Team:BNDS China/Description

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Description Rhamnolipid, a widely used biosurfactant, has played an important role in agriculture, food processing, andoiling industry (Sekhon Randhawa & Rahman, 2014). To be more specific, the usages can be divided into three major aspects: as fertilizers to boost plants growth, to clean stains on cloth, or increase the effect of pesticides and fertilizers. (Du et al., 2017) Currently, there are many companies that produce a large amount of biosurfactant including rhamnolipid, and use them in those above-mentioned aspects in reality. Fig. 1 Spraying rhamnolipids as fertilizers in agricultural production Water flooding, an approach for Mechanical Oil Recovery, and other conventional oil extraction techniques result in a large variety of environmental issues and pollution such as soil acidification, draining leakage, air pollution, and so on. In addition, approximately 70% of the original oil in place is left in a reservoir at the end of water flooding(Yen, 1990), causing a waste of oil. Thereby, Microbial Enhanced Oil Recovery is considered to be a better substitute for MOR, thus receiving a great deal of research and investigation. Fig. 2 Oil field in Northeastern China(“ZFA Technologies - ZFA Technologies Inc.: Biosurfactant Enzyme to Enhance Oil Recovery”) One of the significant reasons accounting for the popularity of rhamnolipid is that it is ecologically friendly, since it is non-toxic and biodegradable. (Du et al., 2017) This property of rhamnolipid also enables it to be used in Microbial Enhanced Oil Recovery. Most oilfields in China are distributed in continental sedimentary basins, in which both the changes of reservoir physical properties and the distribution of sand bodies are complex, which increase the difficulty of oilfield development in China. And the oil recovery rate in China can only exploit 28% ~ 35% geological reserves under the current technology level. 65%～72% recoverable reserves remain under the ground. In addition, the national onshore oilfield has entered into high water cut stage with water cut as high as 82% in recent years in China. Oil-water separation and sewage recycle greatly increase the energy consumption, which makes oil production cost rise to almost no profit and force part of mature oilfields to reduce production. (“ZFA Technologies - ZFA Technologies Inc.: Biosurfactant Enzyme to Enhance Oil Recovery”) Nevertheless, Microbial enhanced oil recovery (MEOR) enables us to solve these challenges very easily. In the oil industry, rhamnolipid can be produced by microorganisms to maximize their role in synergistic oil recovery; its function of reducing the oil-water interfacial tension is still remarkable (decrease the tension to 10-3mN/m), which can highly improve oil displacement efficiency. At present, since the demand of oil in the world is still increasing despite regulations of oil extraction, most of the oiling companies are using P. aeruginosa to produce rhamnolipids for MEOR( citing Sinopec if you like but we need a citation here). Consequently, the objective for scientific research is to figure out the pathway allowing themixmized synthesis of rhamnolipid. Three major materials that can compound to form rhamnolipids: Glycolysis or Gluconeogenesis, ß-oxidation, and Fatty acid. The synthetic processes are shown below in fig. 2. Fig. 3 Rhamnolilpids biosynthetic pathway. rhaBDAC: dTDP-L-rhamnose synthesis gene cluster; rhlA: rhamnosyltransferase A; rhalB: rhamnosyltransferase B; rhlC: rhamnosyltransferase C; FASII: fatty acid de novo synthesis. (Du et al., 2017) Glucose-1-phosphate can be synthesized into dTDP-L-rhamnose with the assistance of RhaBDAC. Meanwhile, 3-hydroxyacyl-CoA that is derived from ß-oxidation can compound with 3-hydroxyacyl-ACP under a special enzyme——RhlA to form 3-hydroxyalanoyl-3hydroxyalkanoyl(HAA)-ACP or S-CoA. Eventually, dTDP-L-rhamnose and HAA will combine together under the catalytic effects of RhlB to forge Mono-rhamnolipids. And then, with the assistance of RhlC, Mono-rhamnolipids can be turned into Di-rhamnolipids which are experimentally proved to be a better biosurfactant In order for a more efficient production of Rhamnolipid, our team, BNDS China 2021 aimed to increase the productivity of utilizing P.Aeruginosa to produce rhamnolipids by applying directed evolution (we are not sure about the model for pointed mutation so I would consider a more general approach by DE) and protein scaffolds. Protein scaffold is a technique allowing proteins to carry all enzymes such as RhlA, RhlB, and RhlC together so all the reactions can happen spontaneously instead of transferring the substance around the cell. EvolvR, a directed evolution system including guide RNA, Cas9 enzyme, and low fidelity polymerase, simulates an accelerated natural selection process to obtain the strain with highest productivity.To sum up, with the help of protein scaffold, EvolvR directed evolution, and biosensors to screen the mutant with the greatest amount of manufactured rhamnolipids, we sought to achieve the high-efficiency and increased amount of production of rhamnolipid in P. aeruginosa. 1. Sekhon Randhawa, K. K., & Rahman, P. K. S. M. (2014). Rhamnolipid biosurfactants”past, present, and future scenario of global market. Frontiers in Microbiology, 5. https://doi.org/10.3389/fmicb.2014.004542. Du, J., Zhang, A., Hao, J., & Wang, J. (2017). Biosynthesis of di-rhamnolipids and variations of congeners composition in genetically-engineered Escherichia coli. Biotechnology Letters, 39(7), 1041–1048. https://doi.org/10.1007/s10529-017-2333-23. Yen, T. F. (1990). Microbial enhanced oil recovery. Www.osti.gov. https://www.osti.gov/biblio/6027733-microbial-enhanced-oil-recovery4. “ZFA Technologies - ZFA Technologies Inc.: Biosurfactant Enzyme to Enhance Oil Recovery.” Www.zfatech.com, www.zfatech.com/index.php/zh/?option=com_content&view=article&id=86:zfa-technologies-inc-biosurfactant-enzyme-to-enhance-oil-recovery&catid=17&Itemid=244&lang=en. Accessed 18 Sept. 2021.5. Products. (n.d.). Energyremediationsolutions.com. Retrieved September 19, 2021, from https://energyremediationsolutions.com/9-products/8-products6. Our Company | Sinopec Crop. (n.d.). Www.sinopec.com. http://www.sinopec.com/listco/en/about_sinopec/our_company/company.shtml Reference

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