Enhancing production of Yarrowia lipolytica lipase Lip2 in Pichia pastoris

The extracellular lipase Lip2 from Yarrowia lipolytica (YlLip2) has been widely used as a catalyst in industry for biodiesel production, esters synthesis and optical resolution of chiral compounds. YlLip2 was successfully expressed in Pichia pastoris under the control of the AOX1 promoter. To further improve lipase production in P. pastoris, we constructed multi-copy lipase gene Lip2 clones using high Zeocin concentration screening. Two clones which can resist 1000 mu g/mL Zeocin showed higher lipase production in shaking flasks. Southern blot analysis showed that three lipase genes were integrated into the genome of P. pastoris GS115. Low temperature (25 degrees C) could also improve the extracellular lipase yield. A high lipase activity of 42 900 U/mL (3.4 g total protein and 2.16 g lipase per liter) was obtained using the three copy integration clone in fed-batch fermentation using basal salt medium, which was 2.5-fold higher than that using a single copy clone. The high yield of lipase YlLip2 in P. pastoris could decrease the price of this biocatalyst and accelerate its industrial applications.