Mutation Breeding, Genetic Modification Of Rhamnolipid Producing Strain And Heteroexpression Of The KeyEnzyme

With the rapid development of the world economy, the contradiction between supply and demand of energy has become increasingly prominent, especially oil, as the industrial "lifeblood", bring a serious energy crisis due to the dwindling oil storage. The current oil recovery rate is only about30%by conventional techniques, a large amount of oil, especially high viscosity, hypercoagulability and high-wax oil still detained in the reservoir. Seeking an efficient and inexpensive new oil recovery technology has been a research focus. Microbial enhanced oil recovery (MEOR) is considered to be the best technology in depleting of the remaining oil in the reservoir, greatly improving the oil extraction rate and making great significance for full use of the existing reservoir. Moreover, in the process of oil extraction, transportation and use, the oil spill accident or improper emissions often happen, causing serious pollution to the environment. Because of its wide applications, easy operating, excellent effect, low cost and no secondary pollution, hydrocarbon-degrading microorganisms are recognized as the most effective and thorough method to solve complex hydrocarbon pollution.Broadly speaking, MEOR mainly focus on three different strategies:producing the biosurfactant in off-site reactor, and then directly filled into the oil reservoir; injecting alien microbes to the reservoir directly in which these microbes grow, reproduce and produce surfactant in it; transfusing the nutrients into the wells stimulating the indigenous bacteria in the well to produce surfactants. The biosurfactants are widely used in the field of MEOR and biomediation of soils and oceans containing hydrocarbon contaminants. Among them, rhamnolipid is the most efficient and popular one. The reasons hindering the application of rhamnolipid in MEOR mainly are:high cost but low yield; major producing bacteria-Pseudomonas aerugiuosa is a harmful opportunistic pathogen, can not be applied to large-scale industrial production. Aiming at solving the problems above, this study mainly discussed rhamnolipid from the following aspects:1. Mutagenesis of Pseudomonas acruginosa and screening the high-yield rhamnolipid strains.Using UV, DES, plasma and complex mutagenesis method against Pseudomonas aeruginosa SH6(1.2g/L) to screen high-yield rhamnolipid strains. After several rounds of mutagenesis, a high-yield mutant PZAl which gets the highest rhamnolipid yield of4.0g/L was screened. After the formulation of fermentation medium with the orthogonal experiment was optimized, the highest rhamnolipid production of PZAl reached6.7g/L and was of genetic stability, increased nearly five times more than the original strain.2. Multicopy the promoter to build high-yield rhamnolipid engineering Pseudomonas acruginosa.Reported in the previous literature, multicopy promoter of fungi such as Trichoderma reesei and Aspergillus niger can promote the expression of target gene. The key enzyme in the synthesis of rhamnolipid is rhamnosyltransferase. Respectively duplicate1,3,5copies of the functional sequence in promoter of rhamnosyltransferase, i.e.318-479bp. Then the multicopy-promoter was converted into Pseudomonas acruginosa to construct P. aeruginosa1P, P. aeruginosa3P and P. aeruginosa5P. In this way, the rhamnolipid production of the reconstructed Pseudomonas aeruginosa was improved by increasing the expression of the rhamnosyltransferase, the production of rhamnolipid by P. aeruginosa3P was1.6g/L, increased by30%than wide-type stain; P. aeruginosa5P’s production is1.7g/L, significantly increased40%than wild-type strain.3. Knockout of PHA synthetic genes, construction of high-yield rhamnolipid engineering Pseudomonas aeruginosaPseudomonas aeruginosa SH6can simultaneously produce rhamnolipid and polyhydroxyalkanoate (PHA), the production of which partly sharing the same metabolic pathways by using β-hydroxyl fatty acid as the same synthetic substrate. Therefore, blocking the synthesis pathway of PHA by knocking out PHA synthetic gene in Pseudomonas aerugintosa SH6can provide more substrates to rhamnolipid synthesis, supposing the increase of rhamnolipid yield. Triparental mating method was used in knocking out the PHA synthetic gene phaC and phaG in Pseudomonas aeruginosa. The PHA content in AphaG significantly reduced and its rhamnolipid production increased40%than the original strain, while the knock-out of phaC brought little difference to rhamnolipid production.4. Heterologous expression of rhamnosyltransferase in thermophilic bacteria.The thermophilic strains screened from the oil reservoir were identified by morphological observation and molecular biology methods. The two thermophilic strains belong to Geobacillus genus. Plasmid pNW33N was selected as the expression vector, based on the reports on Geobacillus genus. The recombinant plasmid pNWAB was constructed by linking the coding rhamnosyltransferase rhlABRI operon and pNW33N plasmid. During the transformation, electroporation transformation, competent cell chemical transformation and transformation of protoplasts were tried. The expression of rhamnosyltransferase in thermophilic bacteria can not only get a host of non-pathogenic bacteria, but also constructed engineered bacteria that are able to produce rhamnolipid and adapt the special environment in oil reservoir well.