Project Inspiration

If we do not act now, predictions suggest that the increase in global mean temperature will increase from 1 to 4 degrees Celsius. This will lead to devastating effects on wildlife, food production and much more (Nature, 2020). Personall we have seen the rapidly increasing effects of climate change in many areas of Manchester over the past couple of years. Only last year, the Didsbury flood plain was overwhelmed by climate-fuelled rainstorms (which you can read more about on the Government blog). This displaced many people in Manchester and personally affected some members of our team. the floods led to algal blooms in nearby parks and nature reserves, damaging the fragile ecosystems and serving as a dire warning of the effects of our society on the planet. We believe now is the right time to assist in making biofuels a global standard, in order to mitigate the effects of fossil fuels on our biosphere, a key contributor to climate change. As a result of this, we set out to research strategies against climate change. During this research, we came across a paper written by Ao Xia et al. (2016). This paper outlined how strides in microalgae cultivation have allowed for safer and cleaner degradation of digestate (a product of wastewater treatment). We wondered if we could couple lipid (biofuel precursor) production and wastewater treatment, and therefore work on a project which aims to mitigate both floodings caused by climate change and the eutrophication of the streams, resulting from flood water running off treatment plants and farms. Following agreement from our advisors and supervisors of the feasibility of a co-cultivation of E. coli to increase the efficiency of microalgal lipid production as an IGEM project, we decided to pursue this as our final project.

Wastewater Treatment

Water use is essential in our everyday lives; however, domestic, commercial, and industrial use of clean water leads to wastewater production. Wastewater typically contains human waste, food scraps, oils, soaps, and toxic chemicals. The correct treatment of wastewater before it is released into the environment is vital to prevent damaging effects on our ecosystems, wildlife, and human health. A common reason for inadequate wastewater treatment is due to it being a highly costly measure, preventing it from being economically viable for developing countries. It is estimated that approximately 44% of global household water is not safely treated (Unwater, 2021).

We aim to engineer a bacterium that can assist microalgae in using wastewater as a growth medium. Our engineered bacteria will do this by taking up phosphate from the wastewater, which will induce phosphate starvation within the microalgae, consequently increasing the efficiency of lipid production. The removal of phosphate is a necessary step in wastewater treatment and this will be, at least partially, taken care of by our project.

Biofuel production using microalgae

Biofuels can be a great source of renewable energy. Renewable energy sources are vital to help tackle the ever-growing climate crisis and to end our reliance on fossil fuels. A challenge with the widespread implementation of biofuels into society is the high production costs. It is estimated that the production cost of biodiesel is approximately 60% higher than petrol (IEA, 2017) - making this an unattractive way forward for countries. A common source of biofuels is from agricultural crops, such as sugar cane, and this raises concerns over food scarcity. Using microalgae to make biofuels is one of the best alternatives to traditional methods of biofuel production which consists of using agricultural crops such as sugarcane. This is due to the microalgae’s relatively fast ability to grow, in addition to its high productivity (Khan, Shin and Kim, 2018). We believe biofuel production from microalgae, enhanced using synthetic biology, can help reduce production costs and increase productivity, to help bring biofuels to widespread use. Our project would involve increasing the efficiency of biofuel production from microalgae, therefore would help bring down production costs. This is because our engineered bacteria would supply the microalgae with growth hormones and induce phosphate starvation - which would help increase the microalgal growth and subsequent lipid accumulation.

Auxin biosynthesis

In algae, the role of auxin is mainly related to the detoxification of reactive oxygen species (ROS). Auxin can stimulate the antioxidants directly and reduce the levels of peroxide and hydrogen peroxide (Wang et al., 2021). It is because auxin is also associated with cell stress, as during stress, the production of lipids is linked to the production of ROS, which reduces the growth, development, and metabolism of the microalgae (Guo et al., 2019). Adding exogenous phytohormones, such as indole-3-acetic acid (IAA), can prevent the production of ROS by activating antioxidants, such as GSH which catalyzes the conjugation of the reduced form of glutathione (GSH) to xenobiotic substrates for detoxification (Douglas, 2006), and thus can limit cell damage induced by abiotic stress (Guo et al., 2019). Therefore, auxins are used as growth hormones to ensure the enhancement of the growth of the microalgal population. Within the auxin class, IAA is the most common naturally occurring plant hormone and is the best one characterized (Guo et al., 2019). One of the dominating substances from the auxins family that can be found in microalgae is IAA (Han et al., 2018). We are going to use Scenedesmus sp. We are going to use Scenedesmus sp. LX1 as it is a species of interest towards biofuel production (Lin et al., 2020), ensuring the coupling of biofuel production and wastewater treatment.Various concentrations of IAA are identified in microalgae extracts and supernatants, inducing both stimulatory and inhibitory effects on the growth and also metabolism of the microalgae (Han et al., 2018). Growth and increase in biomass and biosynthesis of high-value biomolecules (such as fatty acids) are facilitated by low concentrations of auxin, but higher concentrations of auxin may inhibit cell growth (300 µM for the Desmodesmus species, which are part of the class of Chlorophyceae to which our strain of microalgae belongs(Lin et al., 2020).

To fulfill our goals, we would like to engineer a bacterium to produce IAA whilst being co-cultivated with microalgae. Since E.coli does not naturally make IAA, genes ARO8, KDC, and AldH were transformed into the cells to allow E.coli producing auxin regulated by arabinose (Guo et al., 2019).

