Mutation Breeding And Genetic Modification Of Rhamnolipid Producing Strain

Petroleum is an important non-renewable resource in industrial society, modern economy and society development have been more and more depending on oil, however, with the decrease of oil reserve, the price of crude oil price become higher and higher and are becoming the block for society and economy development. The current oil recovery rate is only about 35-45% by conventional techniques, a large amount of oil, especially high viscosity, hypercoagulability are still detained in the oil reservoir. Above all, how to improve oil recovery to produce more crude oil from underground has become an important research focus in the world. Microbial enhanced oil recovery (MEOR) is widely considered to be the modern technology to improve the current low extraction rate and fully deplete the existing reservoir and plays a significant role in the sustainable development of industrial society. This technology can improve liquidity of the crude oil in the formation pore by using the direct effect of microorganisms and crude oil. Moveover, the secondary metabolites produced by microorganisms, such as biosurfactant, organic acid and emulsifier, can improve the oil recovery rate. In petroleum industry, rhamnolipid is considered as the most popular and efficient biosurfactant. Because rhamnolipids has the best ability of reducing surface activity and is eco-friendly, it has been widely used in MEOR and the degradation of hydrocarbon pollutants. Enhancement of rhamnolipids yield will be the main way to decrease its production cost, therefore, this paper mainly focused on the the following aspects:1. The mutation breeding of Pseudomonas aeruginosa and the screening of high-yield rhamnolipid strainsPseudomonas aeruginosa SH6 (1.29 g/L) was treated by plasma, UV, EMS and different complex mutagenesis methods. After several rounds of mutagenesis, many high-yield rhamnolipid strains were first screened on sheep blood plate and then were re-screened by fermentation in shake flask. Finally, a high-yield mutant EZD3 with the highest rhamnolipid yield of 4.2 g/L was obtained from complex mutagenesis. After optimization of fermentation medium including carbon and nitrogen sources, the rhamnolipid production of EZD3 reached 4.8 g/L and was nearly four times more than that of the Pseudomonas aeruginosa SH6. The mutant EZD3 has good genetic stability, can be applied to further genetic modification.2. Construction of high-yield rhamnolipid engineering strains of Pseudomonas aeruginosa with strong endogenous promoterAcoording to the previous reports, strong promoters such as lac and tac can promote heterologous expression of key rhamnosyltransferase gene rhlABRI in Gram-negative bacteria, such as Escherichia coli. However, the yield of rhamnolipid is always very low. Firstly, we also used the exogenous promoters such as tac, lac to investigate their effects on the expression of gene rhlABRI in Pseudomonas aeruginosa, however, rhamnosyltransferase gene rhlABRI were not well expressed in Pseudomonas aeruginosa. Secondly, endogenous promoters of czcC and pilG gene were respectively cloned and connected to structure genes to construct a recombined plasmid. Then the recombined plasmid was converted into P. aeruginosa to construct recombined strains with the target gene converted in genome. In this way, a recombined strains with the highest yield of rhamnolipid 7.25 g/L was obtained, thus, after several rounds of mutagenesis and genetic modification, the rhamnolipid yield of recombinant strain was increased by 462% than the original strain SH6. After optimization of fermentation medium including carbon sources, the rhamnolipid production of recombined strains reached 7.58 g/L.