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Our Solution: Curdlan

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 [1]. 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 for the bacteria 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

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 bacteria that can be grown and cultured easily and inexpensively in a laboratory unlike Agrobacterium .

Since E. coli naturally produces UDP Glucose (metabolite number 3 on Figure 5), adding the β 1,3 glucan synthase would theoretically allow curdlan production. We therefore inserted the three genes which code for the β glucan synthase and metabolic transporters in Agrobacterium (crdASC) into E. coli . However, since our gene sequences for crdA, crdS, and crdC originally come from Agrobacterium we decided to optimize our gene codons for E. coli with IDT's codon optimization tool in order to make sure that our gene would correctly be expressed. Furthermore we decided to place the genes under an easier control than N-starvation by using a promoter which is activated in stationary phase. (osmY, BBa_J45992 characterized by MIT in 2006). This should theoretically allow maximum production in simple conditions.

All three genes were synthesized by IDT separately with the correct ends to allow integration in the iGEM plasmids and an easy creation of our biobricks. We planned on amplifying our fragments by PCR and creating different biobricks with different assemblies of our genes and plasmids in order to find the effect of each gene on Curdlan production and to verify the effectiveness of our promoter.

In order to have control over the different culture perameters we also decided to use M63 culture medium. This is the medium which is used for curdlan production in Agrobacterium and is also interesting since it is a minimal medium which allows us to easily vary parameters and optimize production. Furthermore, since Curdlan is a glucose derivative, being able to control the amount of sugar in the medium is interesting for production optimization. We plan on testing the following parameters by looking at Curdlan production:

❃ Different mediums (minimal and complete)

❃ Different stationary phase temperature: by reducing the temperature we might be able to reduce growth and obtain more Curdlan

❃ Glucose shot during stationary phase at different times

❃ Lowering pH in stationary phase to reduce cell division

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

Figure 7: Curdlan properties and applications www.link.springer.com

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, curdlan gels have medical and pharmacological potential, for example in drug delivery through sustained and diffusion-controlled release of the active ingredient. [10]

Furthermore, curdlan derivatives are members of a class of compounds known as biological response modifiers that enhance or restore normal immune defences. Useful properties include antitumor, anti-infective, anti-inflammatory, and anticoagulant activities [11] Hydrolysed curdlans with a degree of polymerisation <50 are not effective anti-tumour agents but the carboxymethyl ether and the sulphate and phosphate esters of curdlan, show an enhanced biological activity [12]. Furthermore, curdlan sulphate has anti-HIV activity [13] and inhibitory effects on the development of malarial parasites in vitro [14]. All the other curdlan clinical applications in cancer, diabetes, hypertension, hypertriglyceridemia etc. are listed here. Curdlan also has potential for exploitation as a new biomaterial based on the self-assembling ability of (1→3)-β-glucan-megalosaccharides (DP 30–45) to form single, hexagonal, lamellar nanocrystalline structures (∼8–9 nm thick) containing water of crystallization after heating to 90°C [15]. Manipulation of the conditions for self-assembly may allow the engineering of new materials.

However, more research is needed for the further development of these useful properties, in particular by reducing the cost of production. This may involve the use of cheaper C sources, optimisation of fermentation conditions, development of higher curdlan-yielding strains, or manipulation of curdlan synthesis and/or regulatory genes. [1]

Literature Cited:

[1] M. McIntosh (2005) Curdlan and other bacterial (1→3)-β-D-glucans mini review. Appl Microbiol Biotechnol 68: 163–173

[2] R. Zhang and K. J. Edgar (2014) Properties, Chemistry, and Applications of the Bioactive Polysaccharide Curdlan. American Chemical Society

[3] Zhang HB, Nishinari K, Williams MAK, Foster TJ, Norton IT (2002) A molecular description of the gelation mechanism of curdlan. Int J Biol Macromol 30:7–16

[4] Nishinari K, Zhang H (2000) Curdlan. In: Phillips GO, Williams PA (eds) Handbook of hydrocolloids. CRC, Boca Raton, pp 269–286

[5] Spicer EJF, Goldenthal EI, Ikeda T (1999) A toxicological assessment of curdlan. Food Chem Toxicol 37:455–479

[6] (2000) WHO food additives series. In: WHO (ed) 53rd Meeting of the joint FAO/WHO expert committee on food additives. JEFCA/WHO, Geneva

[7] 21 CFR 172. Food additives permitted for direct addition to food for human consumption: curdlan. Federal Register 61:65941

[8] (1996) Bioproducts: bio-concrete. BioIndustry 13:56–57

[9] Harada T (1992) The story of research into curdlan and the bacteria producing it. Trends Glycosci Glycotechnol 4:309–317

[10] Kanke M, Tanabe E,(1995) Application of curdlan to controlled drug delivery. III. Drug release from sustained release suppositories in vitro. BiolPharm Bull 18:1154–1158

[11] Janeway CA, Medzhitov R (2002) Innate immune recognition. Annu Rev Immunol 20:197–216

[12] Toida T, Chaidedgumjorn A, Linhardt RJ (2003) Structure and bio- activity of sulphated polysaccharides. Trends Glycosci Glycotechnol 15:29–46

[13] Jagodzinski PP, Wiaderkiewicz R (1994) Mechanism of the inhibitory effect of curdlan sulphate on HIV-1 infection in vitro. Virology 202:735–745

[14] Evans SG, Morrison D, Kaneko Y, Havlik I (1998) The effect of curdlan sulphate on development in vitro of Plasmodium falciparum. Trans R Soc Trop Med Hyg 92:87–89

[15]Chanzy H, Vuong R (1985) Ultrastructure and morphology of crystalline polysaccharides. In Atkins EDT (ed) Polysaccharides: topics in structure and morphology. Macmillan, London, pp 41–71

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