Cyanobacteria are oxygenic phototrophs. They only require water, carbon dioxide, inorganic compounds (like nitrogen, phosphorus) and light. Due to the changes in the environment associated with human intervention, cyanobacterial overpopulation is affecting people and ecosystems all over the world.
There are two main contributing factors. Increased levels of phosphorus are a major factor as it is an essential substance, which cyanobacteria need to gain form the environment. The concentration of phosphorus in the water directly correlates to the size of its cyanobacterial population. Cyanobacteria also have a substantial storage capacity for phosphorus. They can store enough to perform two to four cell divisions. Another issue is the slow spontaneous degradation of phosphorus in water, so the effects of the increase in its concentration are present for a long time. Because phosphorus is released from dead biomass and can be again reused by living cyanobacterial cells.
Second factor is temperature as cyanobacteria multiply faster at temperatures over 25 ℃. However, 15 ℃ is enough for them to peacefully exist. even some of them have remarkable ability to survive at extremely low or high temperatures. They can be found at hot springs, mountain streams or even at Arctic and Antarctic lakes. It just shows how cyanobacteria are a global problem [1; 2; 3].
So what issues does cyanobacterial overpopulation cause? Overpopulated cyanobacteria create so-called water blooms on the water surface. This can cause many issues for local animals, for example diving birds who are thus unable to hunt fishes and have to migrate to a new place in order to hunt food. Major producers of cyanobacterial blooms include Aphanizomenon, Cylindrospermopsis, Dolichospremum, Microcystis and Planktothrix, but the most abundant pest is Microcystis. In Czech Republic for example, Microcystis aeruginosa represents 75-93% of total cyanobacterial population mid-august[3;5].
Another problem is the odor associated with the decomposition of the accumulated biomass, which drives people away from natural waters. Cyanotoxins, their secondary metabolites, are however the main issue. They are not only harmful to aquatic life, but also to mammals. There is a great number of cyanotoxins with varying negative effects. Some of them are neurotoxins (anatoxins and saxitoxins), some are cytotoxic alkaloids (debromoaplysiatoxin), some are hepatotoxins (microcystins) or irritant toxins and much more.
Now, let's explore one of the most common cyanotoxin - microcystin.These cyclic peptides are mainly produced by Microcystis sp. and pose a major challenge during production of safe drinking water. Microcystins are soluble in water. They usually remain inside of the cyanobacterial cell and thus are mainly released after cell lysis. They are mostly unable to directly penetrate the phospholipid bi-layer, and must be transported through membrane transporters. Fortunately for us, there are not many reported examples of liver damage after short exposure in water infested by Microcystis sp., in this case the skin irritation is more occured. Long-term exposure could however cause liver damage and other issues. Mycrocysin induced toxicity can also cause a financial burden as it is quite deadly for fish [3; 5].
These are the reasons why we are focusing on degradation of cyanobacteria and microcystin.
Bacillus subtilis is a commonly used model organism for Gram-positive bacteria. Its rod-shaped cells are usually 4-10 μm long and their diameter varies between 0,25 and 1 μm. In an adverse environment, it is able to form endospores and thus survive until more favourable conditions occur. It can be found in the soil and in the gastrointestinal tract of some mammals, including humans. Bacillus subtilis is also widely used in biotechnologies and molecular biology. It is considered to be the Gram-positive equivalent of Escherichia coli, as there is a large amount of information and genetic tools available for this organism.
As Bacillus subtilis is known for its high protein secretion and the concept of CYANOTRAP is based on extracellular display of proteins, it is no surprise we have decided to use this particular bacterium. Yet another argument for choosing this bacterium for our project is that it has been already engineered to carry a synthetic protein scaffold - cellulosome .
Unfortunately, there is no laboratory that routinely works with Bacillus subtilis at Masaryk University. Therefore, we approached Dr. Libor Krásný and his colleagues from the Laboratory of Microbial Genetics and Gene Expression which operates under the Czech Academy of Science in Prague. Dr. Krásný provided us with the most commonly used laboratory strain of this bacterium - Bacillus subtilis 168. He and his colleague Dr. Hana Šanderová also gave us many useful tips on how to work with this bacterium. During our attempts to establish this organism at our university, we ran into some problems while learning to cultivate and transform Bacillus subtilis, which cost us a considerable amount of time to solve. Nevertheless, during the process we also learned a number of useful lessons. To share our experience with any future iGEM teams that intend to work with B. subtilis, we created a short handbook that contains some basic tips and useful information on handling this bacterial species. You can find the details here.
Cellulosome is a multi-enzyme protein complex that is found on the cell surface of some cellulolytic bacteria. It was first described in 1983 [7;8] and since then it has been thoroughly studied and used in many projects focused on biotechnological processing of polysaccharides found in the plant cell wall. Current knowledge on cellulosomes has been reviewed by Artzi et al. in 2017 .
