Improving Expression Levels Of The Mutants Of Yarrowia Lipolytica Lipase 2 In Pichia Pastoris

Yarrowia lipolytica is an important industrial lipase with many excellent properties.There are 16 lipases in its family.Lip 2 has high hydrolysis,esterification and transesterification activity,which has been used in many fields.Pichia pastoris is the most widely used expression system,which has many advantages of short cultivation cycles,convenient operation,low cost and overexpression.Therefore,it is paid more and more attention in various fields.In this study,we had combined disulfide bonds on Lip2 gene that laboratory screened out in the early period,to improve the expression level and keep thermostable effect form decreasing.After getting the mutant with high expression level,we optimize it again by reducing the number of disulfide bonds,co-expressing chaperones and adjusting the gene dosage.This paper is divided into four experiments:In experiment I,we combined strongly effective disulfide bond mutation sites(S8-214,S60-69,S122-196,S118-177 and S2-210)that our laboratory screened previously,to probe the expression level of four disulfide bonds at different sites on Lip2.In experiment II,we investigated the effect of the number of disulfide bonds on the expression level of mutant by eliminating the original disulfide bonds.In experiment ?,we co-expressed chaperones(PDI)and signaling molecules(HAC1)with mutants of Lip2 in engineered Pichia strains to increase the production.In experiment ?,we screened the recombinant strain with the highest expression and determine the copy number.The results showed as follows:1.We combined the mutation sites S8-214,S60-69,S122-196,and S2-210 to obtain the lipase mutant 4sEx.We combined the mutation sites S60-69,S122-196,S118-177 and S2-210 to obtain the lipase mutant 4sSp.They can form the bond respectively.Compared with 4s(S8-214+S60-69+S122-196+S118-177),the lipase actibity of 96 h shake flask fermentation was increased by 26%,to 613 U/mL(4sEx).4sSp was decreased by 37%,to 307 U/mL.There was no significant difference in thermal stability.Td and T5015 of 4sEx was 68.24?and 68.61?,Td and T5015 of 4sSp was 67.0? and 68.31?,respectively.2.Using 4sEx as a template,we eliminated the original disulfide bonds S30-299?S43-47?S120-123 and S265-273.Results show that,removal of original disulfide bond S120-123 was beneficial to improve the expression level of 4sEx,and the lipase activity was 653 U/mL.But there was no significant difference between 4sEx-123 and 4sEx.On this base,we mutate Cys at the 244 site into Ala and get a new mutant 4sEx-123-244.The lipase activity of 96 h fermentation of 4sEx-123-244 was 516 U/mL.3.Co-expressing Y and mutant 4sEx with chaperones recombinant vector pA0815-PDI,we found that PDI could significantly improve the expression level of Y and 4sEx,increased by 32%and 51%,to 920 U/mL and 889 U/mL,respectively.While co-expressing Y and 4sEx with signaling molecules recombinant vector pA0815-HAC1 and combined recombinant vector pA0815-PDI/HAC1,and we found that HAC1 had no significant effect of the expression level of Y and 4sEx.4.In the case of co-expressing with chaperones PDI and signaling molecules HAC1,we use high Zeocin concentration to screen multi-copy clones and use Q-PCR to identify the copy number of the recombinant strain GS115-4sEx-PDI/HAC 1-3.Through optimization of gene dosage,the lipase activity of recombinant strain GS115-4sEx-PDI/HAC 1-3 was 1182 U/mL,which was 18%higher than GS 115-4sEx-PDI/HAC 1.