Team:Peking/secondtry/Degradation

Introduction

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

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

Based on utility of MlrA, we measure the degradation efficiency of the living bacteria, the periplasmic protein and the lysed whole cell production. The results indicate that our engineered bacteria could degrade MC-LR to a certain extent.

Design

Microcystin and MlrA enzyme

MCs are widespread toxic cyclic heptapeptides produced by many species of algae 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.

The most known mechanism of 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 cell. 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 hepatoxin 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. [3] (Fig. 2)

Secretion System

In order to enhance the degradation effect, location of MlrA should be considered. There are some porins proteins 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.

Result

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. 4) 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.

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

Based on the premise of linear relationship between product and absorbance, we choose 0.05unit/ul as the working concentration of PP1 and then test 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.

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 function well in E. coli expression system.

3. Attempting to secreting MlrA

MlrA exhibits high degradation activity in lysis culture. Its activity, however, has no difference with control group.(Fig. 6b) The 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.

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.