초록
<P><B>Abstract</B></P> <P> <I>Rhizopus oryzae</I> 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 <I>Pichia pastoris</I>. An efficient method based on bio-brick was developed to construct a series of vectors harboring different copy numbers of <I>ROL</I> gene cassettes, which were then transformed into <I>P. pastoris</I> GS115 to generate a strain with specific copy numbers of <I>ROL</I>. An optimized gene-dosage recombinant strain of GS115/pAOα-5ROL 11# harboring five copies of <I>ROL</I> 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 <I>Ubc1</I> or/and <I>Hrd1</I> were co-expressed with <I>ROL</I> to further increase <I>ROL</I> 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, <I>ROL</I> 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 <I>ROL</I> in <I>P. pastoris</I> using this combined strategy is feasible for large-scale industrialization.</P> <P><B>Highlights</B></P> <P> <UL> <LI> An efficient method was developed to construct multi-copy genes biobricks </LI> <LI> A 5-copy-<I>ROL</I>-gene recombinant with the highest activity was screened. </LI> <LI> ROL co-expressed with two ERAD-related proteins further enhanced expression level. </LI> <LI> The fermented conditions of the derivative recombinants were totally optimized. </LI> </UL> </P>