Availability MC Yeast : Stress-based detection and enzymatic degradation of the cyanobacterial toxin microcystin

Cyanobacteria, also known as blue-green algae, are an annual problem in many water systems worldwide. During the summer, the cyanobacteria release hepatotoxins called microcystins which pose health risks to humans and animals. Our goal was to build a two-part system to detect and then degrade these toxins. We based our detection system on the natural oxidative stress response of the yeast Saccharomyces cerevisiae. Exposure to microcystins is linked to higher levels of oxidative stress, and we coupled the oxidative stress response to the expression of yellow fluorescent protein. Thus, fluorescence levels are expected to indicate the amount of microcystins present in a sample. To understand and validate our toxin detection mechanism, we also created mathematical and molecular models. To degrade the detected toxins, we expressed the enzyme microcystinase, which is naturally found in some gram-negative bacteria. The enzyme renders microcystins 160 times less toxic by linearizing their structure. Our results indicated that coupling promoter regions of genes involved in oxidative stress to reporters molecules could be used to measure the amount of stress factors in yeast cells. We were able to show that the promoters we chose are a part of the yeast’s rapid stress response, which means they were a good choice for experiments such as this. In addition, we demonstrated that active microcystinase can be produced in Saccharomyces cerevisiae. Financial Disclosure Academic sources of funding include Aalto University’s School of Chemical Engineering, School of Science, School of Electrical Engineering, School of Engineering, and the HYBER Centre of Excellence, and also the University of Helsinki and its Faculty of Agriculture and Forestry, Faculty of Biological and Environmental Science, Faculty of Science, and Biocentrum Helsinki. Individual grants were received from Aleksander ja Lucie Lampénin rahasto (P.S., A.K.), Oiva Allan Pölkkysen rahasto (H.H.) and Tekniska Föreningen i Finland rf (E.B.). Funding and support was also provided by Algol Oy, CA Flowdock, IDT (Integrated DNA Technologies) Inc., Kemianteollisuus ry, LAL ry, Thermo Fisher Scientific Oy, Teollisuuden Vesi Oy, Geneious (Biomatters Ltd.), and Veikko Laine Oy. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests The authors have declared that no competing interests exist. Ethics Statement N/A Data Availability MC Yeast: Stress-based detection and enzymatic degradation of the cyanobacterial toxin microcystin Data Availability (http://www.protocols.io/groups/aaltohelsinki-2016) All data are fully available without restriction. More detailed descriptions of experiments, results and conclusions can be found at our iGEM wiki page: http://2016.igem.org/Team:Aalto-Helsinki (http://2016.igem.org/Team:Aalto-Helsinki). Protocols are available at www.protocols.io/groups/aaltohelsinki-2016 (http://www.protocols.io/groups/aaltohelsinki-2016) Introduction Cyanobacteria, also known as blue green algae, produce toxins that pose serious health risks to humans and animals worldwide. The most common class of cyanotoxins is the microcystins (MCs), which are potentially lethal for liver cells. Microcystins are cyclic peptides that consist of seven amino acids. They contain a unique β-amino acid Adda, and two of the seven amino acids vary between different variants of microcystin. Due to the variation in these two amino acids, more than a hundred different variants of microcystin are known. The most common variant is microcystin-LR (MC-LR), where the variable amino acids are leucine and arginine. It is also the most potent of all microcystins [1].