A cassette of proteins endogenous to Sphingopyxis sp. has been shown to sequentially break down microcystin-LR (Figure 1). The proteins have been named mlrA, mlrB, mlrC, mlrD, mlrE, and mlrF [2]. The genes for these proteins are the basis of our composite biobricks.
MlrA is the first enzyme in the microcystin-degradation pathway. It is a microcystinase that degrades cyclic microcystin-LR to form a linear intermediate. To do so, mlrA hydrolyzes the peptide bond between Adda at position 5 and the amino acid residue at position 4 [2].
MlrB is the second enzyme in the microcystin-degradation pathway. MlrB is a serine protease that catalyzes the hydrolysis of linearized MCs via the cutting of the peptide bond between alanine at position 1 and a variable amino acid (e.g. leucine) residue at position 2. This results in the formation of a tetrapeptide that is subsequently degraded by MlrC [2].
MlrC is the third enzyme in the microcystin-degradation pathway. MlrC is assumed to be a metallopeptidase. It cleaves the tetrapeptide intermediate into an Adda amino acid and other smaller peptides. The exact smaller peptides that result are still unknown [2, 3, 4, 5].
MlrD codes for what is most likely a transmembrane transporter protein responsible for transporting the cyclic microcystin-LR toxin from the extracellular environment into the cell. However, its exact function is yet to be characterized. Although mlrD does not ultimately play a role in our project, we have added this part so that the entire mlr cassette is documented in the Registry [2].
MlrE has recently been identified in the genome of Sphingopyxis sp. strain C-1 and may be involved in microcystin degradation. Its exact function is still unknown. The gene for mlrE is, however, close in proximity to the gene for mlrB, so it is hypothesized that mlrE enhances the function of mlrB (or vice versa), [6].
MlrF has recently been identified in the genome of Sphingopyxis sp. strain C-1 and may be involved in microcystin degradation; is the expected sixth enzyme in the microcystin-degradation pathway. Its exact function is still unknown but it likely is involved with catalyzing reactions downstream to those of mlrA through C [6].
For the exception of mlrD, the transporter protein, we have also included the TorA twin-arginine translocation tag in each composite part. The twin-arginine translocation (Tat) pathway is responsible for the export of folded proteins across the cytoplasmic membrane of bacteria into the periplasmic space (Figure 2). The Tat pathway acts separately from the general secretory pathway, which transports proteins in an unfolded state. A specific signal peptide, which contains three domains: a positively charged N-terminal domain, a hydrophobic domain, and a C-terminal domain, is necessary to initiate protein export by the Tat pathway [1].
We have included this tag with the goal of transporting the microcystin-degrading enzyme into the bacterial periplasmic space. This biobrick was designed for expression in bacteria contained in a bioreactor. Moving the enzymes closer to the periphery of the bacterial cell, and therefore closer to the flow of contaminated water passing through the bioreactor, increases the efficiency of the degradation.
As an additional note, we decided to utilize the Twin-Arginine Translocation pathway because of its ability to transport fully folded proteins. Many proteins (such as GFP), are unable to fold properly if exported unfolded into the periplasm. The periplasm is an oxidizing environment, which promotes the formation of aberrant disulfide bonds. This can cause proteins to misfold, aggregate, and become inactive. By exporting our enzymes fully-folded into the periplasmic space, we avoid this problem.
Moreover, inclusion of the tag negates the need to transport individual MC-LR proteins into the cell with mlrD, which may further slow the degradation process. Furthermore, we do not need to express the entire mlr casette in each cell, which would place a high metabolic strain on the cell. Instead, we can engineer the cells to produce only one of the mlr protein species. These different cell types are then encapsulated in alginate beads and packed sequentially in the bioreactor. When microcystin-contaminated water is passed through the reactor, it is first degraded by mlrA, then these byproducts digested by mlrB, and so on.
Finally, we have also included a 3X FLAG tag at the end of the sequence. This permits protein expression to be detected, quantified and/or purified by western blot, SDS-PAGE, and other methods.
Aptamers are oligonucleotide or peptide molecules that bind to a target molecule with high affinity, specificity and selectivity. They are widely used for environmental monitoring as “chemical antibodies.” We chose to use a DNA aptamer because it is easy to synthesize, small in size, and of excellent chemical stability.
Our aptamer codes for a ssDNA aptamer which binds with high specificity to microcystin-LR.
The aptamer was used to create a rapid colorimetric microcystin-detection sensor. Gold nanoparticles (AuNPs) were adsorbed onto the aptamer surface. Normally, in the presence of NaCl, AuNPs aggregate, shifting the solution color from red to blue. Adsorption of the AuNPs onto the aptamer, however, prevents aggregation.
In the presence of microcystin, the aptamer dissociates from the AuNPs and preferentially binds to the microcystin. Thus, the AuNPs will again aggregate when induced by salt. This results in a predictable shift in the absorption spectra of a solution containing microcystins [7, 8].
[1] Lee, P. A., Tullman-Ercek, D., & Georgiou, G. (2006). The bacterial twin-arginine translocation pathway. Annual review of microbiology, 60, 373–395. doi:10.1146/annurev.micro.60.080805.142212
[2] Zhang, J., Lu, Q., Ding, Q., Yin, L., & Pu, Y. (2017). A Novel and Native Microcystin-Degrading Bacterium of Sphingopyxis sp. Isolated from Lake Taihu. International journal of environmental research and public health, 14(10), 1187. doi:10.3390/ijerph14101187
[3] Family M81. (n.d.). Retrieved from https://www.ebi.ac.uk/merops/cgi-bin/famsum?family=M81.
[4] Cerdà-Costa, N., & Gomis-Rüth, F. X. (2014). Architecture and function of metallopeptidase catalytic domains. Protein science : a publication of the Protein Society, 23(2), 123–144. doi:10.1002/pro.2400
[5] BLAST: Basic Local Alignment Search Tool. (n.d.). Retrieved from https://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_BAI47772.
[6] Wang, J., Wang, C., Li, J., Bai, P., Li, Q., Shen, M., … Zhao, J. (2018). Comparative Genomics of Degradative Novosphingobium Strains With Special Reference to Microcystin-Degrading Novosphingobium sp. THN1. Frontiers in Microbiology, 9. doi: 10.3389/fmicb.2018.02238
[7] Li, X., Cheng, R., Shi, H., Tang, B., Xiao, H., & Zhao, G. (2016). A simple highly sensitive and selective aptamer-based colorimetric sensor for environmental toxins microcystin-LR in water samples. Journal of Hazardous Materials, 304, 474–480. doi: 10.1016/j.jhazmat.2015.11.016
[8] Aptamer-Based Biosensors to Detect Aquatic Phycotoxins and Cyanotoxins Isabel Cunha 1,*, Rita Biltes 1 , MGF Sales 2,3 and Vitor Vasconcelos 1,4