Cloning And Expression Of Cryophilic Lipase Genes And Preliminary Establishment Of A Lipase Highthroughput Screening Method

Lipase is a special triglyceride hydrolase.Its acting condition is mild and the reaction has few by-products.It can act on the oil-water interface to catalyze the hydrolysis of glycerides into fatty acids and glycerol.It can catalyze hydrolysis,transesterification,esterification,alcoholysis,acid hydrolysis and ammonolysis.Low temperature lipase refers to those that still have a certain catalytic activity at 0oC,and have an optimum temperature lower than 30oC.Most of this kind of enzymes have poor enzyme activity at high temperatures.Low temperature lipase has certain tolerance to organic solvents and a preference for C3~C10 long-chain fatty acid esters and has a wider range of acting pH.Therefore,low temperature lipase has a wide application potential in aquatic feed food,biomedicine,leather,environmental management,and biodiesel,washing and other industries.It has been reported that Pseudomonas and Aeromonas are the main source of low-temperature lipase.In order to broaden the low-temperature lipase gene resources,in this study we cloned and expressed three lipase genes from microbes.All three lipase genes were successfully expressed in E.coli,and the recombinant proteins were designated PgLip,PfLip and BcLip,which were from Pseudomonas guguanensis,Pseudomonas formosensis and Bacillus clausii,respectively.The enzymatic properties of the purified lipase genes were determined.The optimal temperatures of PgLip,PfLip and BcLip were all below 20oC.Even at 0oC,they had relatively high enzyme activities and had certain resistance to metal ions.Although the enzyme activity was lost in SDS,the enzyme activity of the enzymes in other detergents inculding Tween20,Tween80 and Triton X-100 was greatly improved.The enzymes were also activated at high concentrations of some organic solvents,such as ethanol,iso-amyl alcohol,and isopropanol.Therefore,the cloning,expression and biochemical characterization of three cryogenic lipases enriched our scientific understanding of lipase biodiversity.We tried to preliminarily establish a high-throughput screening method for lipase by combining the sensitivity of the biosensor with the rapidity of flow sorting.In this method,first a gene expression cassette is constructed that responds to intracellular Acyl-CoA.This cassette uses the AR promoter so that the expression level of the downstream red fluorescent protein gene that it controls can reflect the intracellular concentration of Acyl-CoA.Fatty acyl CoA will be degraded into the metabolic pathway by the fatty acid ?-oxidase system in the cell,so the sensitivity of the biosensor decreases.In order to increase the sensitivity of the sensor,the genes related to ?-oxidation need to be knocked out or knocked down.Therefore,we used the CRISPR/Cas9 technology to knock out the fadE gene in BL21(DE3).The fadE of E.coli is an ester Acyl-CoA dehydrogenase,and knocking out the fadE gene can block the catabolism of fatty acids without affecting the growth of the strain.Therefore,the knock-out of this gene allows the fatty acid in the cell to be maintained at a stable concentration.Based on the fadE knockout E.coli strain and using BcLip lipase as a model,a cell system responding to fatty acids(precursor of fatty acyl CoA)was constructed.The water,oil,and water emulsification system was optimzied so that the bacteria could form a good emulsification system.Finally,through high-throughput screening,the E.coli expressing the lipase BcLip was isolated by flow cytometry,and the gene was isolated.Based on this,a soil metagenome plasmid library was constructed.Using the high-throughput screening method combined with biosensor and flow sorting,a lipase producing strain was initially screened from the soil sample.The results of this study are as follows: through homology alignment,three low-temperature lipase genes were cloned.These genes were successfully expressed in E.coli and their enzymatic properties were biochemically characterized which enrichs the lipase gene resources and scientific understanding of lipase biodiversity.At the same time,by combining biosensors and flow sorting,we also established a preliniminary high-throughput flow screening method for lipase genes using the obtained lipase BcLip as a model.Using this method,a recombinant strain expressing lipase was successfully isolated from the soil metagenome plasmid library.After optimization this method is expected to be used for lipase screening,so it provides new ideas for lipase screening.Because the method is based on function screening,the biosensor element in the method may be modified for use in screening of other feed enzymes such as xylanase,mannanase.This can also lead to obtaining of brand-new enzyme gene resources.