Team:BNDS China/Engineering

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EngineeringSuccess To optimize the efficiency of rhamnolipid production in our host, Pseudomonas, we transcribed the plasmid originally used to manufacture rhamnolipid into E.coli by heterologous expression and then re-expressed the modified plasmid into Pseudomonas. To begin, the lactose promoter, lactose operator, and lacI are fused to the C3591GF270 backbone (see features and sequence in BBa_K3745043) with three unique enzymes that are responsible for synthesizing rhamnolipids via three different processes: glycolysis or gluconeogenesis, beta-oxidation, and fatty acid synthesis (see synthetic pathway for rhamnolipids on the main page of BBa_K3745043) Additionally, IPTG is used in our research because it is a chemical that is similar to lactose. In other words, it can be used to induce Plac and further propel the production of rhamnolipids by activating rhlA, rhlB, and rhlC. After successfully constructing our plasmid for synthesizing rhamnolipids, a quantification analysis of rhamnolipid production is carried out by using the HPLC-MS method. The particular quantification procedures are detailed on the BBa_K3745043 design page. According to a prior study (Liu et al., 2014), since the peak of impurities and substances other than dirhamnolipids may occur in the time period of 6.5 minutes to 13.35 minutes due to the appearance of shorter fat chains (Rha2C10C10, Rha2C10C12-H2, and Rha2C10C8-H), HPLC-MS of rhamnolipids is measured in the time period between 13.937 minutes and 14.004 minutes for higher accuracy for quantitative analysis. The results are shown in the figure below: the high intensities at 503.0 and 677.7 Da correspond to pure rhamnolipid, monorhamnolipid with a C10-C12 tail, and dirhamnolipid with a C10-C10 tail, respectively. Thereby, the manufacture of rhamnolipids is successful. Fig 1: HPLC-MS of culture induced with IPTG between13.937 min and 14.004 min LIU, Y., ZHONG, H., LIU, Z., JIANG, Y., TAN, F., ZENG, G., LAI, M., & HE, Y. (2014). Purification and characterization of the biosurfactant rhamnolipid. Chinese Journal of Chromatography, 32(3), 248. https://doi.org/10.3724/sp.j.1123.2013.10026 Reference
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