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Our Solution

What is Curdlan?

To start with, let's talk about glucans. Glucan molecules are polysaccharides of D-glucose monomers linked by glycosidic bonds. One of them is called Curdlan, a (1→3)-β-D-glucan. This molecule is a linear homopolymer which may have as many as 12,000 glucose units. It is naturally produced by Agrobacterium sp. ATCC31749 which uses it as an Extracellular PolySaccharides (EPS) in it's capsule (REF). The capsule formation is correlated with cell aggregation (floc formation) and it is suggested that the capsule and floc formation together function as protective structures in cases of Nitrogen-starvation of the post-stationary phase. The protective effects are due to the fact that Curdlan forms a capsule that completely surrounds the outer cell surface of bacteria.

Figure 4: Representation of the Curdlan molecule showing the beta 1,3 links between the glucose units

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. (REF) As a matter of fact, this β1,3 glucan can stimulate the plant's immune system. More precisely, applied to grapevine plants, sulfated Curdlan induces the accumulation of phytoalexins (organic antimicrobial substances) and the expression of a set of Pathogenesis-Related proteins .

However, non-sulfated Curdlan doesn't trigger the hypersensitive response characterized by the rapid death of cells in the local region surrounding an infection, avoiding a complete contamination of the plant. This response has been studied in Arabidopsis thaliana through a mutant gene: pmr4 . This mutant is resistant to mildew infections but is unable to induce Pathogenesis-Related proteins expression .

Also, activation of a Pathogenesis-Related protein called PR1 in grapevine is regulated by the salicylic acid signaling pathway . The lack of PR1 expression in non-sulfated Curdlan-treated grapevine could be explained by a negative feedback of glucan. This is demonstrated by the study of a double mutant of pmr4 which restore the susceptibility to mildew. It suggests that linear β-1,3 glucan negatively regulates the salicylic acid pathway. So, sulfation of the glucan would counteract the negative feedback effect.

To conclude, activation of the innate immune system before the invasion of pathogens is a way to improve the resistance of plant against infection and to reduce the use of chemicals products.

« Ok i understand the value of producing Curdlan. How will you proceed? »

« This is a good question i'll answer. »

Figure 5: Schematic representation of the beta 1,3 glucan synthetic pathway (REF)

In Agrobacterium , three genes (crdA, crdS and crdC) are required for Curdlan production. The putative operon crdASC contains crdS, encoding β-(1,3)-glucan synthase catalytic subunit, flanked by two additional genes : crdA and crdC. The first assists translocation of the nascent polymer across the cytoplasmic membrane and the second assists the passage of the nascent polymer across the periplasm. However, all Curdlan biosynthesis is dependent of nitrogen starvation and various parameters. We want to simplify all of this.

Finally we would like to sulfate our Curdlan molecules chemically in order to enhance it's effects on the activation of the plant's imune system. It has been shown that sulfated curdlan is much more effective. (ref)

To sum it up, we would like to produce Curdlan in Escherichia coli or Saccharomyces cerevisiae and then sulfate it to use it as a preventive treatment for the vine against the mildew infection and continue to produce good wine and make everyone happy.

Using Bacteria:
Escherichia coli

Firstly, we decided to produce curdlan with Escherichia coli , because Agrobacterium and it are Gram negative bacteria and have a lot of membrane similarity. Moreover Escherichia coli is a simple pretty good bacteria, it can be grown and cultured easily and inexpensively in a laboratory setting unlike Agrobacterium .

Since E. coli naturally produces UDP Glucose, adding the beta 1,3 glucan synthase would allow curdlan production. We therefore inserted the three genes which code for the glucan synthase in Agrobacterium (crdASC) into E. coli placing the gene under an easier control than N-starvation by using a constitutive promoter.

Figure 6: strains of agrobacterium producing curdlan (green) observed with a microscope using flurescence (???) SOURCE: Goemar

Using Yeast:
Saccharomyces cerevisiae

Yeast cell walls are made up of various layers which are represented in the following diagram. First there is a layer of chitin, then a layer of beta glucans and finally a mixed layer of proteins and mannan. Commonly, the yeast cell wall is made of 5-10% of beta 1,6 glucans and 50-55% of beta 1,3 glucans and beta 1,6 glucans.

