High-level extracellular production of Rhizopus oryzae lipase in Pichia pastoris via a strategy combining optimization of gene-copy number with co-expression of ERAD-related proteins.

Rhizopus oryzae lipase (ROL) is an important industrial enzyme limited in application due to its low production in native strains. Here, we used a new combined strategy to overexpress ROL in Pichia pastoris. An efficient method based on bio-brick was developed to construct a series of vectors harboring different copy numbers of ROL gene cassettes, which were then transformed into P. pastoris GS115 to generate a strain with specific copy numbers of ROL. An optimized gene-dosage recombinant strain of GS115/pAOα-5ROL 11# harboring five copies of ROL was screened, revealing production of the highest activity (2700 U/mL), which was 8-fold higher than that of the strain harboring one copy. The activity of GS115/pAOα-5ROL 11# was then enhanced to 3080 U/mL in a shaking flask under optimized culture conditions. Subsequently, the endoplasmic reticulum-associated protein-degradation-related genes Ubc1 or/and Hrd1 were co-expressed with ROL to further increase ROL expression. The activities of the recombinant strains, GS115/5ROL-Ubc1 22#, -Hrd1 15#, and -Hrd1-Ubc1 1#, were 4000 U/mL, 4200 U/mL, and 4750 U/mL, which was 29.9%, 36.4%, and 54.2% higher, respectively, than that observed in GS115/pAOα-5ROL 11#. Using the combined strategy, ROL expression was improved 15.8-fold, with maximum GS115/5ROL-Hrd1-Ubc1 1# activity reaching 33,900 U/mL via a sorbitol/methanol co-feeding strategy in a 3-L fermenter and resulting in a 1.65-, 1.26-, and 1.14-fold enhancement relative to the activities observed in strains GS115/pAOα-5ROL 11#, GS115/5ROL-Ubc1 22#, and GS115/5ROL-Hrd1 15#, respectively. These results indicated that heterologous overexpression of ROL in P. pastoris using this combined strategy is feasible for large-scale industrialization.