High-level Expression And Industrial Application Of Lipase From Thermomyces Dupontii

Previous studies found that Thermomyces dupontii lipase?TDL?with potential values in many industrial applications such as detergent,oil and fat modification,biodiesel production,etc.Although TDL shows potential values for industrial application,the complicated cultivation process and low production of TDL from T.dupontii limit its further application.Besides fermentation process and activity,temperature property is also important during the industrial application of TDL.The low activity at low temperature and low thermal stability under high temperature hinder its usage in some industry.So it is important for TDL with desired high thermal stability or cold adaptation to make it amenable for industrial applications.In this study,we combined promoter and signal peptide optimization,high level expression,rational design for temperature adaptation and application of TDL and temperature mutants as whole to overcome main hurdles limiting application of TDL,which provide an important basis for further applications of TDL in many industries.The main findings of this paper are as follows:?1?Effect of different promoters on the production of TDL in Pichia Pastoris:Fifteen methanol-inducible and 9 constitutive promoters were used to drive the expression of TDL in P.pastoris.Of the 15 methanol-inducible promoters,P_FLD1LD1 showed the highest efficiency in driving lipase production,followed by P_AOX1 and P_DAS1.The maximum lipase activities of transformants with P_FLD1,P_AOX1 and P_DAS1 in 5 L bioreactor were 27076 U/mL,24159 U/mL and 22342 U/mL,respectively.In the 9 constitutive promoters,P_GCW14 produced the highest amount of lipases,followed by P₀₄₇₂ and P_GAP.The maximum lipase yields in 5 L bioreactors under the control of P_GCW14,P₀₄₇₂ and P_GAP were 17353 U/mL,15046 U/mL and 14276 U/mL,respectively.?2?Effect of different signal peptides on the production of TDL in P.pastoris:Based on the best promoter,seven heterologous signal peptides,eight signal peptides from P.pastoris,two codon optimization?-MFs and eight?-MF mutants were used to express TDL in P.pastoris.Among these 25 signal peptides,D_57-74 produced the highest amount of lipases,followed by Fre and D_57-70.Compared to the strain with?-MF,the maximum lipase activities of transformants with D_57-74,Fre and D_57-70 were improved by 12.9%?9.1%and 8.1%,respectively.The maximum lipase activity of transformant with D_57-74 in 5 L bioreactor was31137 U/mL,which was 1.15 fold than that of the control.?3?Improved the production of TDL in P.pastoris by combined strategies:Based on the best promoter and signal peptide,high copy number,co-expression with secretion helper factor and high cell density fermentation optimization were combined as whole to improve the production of TDL in P.pastoris.Firstly,the effect of gene copy number on the production of TDL was investigated.The recombinant strain designated X33-T23?three copy numbers?with the highest activity amongst 216 recombinant.Compared to the strain with one copy number,the maximum lipase activity of X33-T23 in shake flask and 5 L bioreactor were improved by 47.4%and 26.8%,respectively.To further increase the production of TDL,12different secretion helper factor genes were transformed to X33-T23.The recombinant strain co-expressed with PDI?named X33-T23-PDI?produced the highest amount of lipases,followed by KAR2,HAC1 and ERO1.The maximum lipase activities of transformants co-expressed with PDI,KAR2,HAC1 and ERO1 in 5 L bioreactor were 57521 U/mL,54280U/mL,54234 U/mL and 48156 U/mL,respectively.Additionally,12 different secretion helper factor genes were transformed to X33-T23-PDI which had little effect on the production of TDL in P.pastoris.Finally,the induction temperature and pH were optimized.The highest activity of X33-T23-PDI in 5 L,50 L and 30 cube bioreactors were 81203 U/mL,81062U/mL and 56121 U/mL,respectively.?4?Rational design for temperature adaptation:The cold adaptation of TDL was improved by deglycosylation.The optimum temperature of mutant N55D was 50?,sliding10?towards cold in comparison to TDL with optimum temperature of 60?.Furthermore,the relative activity of N55D at 30?and 40?were improved by 28%and 31%compared to TDL.The thermal stability of TDL at high temperature was improved by disulfide-bridge and site-directed mutagenesis.Firstly,the disulfide-bridge mutant F12C/K237C and G66C/S121C showed better thermal stability than TDL.After water bathing treatment?75?,10 min?,the residual activities of F12C/K237C and G66C/S121C were 1.9 fold and 1.3 fold than that of TDL.Secondly,the site-directed mutant 42/45,98/100 and 103/104 also showed better thermal stability than TDL.After water bathing treatment?70?,10 min?,the residual activities of 42/45,98/100 and 103/104 were improved by 11%?13%and 8%than that of TDL.Finally,based on the disulfide-bridge and site-directed mutagenesis,we combined disulfide-bridge mutant F12C/K237C and site-directed mutant 42/45,98/100,103/104 for further improvement of thermal stability of TDL.Seven combination mutants with higher thermal stability than TDL were obtained.Combination mutant 5 showed the best thermal stability among these combination mutants.The residual activities of combination mutant 5were 6.9 fold and 4.6 fold than that of TDL under bathing treatment at 75?and 80?for 20min.?5?Application of TDL and temperature mutants:The effects of TDL and N55D in deinking of paper industry were studied.TDL and N55D both had positive effect on deinking efficiency.Meanwhile,the application effect of N55D was better than TDL under the temperature from 30?to 50?.The effects of TDL and combination mutant 5 on growth performance of broilers were studied.The corn-soybean meal diets with TDL and combination mutant 5 could improve the growth performance of broilers such as improvement of daily weight gain,crude fat utilization and apparent nutrient utilization,etc.The application effect of combination mutant 5 was better than TDL when the dosage was same.The results of this study not only provide important basis for industrial application of TDL,but also give some clues on high-level expression,rational design for temperature adaptation,application in deinking and broilers of lipase.