The yeast Candida rugosa can secrete a mixture of lipase isoenzymes (Lips), which have been widely applied in industry. Eight Lip genes (LIP1 to LIP8) have been identified and are expressed in Pichia pastoris. However, the expression level was not sufficient for economical industrial application. In this study, two combined processes of antibiotic selection and low-temperature culture efficiently elicited a high-level secretion of recombinant Lip2 in P. pastoris. The LIP2 gene copy number of the Pichia transformants was increased by sequential selections at gradually increasing Zeocin concentrations. After the first selection at 500 mu g/mL of Zeocin, three clones (500-clones) with 2.4-fold to 5.8-fold improvement in Lip2 secretion were identified from 105 survival clones through lipase activity screening. Although the maximum number of LIP2 gene copy was four among these three 500-clones, the lipase secretion of the four-copy clone was not higher than that of the three-copy clone. The effects of multiple gene copy number and low culture temperature resulted in a maximal 32-fold increase in Lip2 secretion. This method could be applied to other Lip isoforms to enhance their yields in P. pastoris. (C) 2015 Elsevier Ltd. All rights reserved.