Cellulosome consists of a non-catalytic protein scaffold termed scaffoldin which contains several domains called cohesins and usually also a cellulose binding module (CBM). Cohesins non-covalently interact with their counterparts - dockerins. This then enables cellulases to interact with cohesins via their dockerin domain and thus attach to scaffoldin. The cellulosome is usually attached to the cell surface and thus so are the cellulolytic enzymes via the interaction between cohesins and docerins. This is a very efficient strategy as the relative closeness between the enzymes with complementary activity enhances the activity of each enzyme and thanks to the presence of CBM the cellulosome can be tightly connected to its substrate .
The cohesin-dockerin pairs possess one feature that is very favourable for their use in synthetic biology. Their interaction is species-specific, which means that the connection will occur only between a cohesin and a dockerin that originated from the same species . This allows us to design a synthetic cellulosome in which the location and quantity of each enzyme is determined precisely.
In CYANOTRAP, we used the basic building blocks of cellulosomes - scaffoldins, cohesins, dockerins and CBMs. CBM can immobilize the cells of our chassis bacteria on a cellulose matrix, and the interaction between cohesins and dockerins is used to display two different sets of proteins on the cell surface. Our scaffoldin ScafL is connected to the B. subtilis surface by LysM and it contains a cohesin from Clostridium thermocellum, which binds to its respective dockerin that is connected to either lysozyme from chicken (Gallus gallus; LysGG) or Bacillus licheniformis (LysBL). ScafL also contains microvirin which functions similarly to CBM - it connects our synthetic cellulosome to its substrate - the cyanobacterial cell wall in our case. This should increase the efficiency of cyanobacterial lysis when compared to lysozyme secreted into the medium.
The purpose of our second protein complex with ScafD, is to degrade microcystin (specifically microcystin LR). ScafD is also connected to the cell wall of Bacillus subtilis and it consists of MlrA (the first enzyme of MC-LR degradation pathway) as well as two cohesins, originating from Bacteroides cellulosolvens and Acetivibrio cellulolyticus. Their respective dockerins are connected to MlrB and MlrC (in this order). Thanks to this system, the first three enzymes of MC-LR degradation pathway can be displayed on the cell surface in a very close proximity which should increase their efficiency.
CBM stands for Cellulose (or Carbohydrate) Binding Module. In nature it is commonly found as a part of cellulolytic multi-enzyme protein complex cellulosome. As CBM is able to bind cellulose, cellulolytic enzymes are brought closer to their substrate and the effectivity of cellulose degradation is increased .
In CYANOTRAP, we use CBM for a different purpose. We decided to anchor this protein to the cell wall of Bacillus subtilis via the LysM domain ( and use it to immobilize the cells on a cellulose matrix. This has already been successfully performed with Escherichia coli by Wang et al. in 2001 . In this study, it has been observed that less than 10% of immobilized cells were disconnected from the cellulose fibers after 24h of washing, which demonstrates the exceptional strength and stability of CBM-cellulose interaction.
The CBM used in our project originates from the Clostridium thermocellum scaffoldin calledCipA. It has been used in many synthetic cellulosomes including a collection of cellulosomes designed by the iGEM team NEAU-China in 2015 .
The LysM domain is a small globular module made up of approximately 40 amino acid residues that got its name after the ever-present lysin motif. It can be found in a wide range of prokaryotic and eukaryotic organisms including Bacillus subtilis and it is one of the proteins involved in binding peptidoglycans or chitin .
When designing our project, we took inspiration from the article written by You and colleagues in 2012 . They used the three LysM domains from the Bacillus subtilis cell-wall hydrolase LytE (GenBank accession number AAC25975.1 ; Margot et al., 1998 ) to connect a synthetic minicellulosome to the cell surface of the same bacterial species. In their work, they describe a simple method of production, purification and analysis of LysM-bound proteins that we chose to adopt for our project.
In CYANOTRAP, we use these three LysM domains as the primary anchoring module for all three fusion proteins that form our system. In all cases, the three LysM domains form C-terminus of the fusion proteins and are connected by a linker to other protein domains. In one case, LysM is connected to a cellulose binding module (CBM). This fusion protein allows us to immobilise B. subtilis cells on a cellulose matrix. In the other two cases, the LysM is connected to two different synthetic scaffoldins which carry cohesins and thus allow dockerin-tagged enzymes to bind to them.
We decided to use this anchoring system as it has already been used for a similar purpose in the same bacterial host  and thus we had some guidance when planning out our experiments. Another argument in favor of us using the LysM module was that in this study a solution of rather high ionic strength (5 M LiCl) was needed to disassociate LysM from the cell surface. This points to a stable bond between the LysM domain and the peptidoglycans of the cell wall, further ensuring that our proteins will be strongly attached to the surface of B. subtilis.
