Expression Of The Yarrowia Lipolytica Lipase LIP2 In Pichia Pastoris Under The Control Of The FLD1 Promoter

Lipase LIP2 from Yarrowia lipolytic (YlLIP2) was one of the important microorganism lipases in industrial application. In this paper, Formaldehyde dehydrogenase promoter (PFLD1) gene was cloned, and a recombinant vector containing both PFLD1 and YlLip2 gene was constructed base on pPCIZαA. Methylotrophic yeast Pichia pastoris X-33 was employed as the host strain to express YlLIP2 under the control of the PFLD1. Meanwhile, different induction fermentation strategies were applied to investigate the expression of YlLIP2 at 10 l fermentor. The main work and the results were as follows:1. PFLD1 gene and Lip2 gene were amplified from P. pastoris GS115/pPIC9K-Lip2- HIS+ by PCR. The PAOX1 of pPICZαA was replaced by PFLD1, and a novel P. pastoris expression vector named pFZαwas constructed. Then, mature Lip2 gene was cloned into pFZα, and the resulting recombinant plasmid was transformed into P. pastoris X-33 by electroporation2. 308 transformants, forming clear fluorescent halos, were obtained after screening three times on rhodamine B plate. Subsequently, the transformants were subjected to flask fermentation to screen by quantitative analysis. As a result, a high production of YlLIP2 transformant No. 83 was obtained, accumulating lipase activity in the supernatant to the level of 672 U/ml after induction for 48 h. According to the most suitable carbon source and nitrogen source screened base on No. 83, two different induction fermentation strategies were studied. In result, the lipase activity of expressed YlLIP2 was accumulated to the level of 2,450 U/ml after 72 h, enhanced 2.65-fold, by using methanol as carbon source and inductor, while to the level of 2,115 U/ml, enhanced 2.15-fold, by using methylamine hydrochloride (MA?HCl) as inductor combined sorbitol as carbon source. Besides, the lipase activity of endocellular YlLIP2 in No. 83 transformants was measured at 147.86 U/ml, only 6.4 percent of the value in the supernatant of fermentation, while 7.77 U/ml in the supernatant of the wild strain X-33.3. Comparison of two induction strategies were conducted in 10 l fermentor with methanol and MA?HCl, respectively. Fermentation was induced by methylamine for 75.34 h. The maximum dry cell weight, the highest extracellular lipase activity, the highest protein content and the highest protein specific activity were 59.08 g/l, 8,400 U/ml, 2.2 g/l and 3,871 U/mg, respectively. Fermentation was induced by methanol for 146.74 h. The maximum dry cell weight, the highest extracellular lipase activity, the highest protein content and the highest protein specific activity were 115.73 g/l, 15,600 U/ml, 6.4 g/l and 4,075 U/mg, respectively. The fermentation time induced by MA?HCl was 51.34 % of that by methanol. The productive activity and the dry cell lipase activity of the fermentation induced by MA?HCl were 111.49 U/(ml?h) and 14.79 U/mg, respectively, while those induced by methanol were 108.95 U/(ml?h) and 14.03 U/mg, respectively. The energy costs of the fermentation induced by MA?HCl was about 50 % lower than that of the other induction strategy. In addition, The pigment content of the fermentation induced by MA?HCl was lower than that of the other induction strategy, which was more conducive to purification.In this paper, the expression of lipase YlLIP2 in P. pastoris under the control of the PFLD1 promoter reached a very high level. We further developed and improved the expression of foreign proteins under the control of the PFLD1 promoter, and offered a theoretical basis for alternative PAOX1 promoter of P. pastoris expression system with PFLD1 promoter.