Phosphate uptake

Nutrient starvation, specifically phosphate and nitrate starvation within microalgae has been known to lead to enhanced lipid accumulation (Roy et al., 2021). The absence of phosphate in the media leads to an increase in lipid accumulation (Yewalkar-Kulkarni et al., 2016), therefore we consider the removal of phosphate from the medium as a good strategy to induce lipid accumulation in microalgae after optimal growth.

We have decided to utilize the Phosphate (Pho) regulon in our engineered bacteria to help produce a phosphate-starved environment for the microalgae, to assist in lipid accumulation. The Pho regulon is a global regulatory mechanism responsible for optimal inorganic phosphate (Pi) uptake in prokaryotic organisms (Santos-Beneit, 2015). This mechanism is composed of three elements: extracellular enzymes conducting the capture of Pi in the medium, Pi-specific transporters, and a set of enzymes responsible for the accumulation of the attained Pi (Lamarche et al., 2008). This complex mechanism is controlled by a two-component regulatory mechanism composed of a cytoplasmic transcriptional regulator and a histidine kinase sensor kinase; in E.coli , these enzymes are named PhoB and PhoR respectively (Marzan et al., 2011). In Pi-deprived environments, the response regulator is phosphorylated by the sensor kinase. Due to this phosphorylation, the response regulator oligomerizes and gains the capacity to activate and repress the expression of a set of genes involved in the increased uptake and accumulation of phosphate (Millan-Oropeza, 2020). These specific gene sequences are known as the PHO box, and it orchestrates the phosphate-starvation stress response. Once the bacteria is subjected to a Pi-replete medium, PhoR begins to act as a phosphatase, dephosphorylation and deactivating the PhoB transcription factors, which greatly reduces the uptake and accumulation of Pi (Lamarche et al., 2008).

In E. coli, the communication between PhoR and PhoB requires five additional proteins. The first four compose the Pi-specific transporter known as Pst(SCAB), and the other one is the metal-binding protein PhoU (Santos-Beneit, 2015). Correct expression and performance of these proteins ensures phosphate homeostasis in bacteria, with mutations leading to abnormal Pi-uptake rates. Our design utilizes this principle in order to maximize phosphate accumulation, turning PhoR into a constitutive kinase by disruption of the Pi-sensing pathway. We seek to achieve this through the silencing of the PhoU-encoding gene.

Previous literature presents how the silencing of the PhoU gene leads to an increased uptake and accumulation of Pi. De Almeida et al. (2015) presented a study on the effects of inactivating phoU in Pseudomonas aeruginosa, concluding that the mutation greatly enhances Pi removal (depleting 93% of the initial mM Pi solution) and its accumulation as the insoluble polymer polyphosphate (with the mutant containing 11 times more polyphosphate than its WT analog). Morohoshi et al. (2002) performed a similar study with promising results in E. coli, with Polyphosphate accumulation in the GM E. coli being at least 100-fold higher when compared with its wild-type analog and its Pi-removal capacity being twofold higher. It is for this reason that we seeked to target the PhoU gene, silencing it with a dCas9 mechanism to avoid any form of permanent activation in case of leakiness.

Light switch

Our project has two aims: the first one being to increase microalgal growth through the secretion of auxin from our engineered bacteria and the second aim being to increase lipid accumulation via phosphate starvation of microalgae. Unfortunately, high levels of auxins are shown to disrupt lipid accumulation within the microalgae (Dao et al., 2018). For this reason, we needed a control mechanism to allow us to switch off auxin production and subsequently switch on phosphate accumulation within our engineered bacterium. Our chosen control mechanism is light-activated switch, as this methodology allows for a smooth transition from monochromatic photoreactors to open raceway ponds, enabling a close regulation of the system while adapting to conventional industrial biodiesel-substrate production processes (Rafa et al., 2021).

Cre recombinase has emerged as a useful tool for controlling gene expression within a variety of model species(Feil et al., 2009). It allows for precise spatio-temporal control of gene expression within mammalian hosts, and has more recently seen use within bacterial systems to alter metabolic routines (Eroshenko et al., 2013). Within this system, a restriction enzyme known as Cre recombinase acts to recognize specific sequences within the DNA called Lox sites, and can either invert or excise the sequence between the Lox sites, depending on the orientation of the Lox sites. Different types of Lox site have been developed for this purpose, such as LoxP and Lox551 (Suzuki et al., 2011).

Researchers have been able to generate powerful Cre-Lox recombination systems, which make use of multiple types of Lox sites where recombination occurs only specific to each type (Feil et al., 2009). Also, unique combinations of these sites allow genes to be manipulated in various ways. One combination of these sites is the FLEx cassette, which allows the expression of two genes to be swapped, stopping the expression of one while starting the expression of another. We decided to couple this powerful system with blue light-inducible Cre recombinase. For our purposes, the first set of genes are the Auxin genes, and the next set of genes are dCas9 genes sequenced in the opposite orientation, making it inactive. Therefore, the genes involving the auxin production will be expressed first, and the dCas9 genes are kept inactive as long as the system is kept out of blue light. Under light activation at 460 nm, Cre recombinase protein within the bacteria undergoes conformational change and becomes active. Activated Cre will then recombinase the cassette leading to the excision of auxin genes and the inversion of dCas9 gene, putting it in a correct orientation. Transcribed dCas9 gene will then target and silence the PhoU gene within the bacterial chromosomal genome and induce lipid accumulation.