Microvirin is a mannan-binding lectin protein composed of 108 amino acids with a molecular weight of 12.16 kDa . In nature, it was found to be produced by cyanobacterium Microcystis aeruginosa PCC7806 where it binds to the lipopolysaccharides (LPS) in the cell wall of cyanobacteria. This bond is mediated through its mannan-binding domain. Microvirin is used by Microcystis aeruginosa for cell-cell recognition and attachment and its production seems to relate with the levels of microcystin produced by the cyanobacterium . Carbohydrate microarrays were used to showcase that Microvirin interacts with high-mannose structures containing alpha(1-->2) linked mannose residues with high specificity .
We first considered using microvirin when reading about a project done by iGEM team Peking 2014. To improve the ability of their modified E. coli to lyse cyanobacteria, they used microvirin, displayed on the cell's surface via truncated Ice Nucleation Protein , to bring cyanobacteria closer to the enzyme lysozyme. They successfully designed their E. coli to attach itself to Microcystis aeruginosa which was proven by their binding assay using fluorescent signals from GFP produced by their E. coli and chlorophyll A in Microcystis aeruginosa .
In CYANOTRAP we also take advantage of the ability of microvirin to bind Microcystis aeruginosa in order to bring them closer to lysozyme C, displayed on the same scaffoldin. We believe that spatial proximity as well as prolonged time in the presence of lysozyme will improve lyzing rates and make our device more effective.
Lysozyme is an enzyme which hydrolyses the ß-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine. This leads to the disruption of the bacterial cell wall, which allows it to act as an antimicrobial agent. There are several types of lysozyme, which differ in the primary amino acid sequence and in molecular weight. These can each target a different range of microorganisms .
The purpose of lysozyme in our project is to lyse the cyanobacterial cell wall and thus enable the release of toxins from the cells. To increase its efficiency, the lysozyme is expressed extracellularly and immobilized on the scaffoldin in close proximity to microvirin, which is able to attach to the cyanobacterial cell wall. The immobilization is enabled by fusing the lysozyme with dockerin, which can bind to the cohesins of our scaffoldin complex.
Gram-positive bacteria, including B. subtilis, do not possess the ability to protect their cell walls by producing lysozyme inhibitors into their periplasm. To make sure our bacteria won't be negatively affected by lysozyme production, we decided to use bacterial lysozyme from Bacillus licheniformis as it has been previously produced in B. subtilis without significantly affecting its vitality by Zhang and colleagues in 2014 . Lysozyme from B. licheniformis consists of 317 amino acids and possesses antimicrobial activity against various strains, including gram-negative bacteria. Its activity against cyanobacteria, however, has not yet been tested .
Lysozyme C on the other hand, is the most extensively studied lysozyme and its ability to lyse Microcystis aeruginosa was described by Peking iGEM team . Lysozyme C is present in many vertebrates and insects and with its length of 147 amino acids it is smaller than the previously described bacterial lysozyme. In our B. subtilis we would like to be able to produce both lysozyme C from chicken (Uniprot number: P00698; ) and lysozyme originating from B. licheniformis (Uniprot number: Q65KM7; ) so we can compare the their efficiency in destroying M. aeruginosa. We also plan to compare the resistance of B. subtilis to these enzymes.
Bacterial degrading cyanotoxins are quite common in nature. The metabolic pathway responsible for microcystin degradation consists of several Mlr enzymes , which work together to degrade microcystin into a less toxic compound.
In CYANOTRAP, we used Mlr enzymes fromSphingomonas sp. to create a complex gene cassette. We decided to produce 3 genes from this degradation pathway - MlrA, MlrB and MlrC as others are not necessary in our design. MlrD, for instance, mediates the transports of microcystin into bacterial cells. As our enzymes would be displayed on the surface of Bacillus subtilis, the microcystin transportation is not needed.
One of the more significant products of this degradation pathway is a non-proteinogenic amino acid Adda. This compound may contribute to the microcystin-induced toxicity. Adda is the last step, to which we are able to degradete microcystin by MlrA, MlrB and MlrC. Mechanism of Adda degradation is not well understood .
MlrA (GenBank AF411068; ) – Microcystin is a partially cyclic molecule and this enzyme catalyzes its linearization by breaking the bond between Adda and L-arginin. The product of this reaction is linear heptapeptide, which is more accessible for enzymes mlrB and mlrC. (Part:BBa_K1378001; )
MlrB (GenBank AF411069; ) – This enzyme breaks down linearized microcystin into smaller fragments by hydrolysing the bonds between alanine and other arbitrary amino acids .
MlrC (GenBank AF411070; ) – This tripeptidase is able to digest products of enzymatic activity of both MlrA and MlrB. Bonds between Adda and glutamic acid are hydrolysed. Adda, the non-proteinogenic amino acid, is one of the products of this reaction .
By producing these enzymes in our B. subtilis, we can reduce the microcystin-induced toxicity of cyanobacteria infested waters.
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