Improvement Of Molecular Manipulation Method Of Pichia Pastoris And Its Application In High-level Expression Of Rhizopus Oryzae Lipase

Rhizopus oryzae lipase?ROL?is a 1,3-regiospecific lipase with good hydrolysis and esterification activities,thus,it is widely used in food processing,biodiesel production,chiral compound resolution and other industrial fields.However,its natural yield is too low to meet industrial demand.The use of genetic engineering technology to improve the expression level of ROL is of great significance for promotion of its industrial application.At the same time,Pichia pastoris is widely applied for high-level expression of various heterologous proteins owing to its following advantages: ease of genetic operation,capability to execute eukaryotic post-translational modifications,ability to grow to a high cell concentration with low nutritional requirement.Generally,the strategy of increasing gene copy number and co-expressing chaperones is often adopted to improve the expression level of the recombinant protein in P.pastoris.Nevertheless,owing to the limited availability of screening markers for P.pastoris,it is rather difficult to conduct multiple genetic manipulations via existing molecular biology methods,which to some extent limits the coupling application of multiple genetic engineering strategies.Therefore,several molecular manipulation methods of P.pastoris have been explored and developed in this study.Furthermore,a series of high-producing-ROL engineered strains were obtained by appropriately using the developed methods coupling with selecting strong promoters,optimizing gene dosage,and co-expressing multiple expression-related helper proteins.The higher level expression of ROL was further achieved via 3-L high density fermentation.This study would lay a consolidated theoretical and methodological foundation for the large-scale production and industrial application of the recombinant ROL.The main research contents and results are summarized below:1.Exploration of key factors affecting the high-level expression of ROL in P.pastoris indicated that the gene dosage had the greatest effect.The effects of prosequence,vector integration site,promoter and gene dosage on the expression of the recombinant ROL were preliminarily investigated,and it was found that the prosequence and gene dosage had the biggest effect on ROL expression.Thus,using ROL gene with prosequence?proROL?as starting gene,a recombinant strain GS115/9K-Z?-H?-proROL 3# with the activity of 1200 U/mL was screened by increasing the gene dosage.The fermentation conditions of the strain GS115/9K-Z?-H?-proROL 3# in shaking flask and 3-L fermenter were further optimized,the highest lipase activity reached 11,500 U/mL.2.Investigation on the mechanism of increasing gene copy number by posttransformational vector amplification?PTVA?in P.pastoris found that PTVA did not always lead to an increase in the copy number of the entire exogenous vector.The recombinant strain GS115/Zeocin harboring the antibiotic gene without any repeat DNA fragment at both sites and the recombinant strain GS115/Z?-ROL-5'AOX with the antibiotic gene flanked by multiple homologous arms of pAOX1,AOX1-TT and 5'AOX were respectively constructed.It was found that the recombinant strain GS115/Zeocin was not suitable for PTVA.Meanwhile,all of the repeat fragments pAOX1,AOX1-TT and 5'AOX surrounding the antibiotic gene in the recombinant strain GS115/Z?-ROL-5'AOX could mediate the replication amplification of antibiotics gene in PTVA.3.Improving the expression level of ROL in P.pastoris by optimizing gene copy number based on multi-copy vectors.A plasmid pAO?-ROL harboring a single copy of ROL gene cassette was firstly constructed,and the recombinant vectors harboring 2 to 8 copies of ROL gene expression cassettes were successfully generated based on the pAO?-ROL via the optimized “bio-brick” method.On this basis,the recombinant strains GS115/pAO?-nROL?n=1,2,3...8?harboring 1 to 8 copies of ROL gene expression cassettes were then successfully obtained.The results of shaking flask fermentation indicated that the recombinant strain GS115/pAO?