Part:BBa_K4125000

mlrA from Sphingopyxis sp.m6

MlrA is among the mlr gene cluster that is responsible for the natural microcystin degradation pathway. MlrA (microcystinase protein) can cleave the peptide bond between amino acid Adda and Arg, and thus linearizing the cyclic microcystin. This is the first and most efficient step in the enzymatic cascade, causing a 2100-fold decrease in toxicity and essentially rendering a non-toxic product.

The mlrA gene sequence that we used was from Sphingopyxis sp.m6, a strain separated from Lake Taihu in China. It is one of the most efficient types of mlr genes that have been tested yet.

FIG.1 MlrA Catalyzed Reaction (Shimizu et al, 2012)

Sequence and Features

In order to avoid an illegal EcoRI restriction site, we performed a point mutation at Base Pair 870 and changed T to C. We ensured compatibility with RFC 10 Standards.

Assembly Compatibility:

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

  Illegal NgoMIV site found at 708
  Illegal AgeI site found at 243
  
• 1000
  COMPATIBLE WITH RFC[1000]

The mlrA sequence vary among microcystin degrading bacteria, and the enzyme structure may have minor differences. We used Phyre2 to predict the structure of our MlrA from Sphingopyxis sp. m6 using its full sequence.

FIG.2 Structure of MlrA

Usage by 2022 Nanjing_NFLS

2022 Nanjing_NFLS constructed an extracellular enzyme display system to exhibit MlrA enzyme at the surface of E. coli. The InaK protein served as an anchoring motif. This way, the enzyme can directly degrade toxins present in the environment without toxin intake. The genes were transformed on a pET23b plasmid vector.

FIG.3 Schematic of Plasmid, Created with SnapGene

Restriction Endonuclease Digestion

The recombinant plasmid was then extracted to be identified with restriction endonuclease digestion. We separated the targeted gene inaK (537 bp) from the plasmid, and performed 20 rounds of PCR on it. This way, we obtained a larger quantity of the targeted gene, and the gel electrophoresis graph would be of better clarity. The sequence of the primers we used are listed as follows:

Primer	Sequence (5’ to 3’)	Restriction Site
InaK-F	TCGAGCTCCGTCGACAAGCTTATGACTCTCGACAAGGCGTTG	Hind III
InaK-R	TTCCGCATGGTCTGCAAATTCTGCGGC	/
MlrA-F	AATTTGCAGACCATGCGGGAGTTTGTCAAACAG	/
MlrA-R	GTGGTGGTGGTGGTGCTCGAGCGCGTTCGCGCCGGACTT	Xho I
Total-F	AACGGTTTCCCTCTAGAAATAATTT	Hind III
Total-R	CCTTTCGGGCTTTGTTA	Xho I

TABLE 1 Primer sequences and Restriction Sites

We used a 200 bp ladder marker in gel electrophoresis. As shown in FIG.4, there were 3 significant bands respectively around 550 bp, 1000 bp and 1550 bp, each corresponding to inaK, mlrA and combination of the two. This indicated that the plasmid construction had been successful.

FIG.4 Restrictive Endonuclease Digestion Results

Identification of Protein Location

We separated different components of the cell by cell fractionation with ultracentrifuge. The samples of outer membrane, inner membrane and cytoplasm were obtained and stored at -4°C overnight. SDS-PAGE gel electrophoresis was performed the next day. We then used Coomassie Bright Blue to stain the gel and observe the proteins in each sample.

FIG.5 SDS-PAGE Results after Coomassie Staining

The composite InaK + MlrA protein (56 kDa) was found in the outer membrane fraction of pET23b-inaK+mlrA transformed E. coli, verifying our construction. MlrA only (37 kDa) was found in the inner membrane fraction of pET23b-mlrA transformed E. coli, which matched previous literature’s observations. Overall, we verified the compatibility and location of the inaK-based enzyme display system.

Degradation Assay

We first decided to measure the concentration of cyclic microcystins using high performance liquid chromatography. Using samples of known cyclic microcystin-LR concentrations (0.1 mg/L, 0.25 mg/L, 0.5 mg/L, 1 mg/L, 2.5 mg/L, 5 mg/L, 10 mg/L), we derive a standard curve that correlates HPLC area to mass concentration. The R Squared value =0.997, indicating a reliable fit.

FIG.6 HPLC Standard Curve for Cyclic Microcystin-LR

We cultured the reaction systems at 37 °C, and retrieved samples of 1 milliliter hourly. The intensity of both the cyclic Microcystin-LR and the linearized Microcystin-LR were measured.

FIG.7 Microcystin-LR Degradation Curve (error = standard deviation)

From mass spectrometry, and comparison with standards derived in previous studies, we also verified the degradation product: linearized microcystin-LR (m/z = 1013).

FIG.8 Mass Spectrometry of Degradation Product

References:

[1]. Qin, L., Zhang, X., Chen, X., Wang, K., Shen, Y. and Li, D., 2019. Isolation of a novel microcystin-degrading bacterium and the evolutionary origin of mlr gene cluster. Toxins, 11(5), p.269.

[2]. Shimizu, K., Maseda, H., Okano, K., Kurashima, T., Kawauchi, Y., Xue, Q., Utsumi, M., Zhang, Z. and Sugiura, N., 2012. Enzymatic pathway for biodegrading microcystin LR in Sphingopyxis sp. C-1. Journal of Bioscience and Bioengineering, 114(6), pp.630-634.

[3]. Liu, M., Feng, P., Kakade, A., Yang, L., Chen, G., Yan, X., Ni, H., Liu, P., Kulshreshtha, S., Abomohra, A.E.F. and Li, X., 2020. Reducing residual antibiotic levels in animal feces using intestinal Escherichia coli with surface-displayed erythromycin esterase. Journal of hazardous materials, 388, p.122032.