Team:Peking/Degradation

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Introduction

Apart from the lack of sunlight in the water and anoxia caused by cyanobacteria itself, the potential detrimental effect of toxin secreted by cyanobacteria should be noticed. One of the most harmful toxins is called microcystin (MC), which has severe hepatotoxicity. The work in this part aims at degrading MCs in aquatic environment during an algal bloom.

To accomplish this work, the potent microcystin-degrading enzyme-MlrA, originally from Sphingomonas, is utilized. This enzyme can cleave the ring structure in microcystin, significantly reducing the toxicity of the peptide. Since MCs are released into water by cyanobacteria, secretion for MlrA is also necessary to facilitate the degradation of MCs.

Based on utility of MlrA, we measure its degradation efficiency expressed by E. coli. The results indicate that our engineered bacteria could express functional MlrA and noticeably degrade MC-LR. Moreover, secretion signal peptide is considered to be introduced for better degradation performance.

Design

Microcystin and MlrA enzyme

MCs are widespread toxic cyclic heptapeptides produced by many species of cyanobacteria with different variants (Fig. 1). MCs are synthesized by polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) pathway. Among different variants, MC-LR is a widespread and deleterious one.

Figure 1. Structure of MCs. MCs share cyclic structure of cyclo-(-D-Ala-L-X-D-MeAsp-L-Z-Adda-D-Glu-Mdha), where X and Z are variable [1].

The most known mechanism for its toxicity is that MCs can inhibit protein phosphatase 1 (PP1) and 2A (PP2A) specifically and efficiently [2]. The inhibition can lead to a severe disorder of biochemical reaction and disorganization of cytoskeleton in many eukaryotic cells. Many routine tools of decontamination cannot significantly reduce activities of MCs. Here, we propose a new idea of biodegradation, which could degrade MCs effectively without apparent side effects.

Many bacterial species have been reported to have ability to degrade MCs. Among them, a gene cluster in Sphingomonas has been found and sequenced. The cluster includes four genes, mlrA, mlrB, mlrC and mlrD, which can hydrolyze MCs and facilitate absorption of the products as carbon source. During the degradation process, the first-step linearized product, which is catalyzed by MlrA, shows much weaker hepatoxicity compared with MCs. In the experiment of mouse bioassay, up to 250 mg/kg of linearized MC-LR shows no toxicity to mouse, much higher than 50% lethal dose 50mg/kg of cyclic MC-LR. Furthermore, the linearization also raises the median inhibition concentration to 95nM, around 160 times higher than original 0.6nM (Fig. 2) [3].

Figure 2. First step of biodegradation of MC-LR. MlrA mediates breaking peptide bond between Adda and Arg, which leads to significant decrease of toxicity [3].

Secretion System

In order to enhance the degradation effect, location of MlrA should be considered. There are some porins on the outer membrane of E. coli, which allow small molecules, including MCs, to penetrate the membrane. Consequently, it is sufficient to secret MlrA into periplasm for decontamination.

Sec pathway, which belongs to Type II secretion system that exports proteins to periplasm, enters our sight. During the exporting process, target protein is translocated across inner membrane in unfolded conformation and is refolded in the periplasm [4]. A signal peptide is required for the transportation system to recognize the target protein. After export, the peptide is cut off in the periplasm. Particularly, one of them from Pectate lyase B (PelB) holds little limitation to the following protein’s molecular weight and has been widely used in protein secretion. Consequently, we finally decide to use PelB signal peptide to secrete the MlrA protein.

Results

1. Constructing method for analysis of MC concentration

p-Nitrophenyl phosphate (pNPP) is a widely used non-specific substrate to test protein phosphatase activity and it can be hydrolyzed to p-Nitrophenyl (pNP) with characteristic absorption at 405nm. The measurement of PP1 activity is based on the accumulation of pNP. Considering the microcystin (MC) is the inhibitor of PP1 and MlrA can disrupt MC’s structure to disrupt its inhibitory effect, the MlrA activity can be detected by quantification of absorption at 405nm (Fig. 3). So the concentration of MCs after degradation can be finally measured by absorption spectrophotometry method with all the calibration curves for all the interactions above.

