The emerging threat of water scarcity
With a growing world population, the need for freshwater for agricultural use is greater than ever and it will only keep increasing. Today, farming alone accounts for almost 70% of the water withdrawals, putting major stress on the freshwater sources at our disposal.
CyaSalt is a novel synthetic biology method to solve the freshwater shortage. Our aim is to desalinate seawater using genetically modified phototrophic organisms to create freshwater for agricultural use. Our solution is to exploit the photosynthetic properties of phototrophic organisms and therefore use sunlight as the driving force in the desalination process.
By using halophilic organisms that not only survive, but thrive, in salty conditions, we ensure that this method does not require the cultivation of phototrophs to occur in a laboratory environment prior to the desalination.
We propose to genetically modify the organisms to express the inwardly directed chloride pump halorhodopsin originating from the archaea Natronomonas pharaonis. Halorhodopsin is a light-activated ion pump and when induced, it facilitates the transport of chloride ions to the inside of the cell, against the concentration gradient.
To facilitate the influx of sodium ions, we also express channelrhodopsin in the phototrophic organisms, which is a light-induced ion channel originating from Chlamydomonas reinhardtii. The previously created negative membrane potential then aids channelrhodopsin to transport sodium ions into the cells.
An illustration of our complete desalination and separation system, that is further described here can be seen in Figure 1.
Halorhodopsin, seen in Figure 2, is an inward directed chloride pump originating from the archaea Natronomonas pharaonis. It is located in the cell membranes of halobacteria and has the function of maintaining the chloride content of the cell. Halorhodopsin is a light-activated ion pump and when activated by light it facilitates the transport of chloride ions to the inside of the cell against the concentration gradient .
Channelrhodopsin, seen in Figure 3, is a nonspecific cation channel originating from the green algae Chlamydomonas reinhardtii. It is activated by light, like the ion pump halorhodopsin. When activated, a conformational change occurs allowing positively charged sodium ions to diffuse down their concentration gradients. The negative membrane potential created by the influx of chloride ions induced by Halorhodopsin is exploited so that channelrhodopsin can transport sodium ions into the cells [8, 9].
The activities of halorhodopsin and channelrhodopsin are alike in multiple ways. In addition to them both being activated by light, they require All-trans retinal to be bound to them to function. Thus, the access to All-trans retinal is a limiting factor for the chloride and sodium ion influx created by halorhodopsin and channelrhodopsin. All-trans-retinal is formed when beta-carotene is cleaved by beta-carotene 15,15'-dioxygenase. For this reason, our system includes a part that generates an excess expression of beta-carotene 15,15'-dioxygenase from Synechocystis sp. PCC 6803 in addition to halorhodopsin. This way, more beta-carotene can be cleaved so that the chloride ion influx is not constrained by a lack of All-trans retinal .
Large-conductance mechanosensitive channel, MscL, is an ion channel that is endogenous in Synechocystis sp. PCC 6803. It is a 145 amino acid long protein that acts as a sensor of cytoplasmic membrane tension and regulates cell volume under hypoosmotic stress. MscL is responsible for the flow of cations through the cytoplasmic membrane during stress. The purpose of MscL in our project is to regulate osmotic pressure in the cells to hinder cell disruption [11, 12].
We designed a fusion protein containing a carbohydrate binding domain (CBD) and an S-layer protein called slr1272, which is endogenous in Synechocystis sp. PCC 6803. CBDs are proteins derived from cellulose-degrading microorganisms. There is a variety of CBD families depending on their structure and function, CBDCipA, the type of CBD used in our project, belongs to the CBD family 3 and is derived from Clostridium Thermocellum. The reason the slr1272-CBD fusion protein was implemented is because CBD has a high affinity for cellulose. When filtered through a cellulose filter, bacteria expressing the fusion protein on their surface are expected to bind to the filter and get retained, while filtered water is able to flow through .