Difference between revisions of "Team:Bordeaux/Description"

Our Solution: Curdlan

What is Curdlan?

Glucan molecules are polysaccharides of D-glucose monomers linked by glycosidic bonds and 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 1: Representation of the Curdlan molecule showing the beta 1,3 links between the glucose units

More precisely, applied to grapevine plants, sulfated ß-glucans induce the accumulation of phytoalexins (organic antimicrobial substances) and the expression of a set of Pathogenesis-Related proteins . In plants, the fact that oligosaccharides must carry crucial sulfates for their biological function suggests that chemical sulfation of oligosaccharides can improve their biological properties. In recents studies, compared to Laminarin (ß-glucan), its sulfated derivative triggered an enhanced immunity against P. viticola in V. vinifera and a stronger immunity against TMV in Nicotiana tabacum. The results indicate that the chemical modification of an elicitor, such as sulfated derivative of ß-glucans, could improve its resistance-inducer efficiency. Moreover, if a ß-glucan is a substrate for plant (1→3)-β-glucanase, its sulfation clearly protects the molecule from its enzymatic degradation. Thus, a basal activity of plant glucanases can degrade ß-glucans and consequently releases short inactive ß-glucans; whereas sulfated derivatives still remain active molecules during a longer period. This might explain the higher resistance induced by ß-glucan sulfates compared to ß-glucans.

Furthermore, non-sulfated Curdlan doesn't trigger the response 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 (1→3)-β-glucan negatively regulates the salicylic acid pathway. So, sulfation of the glucan would counteract the negative feedback effect. [16]

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

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

« That is a good question, let me tell you how we approached the subject! »

Before starting the project, we took a few weeks to decide which host organism we would use and how they could be useful. To begin with, we looked at three different organisms: Escherichia coli , Bacillus subtilis and Saccharomyces cerevisiae and compared their β-glucan metabolic pathways. We rapidly eliminated Bacillus subtilis from our possible hosts due to its lack of enzymes involved in the metabolic pathway of (1→3)-β-glucans (Figure 5). However, we found that Saccharomyces cerevisiae naturally produces Curdlan in its cell wall, like Agrobacterium. Furthermore, Escherichia coli is only missing one enzyme (the β-Glucan synthase) to synthethize Curdlan. We therefore concluded that we could keep these two organisms: one where we would overexpress the (1→3)-β-glucan synthase using a constitutive promoter and one where we would insert the ability to create Curdlan by adding the enzyme that is needed throught the crdASC putative operon.

Literature Cited:

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

[2] M. McIntosh (2012) Recent advances in curdlan biosynthesis, biotechnological production, and applications. Appl Microbiol Biotechnol 93:525–531

[3] Adrien Gauthier (2014)The Sulfated Laminarin Triggers a Stress Transcriptome before Priming the SA- and ROS-Dependent Defenses during Grapevine’s Induced Resistance against Plasmopara viticola. Plos one 93:525–531

To sum it up, we would like to produce Curdlan in Escherichia coli or Saccharomyces cerevisiae and then sulfate it before spraying it on grapevine leaves as a preventive treatment against Downy mildew infections. That way vineyards can continue to produce good wine and make everyone happy.

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

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

Other useful properties of Curdlan

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 natural defenses. Useful properties include antitumor, anti-infective, anti-inflammatory, and anticoagulant activities [11]. Hydrolysed Curdlan with a degree of polymerisation <50 are not effective anti-tumor 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, optimization 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

[16]Menard R, Alban S, (2004) β-1,3 Glucan Sulfate, but not β-1,3 Glucan, Induces the Salicylic Acid Signaling Pathway in Tobacco and Arabidopsis

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