High-level Expression Of Recombinant Geotrichum Candidum Lipase In Pichia Pastoris And Its High Cell-density Fermentation

The Geotrichum candidum lipase (GCL) is a catalyst having a strong preference forfatty acids with a cis-double bond in the9-position, like oleic acid. Therefore，it is widelyused in industries, such as oil-fat modification, enantiomer resolution and biodieselpreparation. However, in natural conditions, the expression level of GCL is relatively low,which limits its industrial application. Thus, it is of great significance to achieve itshigh-level expression in heterologus host using genetic engineering technique.In this paper, four expression vectors harboring gcl was constructed. Six singlepromoter recombinant Pichia pastoris possessing gcl, three double promoter recombinantP. pastoris possessing gcl, and one recombinant P. pastoris having both gcl and vgb wasalso established. Shaking flask cultivation conditions of P. pastoris possessing gcl wasoptimized. Fed-batch fermentation of five recombinants were implemented in a10-Lbioreactor. The main work and the results were listed as follows:1. Gene gcl was amplified by PCR. Four secretory expression vectors possessing gclwas constructed. They are inducible expression vectors pPIC9K-gcl, pPICZα-gcl,pFLDZα-gcl and constitutive vector pGAPZα-gcl. The linearized plasmids wererespectively transformed into P. pastoris GS115or X-33by electroporation. Six singlepromoter recombinants possessing gcl named GS115/9Kgcl、X-33/Zα-gcl, GS115/Zα-gcl,X-33/FLD-gcl, GS115/FLD-gcl and X-33/GAPgcl were established. Rhodamine B plateand shaking flask cultivation were applied to screen the clones with high lipase production.A clone named GS115/9Kgcl78#was obtained, and its initial lipase activity was up to220U/mL. Then, six key factors of shaking flask cultivation were optimized. The obtainedoptimal conditionss were96h after methanol induction,1%of methanol addition per24h,2%of inoculation concentration, initial pH7.0of culture medium,50mL of culturemedium volume, and induction temperature at25℃. Under these conditions, the lipaseactivity reached735U/mL, which was3.34times more than the initial one.2. Gene vgb was amplified by PCR. The pPICZX-vgb vector was constructed. Thesecond-electroporation was conducted to establish three double promoter recombinants of P. pastoris and one recombinant with both gcl and vgb. Three double promoterrecombinants with high lipase activity GS115/9Kgcl-Zαgcl51#、GS115/9Kgcl-FLDgcl26#and GS115/9Kgcl-GAPgcl63#were selected. Their lipase activities were respectively40.8%，53.8%and27.5%higher than that of pPIC9K-gcl/GS115in shaking flaskcultivation. There is100mL culture medium in500mL shake flask, recombinantGS115/9Kgcl-ZXvgb43#harboring both gcl and vgb can reduce the inhibitory effect oflow dissolve oxygen on GCL expresion.3. Real-time PCR based on TaqMan probe was applied to determine the copy numberof gcl gene in the target clones. The clones GS115/9Kgcl68#, GS115/9Kgcl78#,GS115/9Kgcl86#, GS115/9Kgcl-Zαgcl51#, GS115/9Kgcl-FLDgcl26#, GS115/9Kgcl-GAPgcl63#were all detected. Their gcl gene copy numbers were2,3,1,5,4and4respectively. The clone GS115/9Kgcl-ZXvgb43#had one copy of vgb gene. Targetprotein of63kDa was identified as GCL by mass spectrometry. After Endo H digestion,the molecular weight of GCL decreased to60kDa, much similar to the calculated GCL.Thus, it was demonstrated that GCL had been glycosylated in protein processing.4. In10-L fermentor, the lipase activity of single promoter recombinantGS115/9Kgcl78#was6200U/mL with FM22as basic medium and methanol as inducer.The lipase activity of clones GS115/9Kgcl-Zαgcl51#, GS115/9Kgcl-FLDgcl26#,GS115/9Kgcl-GAPgcl63#and GS115/9Kgcl-ZXvgb43#were respectively45.2%,79.0%,35.5%and18.5%higher than that of GS115/9Kgcl78#. The lipase activity and lipaseproductivity of the clone GS115/9Kgcl-FLDgcl26#were11100U/mL and75.14U/(mL·h)respectively. It was the best clone we had got.Double promoter and VHb coexpresssion strategies were exploied to enhance GCLexpression in P. pastoris. The selected clone had the highest production level of GCL inshaking flask and10-L fermentor. This study would offer a solid basis for large-scaleproduction of GCL.