Over-expression Of Cadida Rugosa Lipase LIP1in Pichia Pastoris And Its High Cell-density Fermentation

Candida rugosa lipases is one of the most widely used lipases in industry, there areeight properties specific isoenzyme, among which, lipase LIP1has higher expression andhydrolysis, esterification and transesterification activities than other isozymes of theoriginal C. rugosa strain. However, the lipase production of of original C. rugosa straincan not meet the practical application. Therefore, it is of great significance to achieve itsheterologus over-expression using genetic engineering. In this paper, a codontransformation and gene synthesis strategy were employed to acquire functional lipaseprotein in Pichia pastoris.19non-nuniversal serine codons CTG of lip1(C. rugosa ATCC14830) were converted into universal serine codon TCT by PCR-based accurate synthesismethod. A variety of strategies were used to improve CRLLIP1expression. Meanwhile,the shaking flask fermentation conditions of the recombinant were optimized, therecombinant CRLLIP1was also characterized. The main work and results weresummarized as follows:1. Gene lip1whose19non-nuniversal serine codons CTG were converted into TCT,was synthesized by PCR-based accurate synthesis. Then, the derived gene was integratedinto the inductive expression vector of P. pastoris expression system, resulting inpPIC9Klip1. The vector was then transformed into P. pastoris GS115，a recombinantGS115/9Klip1139~#with the lipase activity of365U/mL was screened by shaking flaskcultivation. Five key factors of shaking flask cultivation were optimized. The optimalconditions were: culture mediun initial pH=7.5,4.0%(v/v) of inoculation concentration,40mL of culture medium volume,1.0%(v/v) of methanol loading per24h, and inducing96h, the maximum activity reached435U/mL. The emzyme properties werecharacterized, and its optimal temperature and pH were40°C and pH8.0. Furthermore,CRLLIP1retained59%of the original activity after incubation at40°C for4h, and itsoptimum substrate was C10, which coincide with the original lipase LIP1.2. The pGAPlip1and pFZlip1vectors were constructed. Gene fragment5’AOXvgb3’AOXTT harboring gene vgb was amplified by PCR. Based on these genefragment and vectors, pGAPvgb-lip1and pFZvgb-lip1were established. The second-electroporation was conducted to estabalish two different kinds ofdouble-promoter recombinants of P. pastoris and two recombinants with double-promoterexpress CRLLIP1and intracellulariy express VHb. One double-promoter recombinantGS115/9Klip1GAPlip120~#with high lipase activity of437U/mL and the otherrecombinant GS115/9Klip1FZlip139~#of450U/mL were obtained. The activities ofclones harboring double promoters and VHb, GS115/9Klip1GAPvgb-lip171~#andGS115/9Klip1FZvgb-lip11~#respectively attained512U/mL and620U/mL. Their lipaseactivities were1.19,1.23,1.40and1.69flod of GS115/9Klip1139~#, respectively.3. With FM22as basic medium and methanol/D-sorbitol (1:1, v/v) as inducerGS115/9Klip1139~#, GS115/9Klip1GAPlip120~#, GS115/9Klip1FZlip139~#, GS115/9Klip1GAPvgb-lip171~#and GS115/9Klip1FZvgb-lip11~#were fermented in10-L fermentor. Amongall the recombinants, GS115/9Klip1FZvgb-lip11~#had the highest lipase at7,490U/mL,and its cell density and total protein concentration obtained were413.3g/L wet cell weightand4.80g/L after130.7h of cultivation. The lipase activities of recombinantsGS115/9Klip1GAPlip120~#(5,100U/mL), GS115/9Klip1FZlip139~#(4,100U/mL),GS115/9Klip1GAPvgb-lip171~#(6,250U/mL) and GS115/9Klip1FZvgb-lip11~#wererespectively1.81,1.45,2.21and2.65fold of GS115/9Klip1139~#(2,820U/mL). Targetprotein of64kDa was identified as CRLLIP1by mass spectrometry.