Figure 3. Measurement of MlrA activity. The OD405 indicates the concentration of pNP, and the change of pNP level could reflect the PP1 activity(a). MC can strongly inhibit the PP1 activity(b), and the MlrA can cleave the MC and dampen its toxicity(c).

Firstly a calibration curve of PP1 activity was generated. The concentration of substrate pNPP is sufficient overall so the PP1 enzyme is saturated and proportional to the accumulation rate of product pNP. We could select a proper working concentration of PP1 in the range of nearly linear relationship between PP1 and change rate of 405nm absorption.

Figure 4. Calibration curve of PP1. p-Nitrophenyl Phosphate solution was treated with different concentration of PP1 solutions. Absorbance at 405nm was measured after 80 minutes. The absorbance increases in direct proportion to PP1 concentration between 0.02-0.1 unit/ul.

Based on the premise of linear relationship between product and absorbance, we chose 0.05unit/ul as the working concentration of PP1 and then tested the inhibition efficiency of MC-LR. As a result, PP1 activity decreases after the addition of MC-LR and there is a positive correlation between the reduction of absorbance and concentration of MC-LR.

Figure 5. Inhibition efficiency of MC-LR. Working concentration of PP1 is 0.05 unit/ul. Different concentration of MC-LR samples are added to the reaction system. MC-LR shows strong inhibition of PP1 activity and a rapid change of PP1 activity is observed between 10ug/L to 30 ug/L of MC-LR concentration.

2. Verifying the degradation effect of MlrA

To test the degradation efficiency of MlrA expressed by E. coli, MlrA expression plasmid has been constructed and transformed into E. coli strain BL21(DE3) (Fig. 6a). After induction, the bacteria are lysed by lysozyme and incubated with MC solution. Judged by PP1 activity treated by the mixture, the activity in experiment group expressing MlrA is much higher than strain carrying blank vectors, suggesting that MC-LR is degraded (Fig. 6b). Therefore, it could be concluded that MlrA works well in E. coli expression system.

Figure 6. Plasmid construction and results of degradation assays. (a) In our expression plasmid, MlrA is expressed in expression vector pET-21a, while blank vector is used as a negative control. (b) The result shows that MlrA expressed by E. coli has obvious function in degrading MC, which significantly reducing the inhibition effect of MC to PP1.

3. Attempting to secrete MlrA

MlrA exhibits high degradation activity in lysis culture. Its activity in living cells, however, has no difference with control group (Fig. 6b) This result suggests that our bacteria are unable to deal with MC immediately until they commit suicide. Thus, secretion system PelB is introduced. The PelB is linked to N-terminal of MlrA and the fusion protein is inserted into expression vector. We hope this measure would improve degradation effect largely in whole cell level.

Figure 7. Secretion sequence of MlrA. The fusion protein includes Type II secretion peptide pelB and MlrA. The construction as a whole is expressed in pET-21a(+) plasmid.

References

[1] Gehringer, M. M., Milne, P., Lucietto, F., & Downing, T. G. (2005). Comparison of the structure of key variants of microcystin to vasopressin. Environmental toxicology and pharmacology, 19(2), 297-303.

[2] Runnegar, M., Berndt, N., Kong, S. M., Lee, E. Y., & Zhang, L. F. (1995). In vivo and in vitro binding of microcystin to protein phosphatase 1 and 2A. Biochemical and biophysical research communications, 216(1), 162-169.

[3] Bourne, D. G., Jones, G. J., Blakeley, R. L., Jones, A., Negri, A. P., & Riddles, P. (1996). Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin LR. Applied and environmental microbiology, 62(11), 4086-4094.

[4] Choi, J. H., & Lee, S. Y. (2004). Secretory and extracellular production of recombinant proteins using Escherichia coli. Applied Microbiology and Biotechnology, 64(5), 625-635.

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