-5ROL 11# harboring five copies of ROL gene had the highest ROL activity of 2700 U/mL,almost 8-fold higher than that of the one-copy strain.Additionally,the recombinant strain GS115/pAO?-5ROL 11# was performed with a 3-L high density fermentation using methanol/sorbitol co-feeding strategy.After 115 h of fermentation,the highest ROL activity was achieved at 20,500 U/mL.4.Further increasing the expression level of ROL in recombinant strains by co-expression of helper proteins.Twelve different expression-related helper proteins were respectively co-expressed with ROL in strain GS115/pAO?-5ROL 11#.It was found that 9 helper proteins,Ssa4,Bmh2,Sso2,Ubc1,Hrd1,Pdi,Bip,Hac1 and VHb,could promote the expression of ROL.Among them,ERAD?endoplasmic reticulum associated degradation?-related protein Ubc1 or Hrd1 and trafficking-related proteins Ssa4,Bmh2 or Sso2 could enhance 30-45% of the ROL expression level.Then,the ERAD-related helper proteins?Ubc1 and Hrd1?and trafficking-related proteins?Ssa4,Bmh2 and Sso2?were respectively co-expressed with ROL.The strains GS115/5ROL-Hrd1-Ubc1 1# and GS115/5ROL-Ssa4-Sso2-Bmh2 4# with the highest ROL activities were screened,and their ROL activities respectively attained 4750 U/mL and 5230 U/mL.Moreover,other helper proteins with positive promotion effects on ROL expression were further respectively co-expressed with ROL in strain GS115/5ROL-Ssa4-Sso2-Bmh2 4#.The results indicated that only VHb showed synergistic effect with Ssa4,Bmh2 and Sso2,and a recombinant strain GS115/5ROL-Ssa4-Sso2-Bmh2-VHb 9# with the maximum ROL activity of 5650 U/mL was sieved,which was 1.8 times than that of the initial strain GS115/pAO?-5ROL 11#.In addition,high-density fermentation of the recombinant strain GS115/5ROL-Ssa4-Sso2-Bmh2-VHb 9# was conducted in a 3-L fermenter.After 126 h of fermentation,the ROL activity reached 41,700 U/mL.5.Improving the method of gene knock-out in P.pastoris,and through which to disrupt the Gas1 gene to change the permeability of cell wall to enhance the ROL expression.The Pop-in/Pop-out method was improved to allow the positive screening marker to enrich the gene-deficient strain when performing counter-selection.Compared with the double-crossover method,the improved Pop-in/Pop-out method respectively increased the knockout rate of Och1 gene and Gas1 gene from 0 and 20% to 79% and almost 100%.Then,the Gas1 gene of the recombinant strain GS115/pAO?-5ROL 11# was disrupted via the improved method,and the ROL activity was increased from 3080 U/mL to 3900 U/mL.Moreover,the ROL activity was further increased to 4630 U/mL by co-expressing the trafficking-related proteins of Ssa4,Bmh2 and Sso2 in the Gas1-deficient strain.6.Establishment of novel methods for continuous non-resistant integration of target gene in P.pastoris lays a methodological foundation for the future application of recombinant ROL in food,drug and other fields.Based on the improved Pop-in/Pop-out method,a non-resistant integration method of the target gene was developed,and the non-resistant integration of lox71 fragment and ROL gene were performed,respectively.As a result,the Zeocin antibiotic gene and other redundant DNA fragments were successfully eliminated in all of the 3 randomly selected recombinant strains.Additionally,a novel protocol for continuous non-resistant integration of target gene was created based on the Cre/loxP site-specific recombination system.Three rounds of non-resistant integration of ROL gene expression cassette was successfully achieved via alternately repeated use of the 66-R66 type vector pZHCreAOX₆6-R66?ROL?and the R71-71 type vector pZHCreAOX_R71-71?ROL?.Among the resistance-eliminated clones screened after each round of integration,the percentage of positive recombinant strain with all of the useless and redundant sequences,including Cre gene cassette,resistance gene,and redundant His4 gene,were successfully eliminated as expected recombination event could reach 40-67%.