have often been involved in cyanobacterial poisoning incidents. A desirable solution for cyanobacterial management in lakes and ponds would eliminate both excess cyanobacteria and the MC that they potentially produce and release upon lysis. Hydrogen peroxide (H 2 O 2 ) has recently been advocated as an efficient means of lysing cyanobacteria in lakes and ponds, however H 2 O 2 (at least when used at typical concentrations) cannot degrade MC in environmental waters. Therefore, mesocosm experiments combining the cyanobacteria-lysing effect of H 2 O 2 and the MC-degrading capacity of the enzyme MlrA were set up in the highly eutrophic Lake Ludoš (Serbia). The H 2 O 2 treatment decreased the abundance of the dominant cyanobacterial taxa Limnothrix sp., Aphanizomenon flos-aquae, and Planktothrix agardhii. The intracellular concentration of MC was reduced/eliminated by H 2 O 2 , yet the reduction of the extracellular MC could only be accomplished by supplementation with MlrA. However, as H 2 O 2 was found to induce the expression of mcyB and mcyE genes, which are involved in MC biosynthesis, the use of H 2 O 2 as a safe cyanobacteriocide still requires further investigation. In conclusion, the experiments showed that the combined use of H 2 O 2 and MlrA is promising in the elimination of both excess cyanobacteria and their MC in environmental waters. Key Contribution:A new concept has been proposed for rapid and efficient mitigation of MC-producing cyanobacterial biomass through a combined treatment with H 2 O 2 and microcystinase (MlrA).

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Section: The Efficiency Of Mlra In Environmentally Relevant Conditionsmentioning

confidence: 75%

Section: Introductionmentioning

confidence: 99%

The Effect of a Combined Hydrogen Peroxide-MlrA Treatment on the Phytoplankton Community and Microcystin Concentrations in a Mesocosm Experiment in Lake Ludoš

Dziga

Tokodi

Drobac

et al. 2019

Toxins

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“…The combination of high expression and low impact on cell growth and final biomass levels suggests promising potential for further optimization and biotechnological exploitation. Previous studies have suggested that cyanobacterial secretion of heterologous proteins may have applications in microcystin degradation [64], heavy metal bioremediation [65] and CO 2 sequestration [63]. We believe this can also be extended to whole-cell biocatalysis where cyanobacteria have the potential to become light-driven cell factories capable of secreting active enzymes to sustain extracellular biotransformations.…”

Section: Discussionmentioning

confidence: 92%

Expression and secretion of a lytic polysaccharide monooxygenase by a fast-growing cyanobacterium

Russo

Zedler

Wittmann

et al. 2019

Biotechnol Biofuels

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Background Cyanobacteria have the potential to become next-generation cell factories due to their ability to use CO 2 , light and inorganic nutrients to produce a range of biomolecules of commercial interest. Synechococcus elongatus UTEX 2973, in particular, is a fast-growing, genetically tractable, cyanobacterium that has garnered attention as a potential biotechnological chassis. To establish this unique strain as a host for heterologous protein production, we aimed to demonstrate expression and secretion of the industrially relevant Tf AA10A, a lytic polysaccharide monooxygenase from the Gram-positive bacterium Thermobifida fusca. Results Two variations of Tf AA10A were successfully expressed in S. elongatus UTEX 2973: One containing the native N-terminal, Sec-targeted, signal peptide and a second with a Tat-targeted signal peptide from the Escherichia coli trimethylamine- N -oxide reductase (TorA). Although the TorA signal peptide correctly targeted the protein to the plasma membrane, the majority of the TorA- Tf AA10A was found unprocessed in the plasma membrane with a small fraction of the mature protein ultimately translocated to the periplasm. The native Sec signal peptide allowed for efficient secretion of Tf AA10A into the medium with virtually no protein being found in the cytosol, plasma membrane or periplasm. Tf AA10A was demonstrated to be correctly cleaved and active on the model substrate phosphoric acid swollen cellulose. Additionally, expression and secretion only had a minor impact on cell growth. The secretion yield was estimated at 779 ± 40 µg L −1 based on densitometric analysis. To our knowledge, this is the highest secretion yield ever registered in cyanobacteria. Conclusions We have shown for the first time high-titer expression and secretion of an industrially relevant and catalytically active enzyme in S. elongatus UTEX 2973. This proof-of-concept study will be valuable for the development of novel and sustainable applications in the fields of bioremediation and biocatalysis. Electronic supplementary material The online version of this article (10.1186/s13068-019-1416-9) contains supplementary material, which is available to authorized users.

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“…MlrA, termed microcystinase, is a metalloprotease linearizing the MC-LR at the Arg-ADDA bond. It is commonly used as marker gene and considered to catalyze the most important step in biodegradation of MCs (Dziga et al, 2012;Dexter et al, 2018;Lezcano et al, 2018). MlrB is a serine protease that drives the hydrolysis of the linearized MC-LR at the Ala-Leu bond, forming a tetrapeptide.…”

Section: Introductionmentioning

confidence: 99%

“…This pathway has been extensively studied in culture (Supplementary Table S1, Bourne et al, 2006;Dziga et al, 2012;Maseda et al, 2012;Dziga et al, 2016;Dexter et al, 2018; as examples), although not all MC degrading isolates have tested positive for these genes. Further, the environmental relevance and distribution of the mlr cassette in the environment remains unclear.…”

Section: Introductionmentioning

confidence: 99%

Insight Into the Molecular Mechanisms for Microcystin Biodegradation in Lake Erie and Lake Taihu

et al. 2019

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Heterologous expression of mlrA in a photoautotrophic host – Engineering cyanobacteria to degrade microcystins

Cited by 29 publications

References 55 publications

The Effect of a Combined Hydrogen Peroxide-MlrA Treatment on the Phytoplankton Community and Microcystin Concentrations in a Mesocosm Experiment in Lake Ludoš

The Effect of a Combined Hydrogen Peroxide-MlrA Treatment on the Phytoplankton Community and Microcystin Concentrations in a Mesocosm Experiment in Lake Ludoš

Expression and secretion of a lytic polysaccharide monooxygenase by a fast-growing cyanobacterium

Insight Into the Molecular Mechanisms for Microcystin Biodegradation in Lake Erie and Lake Taihu

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