Part:BBa_K3699001

MlrA from Novosphingobium sp. THN1

Introduction

This part is microcystin enzyme (MlrA) from Novosphingobium sp. THN1.

MlrA is an enzyme that degrades cyanobacterial toxins, especially microcystin-LR (MC-LR).

By comparing the ability of Sphingomonas sp. ACM-3962, Novosphingobium sp. THN1 and other bacteria’s data, we found that Novosphingobium sp. THN1 shows a favorable toxin degradation activity. Therefore, we plan to characterize the MlrA of THN1, which was regarded as the first enzyme catalyzing the degradation of microcystins.

To promote enzyme expression, we replaced the commonly used strong promoter J23110 with the stronger J23119 in its family. This part successfully expressed MlrA enzyme and showed activity on degrading MC-LR.

Background

Early in 1994, Sphingomonas sp. ACM-3962 was identified as the first bacterium capable of degrading MCs as sole carbon and nitrogen source for its growth. [1] Afterwards, other bacteria of Sphingomonas sp., Sphingopyxis sp., Novosphingobium sp., Stenotrophomonas sp. and Bacillus sp. were verified as able to degrade MCs, including Novosphingobium sp. THN1.

These bacteria all have mlr gene clusters. On the gene cluster, four mlr genes are located sequentially as mlrC, A, D and B, where mlrA and mlrD are transcribed in forward direction while mlrC and mlrB in the reverse.

Figure 1. Sketch map of mlr gene cluster responsible for MC-biodegradation. The relative localization of each gene is shown. The direction of the outline borders embracing gene name represents the transcription direction of respective gene (adapted from Bourne et al. (1996, 2001)).

Among them, MlrA is the most important enzyme. The first enzyme encoded by mlrA (i.e., MlrA) gene can hydrolyze cyclic MC-LR at Adda-Arg bond. Linearized MC-LR was reported to be 160-fold less toxic than cyclic MC-LR.[2]

Figure 3. Degradation process of MC-LR

By comparing the ability of Sphingomonas sp. ACM-3962, Novosphingobium sp. THN1 and other bacteria’s data, we found Novosphingobium sp. THN1 shows a stronger activity. So we characterize it.

Figure 1. Comparison of MC-LR degradability of different strains. The original data from previous studies were all equivalent to the degradation effect within 24 h. In the original study, the conditions of each strain were slightly different; however, by comparison, we can find out which strains have been proven to have higher degradation capacity.

Design

We selected the mlrA gene from Novosphingobium sp. THN1 (BBa_K3699001), adding the promoter J23119, RBS (B0034), and terminator. After that, the entire sequence (BBa_K3699005) was introduced into pKMV and yielded the recombinant plasmid pKMV-mlrA-THN1, which was transformed into E. coli JM109.

Figure 2. Schematic map of pKMV-mlrA-THN1. We used the same promoter J23119, and transformed the plasmid into E. coli JM109.

Methods & Testing

We used ELISA kits to detect toxin levels in samples (Ordered from Zhenke Biotec). Detailed methods can be viewed on Contribution and Methods pages.

Figure 3. Plasmid Profile. pKMV-mlrA-C1 (including BBa_K1378001) and pKMV-mlrA-THN1 (including BBa_K3699001).

Result

We measured the concentration of toxin in the reaction system every 6 hours (Fig. 3 and Fig. 4).

Figure 4. Degradation result of pKMV-mlrA-THN1 cell culture.

Figure 5. Degradation result of pKMV-mlrA-THN1 cell extract.

Figure 6. Degradation result table.

●As can be seen from the graph, within 24 h, the toxins were degraded by 25.97% (cell culture) and 28.28% (cell extract). The cell extract of THN1 is efficient in degradation.

●By comparing BBa_K1378001 with our newly designed part, we found that both of them can degrade MC-LR effectively, but BBa_K1378001 possessed slightly higher degradation efficiency under the conditions of this experiment.

●Comparing the degradation rates of 18 h and 24 h, it was found that the degradation efficiency was not close to the maximum at 18 h of reaction (which is different from the experimental results of BBa_K1378001). Therefore, we speculate that the new MlrA enzyme can degrade MC-LR more thoroughly in a longer degradation time, although its results are slightly worse within 24 hours.

●To sum up, we have characterized our new MlrA enzyme from Novosphingobium sp. THN1. It is indicated that it can degrade MC-LR with favorable efficiently.

References

[1] Wang R , Li J , Jiang Y , et al. Heterologous expression of mlrA gene originated from Novosphingobium sp. THN1 to degrade microcystin-RR and identify the first step involved in degradation pathway[J]. Chemosphere, 2017, 184(oct.):159.

[2] Hyung Soo Kim and Young In Park. Isolation and identification of a novel microorganism producing the immunosuppressant tacrolimus[J]. Journal of Bioscience and Bioengineering, 2008.

[3] Ren YY. Synthesis, expression and purification of microcystin-degrading enzyme Mlr A gene optimized by Lactococcus lactis preference codon. [D]. Jilin University, 2015.

[4] Yan H , Wang J , Chen J , et al. Characterization of the first step involved in enzymatic pathway for microcystin-RR biodegraded by Sphingopyxis sp. USTB-05[J]. Chemosphere, 2012, 87(1):12-18.

[5] Xu Q , Fan J , Yan H , et al. Structural basis of microcystinase activity for biodegrading microcystin-LR[J]. Chemosphere, 2019, 236(Dec.):124281.1-124281.9.

[6] Qin, Zhang, Chen, et al. Isolation of a Novel Microcystin-Degrading Bacterium and the Evolutionary Origin of mlr Gene Cluster[J]. Toxins, 2019, 11(5):269-.

[7] Miao H F , Qin F , Tao G J , et al. Detoxification and degradation of microcystin-LR and -RR by ozonation[J]. Chemosphere, 2010, 79(4):355-361.

[8] Liu H , Guo X , Liu L , et al. Simultaneous Microcystin Degradation and Microcystis Aeruginosa Inhibition with Single Enzyme Microcystinase A[J]. Environmental ence and Technology, 2020.

[9] Bourne D G . Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin LR.[J]. Applied & Environmental Microbiology, 1996, 62.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]