Molecular Modification And High-efficiency Expression Of Lipoxygenase From Pseudomonas Aeruginosa

Lipoxygenase(LOX,EC.1.13.11.12)is a fatty acid peroxidase,which catalyzes the specific dioxygenation reaction of polyunsaturated fatty acids with one or more cis,cis-1,4-pentadiene structures,to generate hydroperoxides,these are further transformed into a series of biologically active oxidized lipids.LOX is used in the food,chemical,pharmaceutical and other industries.At present,commercial LOX is mainly produced by extraction of plant tissue,or fruit.However,because of the mixture of isozymes in the raw materials and inefficient extraction methods,the quality of LOX is challenging to control.With the development of biotechnology,LOX production by microbial fermentation has attracted extensive attention because of its low production cost,highly specific,safe,healthy and environmentally friendly catalytic process.However,it still has some disadvantages,such as low production efficiency,low catalytic efficiency and poor stability,which was not suitable for many industrial production and applications.Therefore,efficient and stable production of LOX with high catalytic efficiency and good stability is very important.LOX gene from P.aeruginosa was successful expression in Escherichia coli BL21(DE3).Further mutants with high catalytic efficiency and high stability were obtained by molecular modification.Based on this,the activity and soluble expression of LOX at the industrial operating temperatures(>30?)were optimized and realized to be extracellular production.The main results were as follows:(1)LOX enzymatic properties analysis and catalytic efficiency modificationIn this work,we constructed recombinant E.coli BL21(DE3)to express Pa LOX and Bt LOX from P.aeruginosa and B.thalandensis and compared their enzymatic properties.Enzymatic analysis showed that the optimum reaction temperature of recombinant Pa LOX and Bt LOX was 25? and 35?,respectively.Their optimum reaction pH was 7 and 7.5.Furthermore,the k_cat/K_m of Pa LOX was 16.7 times higher than Bt LOX,which showed potential for industrial application.On this foundation,a mutant library of Pa LOX was constructed via semi-rational design.The k_cat/K_m of mutants increased by 1.4–9.2-fold and the half-life(t_1/2)of Ile159Lys at 50°C increased by 4.6min to 11.6min.Molecular dynamics(MD) simulation indicated that mutation reduced steric hindrance to substrate binding and increased the flexibility of the lid domain that covered the bound unsaturated fatty acid substrate.In addition,van der Waals interactions between the substrate and amino acid residues of the binding pocket increased and alkyl and Pi-alkyl interactions decreased,which might improve the flexibility and substrate binding affinity.These results promoted understanding of the structure-function relationship of LOX and increase its catalytic efficiency and stability for further industrial application.(2)Enhancing plasmid stability to improve LOX expression efficiencyTo reduce the industrial production cost of LOX,it is essential to explore its expression at an industrial operating temperature(>30°C).It was found that plasmid stability was the potential reason affecting LOX soluble expression at high temperatures(>30°C)by analyzing the factors affecting LOX expression at different temperatures.Based on this,the plasmid stabilization system was optimized.The par BCA plasmid stabilization system could effectively improve the LOX activity by 2 times.The carbon source composition of the self-induction medium was further optimized to reduce metabolic pressure,which could effectively improve the expression level of LOX,with the LOX activity by 9 times.Finally,the cell generation stress response caused by inducer and temperature was effectively balanced by the combination strategy,to realize the soluble activity expression of LOX at 30°C and 37°C.This work provides reference value for the research of other"difficult to express"proteins.(3)Transcriptional and translational regulation promotes soluble expression of LOXTo further solve the problem of LOX soluble expression,a soluble expression high-throughput screening system was constructed by the principle of split GFP protein-protein interaction.The linear relationships between soluble expression level,enzyme activity and fluorescence were evaluated.Then,this system was used to screen the?factors and molecular chaperones affecting LOX soluble expression.The different mutant library from GroES,Skp was screened by this system.Finally,the LOX activity reached 4123±201U/g-DCW,and the soluble level increased by 1.7-fold by the regulation of the positive mutant.Meanwhile,this strategy could also be effectively used for the soluble expression of other proteins.(4)Extracellular production of LOX mediated by signal peptide and self-cleavage systemTo achieve extracellular production of LOX,the secreted expression of LOX mediated by the signal peptide was studied.These signal peptides could effectively secrete LOX into the fermentation broth,but the secretion efficiency was insufficient.Surfactants could increase the permeability of the cell membrane by destroyed the phospholipid bilayer resulting promote the ability of extracellular production of recombinant LOX.But extracellular LOX yield cannot reach the ideal level.Therefore,an E.coli strain with self-cleavage function by using bacteriophage lysis gene(?X174-E)was constructed.Furthermore,the extracellular production of LOX was realized by optimized the cell lysis conditions,and the content of inclusion body in the cell was greatly reduced.Finally,the extracellular LOX activity in E.coli arrived at 368U/ml of LOX protein in a 5-L bioreactor by using an optimal fermentation condition.This work provides an efficient protein extracellular preparation strategy that could effectively reduce the content of the inclusion body and enhance the capability of E.coli as a microbial cell factory to produce extracellular proteins.