Figure 5: Schematic representation of the yeast's cell wall

Since the layer of mannan and proteins as well as chitin is insoluble in alkali solutions, beta glucans are easily separated from the rest of the yeast cell wall. Therefore, the only alkali soluble components are a mix of beta 1,6 and beta 1,3 glucans. (aimanianda et al 2009) In order to separate the two we plan on using beta 1,6 glucanases in order to obtain a solution of beta 1,3 glucans and therefore our curdlan molecule.

We therefore decided to over-express the curdlan metabolic pathway by inserting into yeast an inducible promoter (gal1) for the glucan synthase gene (Fks1) hoping that this would allow the cell to produce curdlan in greater quantities. This would allow us to compare our curdlan production in E. coli to the natural production in an organism and the enhanced production through the addition of a promoter.
To do this , we will extract the FKS1 gene from yeast DNA and amplify it by PCR. We will then insert FKS1 in one hand, into plasmid pYES2 to integrate the modified plasmid in Saccharomyces cerevisiae and boost production of curdlan. On the other hand, we will integrate the FKS1 gene into the iGEM plasmid to get our famous BioBrick that we'll send to Boston. However, site-directed mutagenesis may be necessary when integrating the gene into the plasmid because there are restriction sites ( EX and SP) unwanted within the gene.

« It's very clear now. It looks like cool. But, i have a last question: why did you choose this subject? »

« Because, as explained on the problem page, downy mildew is a real problem for the Aquitaine region. This is why we wanted to bring an ecological solution to this problem. And also, we are SWAG (Secretly We Adore Glucan) »

Other useful properties of Curdlan

Curdlan, the linear (1→3)-β-glucan from Agrobacterium, has unique rheo-logical and thermal gelling properties. It is neutral and insoluble in water and if it is heated in an aqueous suspension, it adopts simple helical conformations (55-80°C) or a triple helical connected conformation (80-130°C). [1] It then acts as a gelling agent and form two types of gels (low-set gel or high-set gel which has been documented by Zhang et al [3]). Apart from being tasteless, colourless and odourless, its advantages are that, in contrast to cold-set gels (e.g. gelatin, gellan, carrageenan) and heat-set gels (e.g. konjac glucomannan, methylcellulose), the heating process alone produces different forms of curdlan gel with different textural qualities, physical stabilities and water-holding capacities. Moreover, gels of differing strength are formed depending on the heating temperature, time of heat-treatment and curdlan concentration. This property is widely used in the food industry as a food additive ( E424 ). Curdlan gels have been used to develop new food products (e.g. freezable tofu noodles) and calorie-reduced food, since there are no digestive enzymes for curdlan in the upper alimentary tract, and curdlan can be used as a fat substitute [4}. The safety of curdlan has been assessed in animal studies and in vitro tests [4,5] and it is approved for food use in Korea, Taiwan and Japan as an inert dietary fibre and is registered in the United States as a food additive [7]

Curdlan has also found applications in non-food sectors. Its water-holding capacity is applied in the formulation of “superworkable” concrete, where its enhanced fluidity prevents cement and small stones from segregating [8]. It has also been proposed as an organic binding agent for ceramics [9]. In addition to applications based on its physicochemical properties, e.g. in drug delivery through sustained and diffusion-controlled release from curdlan gels (Kanke et al. 1995), curdlan (Janeway and Medzhitov 2002), other (1→3)-β-glucans and their derivatives have medical and pharmacological potential. They are members of a class of compounds known as biological response modifiers that enhance or restore normal immune defences. These effects are manifested through interactions with soluble or cell-bound (Tolllike) receptors of the innate immune system. Binding to these receptors activates signalling cascades which regulate specific genes concerned with the removal of foreign materials and micro-organisms in both invertebrate and vertebrates and further, through the induction of co-stimulatory molecules, and increased antigen presenting activity, helps to direct adaptive immune responses against antigens derived from the foreign source (Janeway and Medzhitov 2002).

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. All the other curdlan clinical applications in cancer, diabetes, hypertension, hypertriglyceridemia etc. are listed here.

Curdlan is also neutral and insoluble in water. If it is heated in aqueous suspension , it adopts simple helical conformations ( 55-80 ° C) or triple helical connected ( 80-130 ° C). It then acts as a gelling and form two types of gels (low-set gel or high-set gel) . This property is widely used in the food industry since Indeed, curdlan is a food additive ( E424 ).

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