The New Generation of Biosurfactants: Potential Applications of Rhamnolipids

da | Mag 30, 2018 | Biologia Molecolare

Surfactants have a broad range of industrial uses, e.g. production and processing of food, agrochemicals, pharmaceuticals, petroleum, mineral ores, personal care and laundry products, fuel additives, lubricants and many others [1, 2]. This is why it would be very interesting to find alternative biological ways of production instead of depending on the current oil industry. Oil is one of the most used commodity in the world and, at the same time, one of the most expensive and polluting raw materials. Given its negative impact on the environment, identifying alternative raw materials is becoming more and more essential. In this field, biotechnological industries show their ability to provide sustainable products, since biotech industry typically exploits bacteria, yeasts and fungi’s metabolisms to obtain commercially useful molecules. Bio-based sustainable products are manufactured by low cost industrial plants, which cause less environmental impact and avail of cheaper raw materials. This way of production has the useful advantage of biomass valorisation, which allows reducing industrial waste. Moreover, it generally involves the use of more environmentally friendly mechanisms with weak conditions (temperature, pH, and pressure), cheaper raw materials and minor cost of storage and removal. The green industries already use lots of microbial metabolisms to produce alcoholic drinks, medical drugs, pharmaceuticals, biosensors, remediation and purification processes. Surfactants – biological tensioactive agents – are amphiphilic molecules that accumulate at the interface between polar and non-polar solvents. Hence, these molecules possess the ability to reduce interfacial and surface tension, leading to enhanced mixing and interaction between dissimilar phases [3]. In a recent article Mendonça Bahia and collaborators studied the production of rhamnolipids in S. Cerevisiae [4].   Rhamnolipids are glycolipid biosurfactants naturally produced by Pseudomonas aeruginosa and are composed of one or two rhamnose molecules linked to beta-hydroxy fatty acid chains [4]. These compounds are green alternatives to petrochemical surfactants, but large-scale production has not been implemented until today, due to the pathogenicity of the bacteria responsible for their synthetization. Through this study, the authors explain a new system to make the use of large-scale rhamnolipids possible.

Figure 1. Characterizing phases of the transfection process

This technique is based on the functional expression in Saccharomyces cerevisiae of 6 genes from Pseudomonas aeruginosa coding for 6 enzymes involved in mono-rhamnolipids synthesis, the disruption of SUC2 invertase gene and the expression of Sucrose phosphorilase gene Gft instead of it. In particular, episomal plasmids were used as backbone and manipulated via conventional restriction enzyme-mediated cloning methods in order to transfer Sucrose phosphorylase gene Gft from Pelomonas saccharophila to Saccharomyces cerevisiae. Before transfecting the genes, it is necessary to prepare the host (Saccharomyces cerevisiae).  The goal was to achieve the deletion of SUC2 gene coding for an invertase and the functional expression of Gft gene coding for a sucrose phosphorylase. The host internalizes intact sucrose molecules through active transmembrane transport [5] and converts them into gluc-1-P and fructose using an inorganic phosphate (Pi). This strategy has the advantage of reducing the energy supply of the cell by sparing an ATP molecule, but also to lead sucrose phosphorilase to give glucose 1-phosphate, substrate for the first enzyme of the mono-RL’s pathway, RmlA. The selective marker KanMX was used to select strains with deleted SUC2 and Gft expressed obtaining strains CEN-113 and CEN-102. Next the authors   inserted the genes coding for RLs inside specific episomal plasmids. The latter contain primers (TEF, ADH, GPD, CYC1), selection markers (Leu, Ura, Trp) and antimicrobial resistance genes (AmpR). These plasmids have been inserted through electroporation into the storage intermediate E. Coli DH5-a. Electroporation is an electric technique that generates transient holes on cells’ membranes. Bacteria that survive in selective fields have inside complete plasmids, as confirmed by verification PCR and restriction analysis. Correctly formed and selected plasmids are then transfected in CEN.PK113-6B and CEN.PK102-3A strains of Saccharomyces cerevisiae by lithium-acetate protocol. Several tests have been conducted in order to confirm the production of requested molecules, namely dTDP-L-rhamnose and mono-rhamnolipids. In particular, the detection of dTDP-L-Rhamnose involved the separation process by UPLC (Ultra performance liquid chromatography) and the revelation through an electrospray ionization source, coupled with a mass spectrometer. For mono-rhamnolipids detection, fluorescence labelling tests were performed to identify lipid droplets in which the cell stores the product.

Figure 2. Test conduced in order to confirm the production of dTDP-L-Rhamnose (a) and mono-RLs (b); 3D distribution of lipid droplets in cells producing mono-RLs (c)

This strategy allows creating a pathway to produce commercially interesting molecules, such as dTDP-L-rhamnose and mono-rhamnolipids. In particular, the former can be used as a starting molecule to produce cosmetics, skin creams and anti-aging face creams, whereas mono-rhamnolipids are widely used in several processes, such as:

  • bioremediation and Enhanced Oil Recovery (EOR) in order to remove pollutants and heavy metals [6];
  • in agriculture rhamnolipids are employed as biopesticides against vegetal pathogens and pest;
  • they can be used as antibacterial, antifungal or antiviral agents [7];
  • they are useful as immune response modulators, anti-blocking agents, probiotics in immunotherapy or against infections [8].

References

  1. Kamaljeet K. Sekhon Randhawa1 and Pattanathu K. S. M. Rahman: Rhamnolipid biosurfactants—past, present, and future scenario of global market; Frontiers in microbiology, 2014.
  2. Schramm, L. L., Stasiuk, E. N. & Marangoni, D. G. Surfactants and their applications. Annu. Rep. Prog. Chem. 99, 3–48, 2003.
  3. Soberón-Chávez, G. & Maier, R.M. In Biosurfactants: From Genes to Applications (ed Soberón-Chaves, G.) 1–11. Springer, 2011.
  4. F. Mendonça Bahia, G. Carneiro de Almeida et al., Rhamnolipids production from sucrose by engineered Saccharomyces cerevisiae. Scientific Reports, 2018.
  5. Dário, M. G., Effect of changing sucrose uptake in Saccharomcyes cerevisiae metabolism. PhD thesis, University of São Paulo, 2012.
  6. Amar Jyoti Das, Rajesh Kumar: Utilization of Agro-industrial Waste for Biosurfactant Production under Sub-merged Fermentation and its Application in Oil Recovery from Sand Matrix; Bioresource technology, 2018.
  7. Jin-Hyung Lee, Yong-Guy Kim & Jintae Lee (2018): Thermostable xylanase inhibits and disassembles Pseudomonas aeruginosa biofilms; Biofouling, 2018
  8. Paul E. Massa, Aida Paniccia, Ana Monegal, Ario de Marco and Maria Rescigno: Salmonella engineered to express CD20-targeting antibodies and a drug-converting enzyme can eradicate human lymphomas; Blood, 2013

Elia Cioffi

Master Industrial Biotechnology student

Francesca Stella

Master Industrial Biotechnology student