Team:Peking/secondtry/Degradation

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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 decontamination 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

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.

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

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 hydrolysze 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 raise the median inhibition concentration to 95nM, around 160 times higher than original 0.6nM. [3] (Fig. 2)

Fig. 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]

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.

Result

We construct a vector for secretion using Sec pathway(Fig. 3), which belongs to Type II secretion system that exports proteins to periplasm. During the exporting process, target protein is translocated across inner membrane in unfolded conformation and is refolded in the periplasm.[4]

A signal peptide called Pectate lyase B (PelB) in Sec pathway is required for the transportation system to recognize the protein to be export and the signal peptide can be cut off in the periplasm. Since the PelB signal peptide holds little limitation to the following protein’s molecular weight, we finally decide to use PelB to secrete the MlrA protein.

Fig. 3 Secretion vector of mlrA. The fusion protein includes Type II secretion peptide pelB and mlrA. The construction as a whole is expressed in pET-21a(+) plasmid.

The concentration of MCs can be tested in PP1 inhibition assays. As mentioned, MCs can inhibit the activity of PP1 effectively. Thus we constructed a standard curve reflecting the relation between the concentration of MC and the relative activity of PP1. Therefore, the concentration of MCs in any solution could be quantified by measuring corresponding PP1 relative activity.

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)

Fig. 4 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).

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.

Fig. 5 Calibration curve of PP1. p-Nitrophenyl Phosphate solution is 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.

We choose 0.05unit/ul as the working concentration of PP1 and then test the inhibition efficiency of MC-LR because in this region absorbance displays a nearly linear relationship with PP1 concentration less than 0.05 unit/uL. 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.

Fig. 6 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.

To test the efficiency, a degradation assay is performed. MlrA coding sequence and PelB signal peptide is inserted into the pET-21a(+) plasmid. This plasmid is transformed into E. coli strain BL21(DE3) as a secretion vector. Bacteria carrying a blank vector and an expression vector without the addition of signal peptide are used as control.

Fig. 7 Expression vector for degradation assays. Vector (a) is our secretion system. There are 2 negative controls. Blank Vector (b) is used as a negative control of MlrA expression system while vector (c) without any signal peptide is used as a negative control of pelB signal peptide.

First we test if MlrA is expressed in E. coli correctly. MC-LR is co-cultivated with the bacteria and the sample was measured as before to test the degradation efficiency. described above. To accelerate periplasm protein release, we treat bacteria with lysozyme solution. MC-LR is then added to bacteria solution both treated and untreated. After co-cultivation, concentration of MC-LR rest can be tested by spectrophotometry. Without lysozyme treatment, absorbance of group that carrying MlrA sequence is close to that of conctrol group. But after lysozyme treatment, absorbance of bacteria solutions carrying MlrA gene is much higher than control group, indicating that MlrA is correctly expressed.

Fig. 8 Test of periplasm expression. Bacteria carryting vector (a), (b) and (c) are treated with same volume of lysozyme solution and MC-LR is then added, at a final concentration of 100ug/L. After 12 hours of co-cultivation, the sample is diluted and added to the reaction system as describe before. Absorbance of group (b) and (c) is much higher than that of group (a).

The absorbance of bacteria carrying vector(b) is higher than that bacteria carrying blank vectors, suggesting that MlrA exhibits some activity towards MC-LR. But there is no big difference between vector (b) and vector (c), which shows no evidence of effect of pelB signal peptide so far.

Fig. 8 Degradation efficiency of MC-LR. MC-LR is added to E. coli culture at a final concentration of 100ug/L. After 12, 36 and 72 hours of co-cultivation, the sample is sterilized, diluted and added to the reaction system as describe before. 2 Vector described in Fig. 7 are used as control experiment. The result shows that MlrA has some degradation activity toward MCs, while the secretion system seem to be ineffective.





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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