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Our Solution

What is Curdlan?

This year, the team is working on an environmental project, trying to remedy problems associated with the use of copper sulfate by bringing new solutions thanks to synthetic biology. To do that, all the work will be done using non-pathogenic micro-organisms which can easily be grown in our lab: Escherichia coli and Saccharomyces cerevisiae. Therefore our research tries to uncover ways to increase the production of this natural product that may decrease the cost in its industrial production for example.

Our project this year is to produce curdlan, a beta 1,3 glucan. Some (1→3)-β-D-glucans are encountered in some important soil-dwelling, plant-associated or human pathogenic bacteria. (CURDLAN + BACTERIA + IMMUNE S) One of the (1→3)-β-D-glucans is call Curdlan, it is produced as a capsule on Agrobacterium sp. ATTC 31749. the capsule formation is correlated with cell aggregation (floc formation). Capsule formation occurs when the N source in minimal medium is depleted, suggesting that the capsule and floc formation together function as protective structures in nutritional stress. Curdlan is typical of a secondary metabolite in that its biosynthesis occurs in the post-stationary growth phase during conditions of N-starvation. The optimisation of curdlan yield thus depends on the formation of a cell mass in the pre-stationary phase and on the biosynthetic capability of cells in the post-stationary phase. and is used as an Extracellular PolySaccharides (EPS).

Curdlan is a linear homopolymer of the monosaccharide D-glucose molecules and may have as many as 12,000 glucose units. It is a neutral polymer produced by Alcaligenes faecalis var. myxogenes 10C3 strain. Subsequent research on curdlan production focused on selection of bacterial strains and optimization of the culture media. But we would like to produce Curdlan in another bacteria, E.coli, because we are SWAG (Secretly we adore Glucan) <3

Before starting the project, we took a few weeks to decide which host organism we would use and how they coule be useful. To begin with we looked at three different organisms: Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae. We rapidly eliminated bacillus subtilis from our possible hosts due to it's lack of enzymes involved in the metabolic pathway of beta 1,3 glucans. However, we found that yeast naturally produces curdlan and that E. coli is only missing one enzyme to synthethize curdlan. We therefore concluded that we could keep these two organisms: one where we would overexpress the beta 1,3 glucan prodution using a constititive promoter and one where we would insert the ability to create curdlan by adding the enzyme that is needed.

ENZYMES + METABOLIC PATHWAY

Using Bacteria: E. coli

In bacteria, three genes are totally required for curdlan production. These have been identified in Agrobacterium sp. ATCC31749, which produces curdlan in large amounts. This putative operon contains crdS, encoding β-(1,3)-glucan synthase catalytic subunit, flanked by two additional genes : crdA and crdC. The first assists translocation of nascent polymer across cytoplasmic membrane and the second assists passage of nascent polymer across the periplasm. The protective effects are due to the fact that Curdlan forms a capsule that completely surrounds the outer cell surface of bacteria.

Now, we are going to put these three genes into Escherichia coli through the intermediary of plasmids and then do a bacteria culture for curdlan production during its stationary phase. Then we will collect only curdlan by a purification method. Finally, curdlan will be sulfated to be in its active form. This sulfation will be made chemically.

What is curdlan? How is it naturally synthethised? Why is it useful for bacteria? How are we going to produce it? Can we really call it eco-friendly? What organisms are we going to use? Why? What are the advantages and disadvantages of both organisms? How did we choose the genes? Promoters? How are we going to make the biobrick? What is our biobrick?

Effect on the plant

Curdlan is a linear beta-1,3-glucan. This molecule is a polymer composed of many units of glucose, a commonly used sugar. It is produced by bacteria such as Agrobacterium, even by yeast.

Now, why choose curdlan to replace copper sulfate?

In fact, this β1,3 glucan stimulates the plant's immune system. Activation of the innate immune system before the invasion of pathogens is a way to improve the resistance of plant against infection and reduce the use of chemicals products.

Blabla how work curdlan on plant immune system + Schema

Activation of signaling pathways in grapevine by sulfated glucans could be deduced from the recent analysis of the pmr4 Arabidopsis mutant. PMR4 is considered to be the main b(1,3) glucan synthase responsible for curdlan deposition upon biotic and abiotic stresses. This pmr4 mutant is resistant to powdery mildew infection and is unable to produce a pathogen-induced curdlan response. The susceptibility to powdery mildew was restored in the pmr4 nahg double mutant, suggesting that linear β-1,3 glucan negatively regulates the SA pathway. Because PR1 activation in grapevine is regulated by the SA signaling pathway, the lack of PR1 expression in curdlan-treated grapevine could be explained by a negative feedback of the glucan. Consequently, sulfation of the glucan would counteract the negative feedback effect.

Other useful properties of Curdlan

Curdlan belongs to the class of biological response modifiers that enhance or restore normal immune defenses, including antitumor, anti-infective, anti-inflammatory, and anticoagulant activities. CrdS is an integral inner membrane protein with seven transmembrane (TM) helices, one non-membrane-spanning amphipathic helix and a Nout–Cin disposition

Sulfation of Curdlan. Acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (HIV) is a severe disease that can destroy the body’s immune system, so the discovery of methods to prevent AIDS infection is of great importance.