초록
<P><B>Highlights</B></P><P>► Lipase gene and <I>Vitreoscilla</I> hemoglobin gene were co-expressed in <I>Pichia pastoris</I>. ► Recombinant strain (VHb<SUP>+</SUP>) exhibited high specific oxygen uptake rate. ► VHb<SUP>+</SUP> strain displayed higher biomass and lipase activity than control strains. ► The maximum lipase activity of VHb<SUP>+</SUP> in a 10-L fermentor reached 33,000U/mL. ► VHb<SUP>+</SUP> strain also achieved higher cell viability than control strain.</P> <P><B>Abstract</B></P><P>The <I>Yarrowia lipolytica</I> lipase LIP2 (YlLIP2) gene <I>lip2</I> and <I>Vitreoscilla</I> hemoglobin gene <I>vgb</I> were co-expressed in <I>Pichia pastoris</I>, both under the control of AOX1 promoter, in order to alleviate respiration limitation under conditions of high cell-density fermentation and enhance YlLIP2 production. The results showed that recombinant <I>P. pastoris</I> strains harboring the <I>lip2</I> and <I>vgb</I> genes (VHb<SUP>+</SUP>) displayed higher biomass and YlLIP2 activity than control strains (VHb<SUP>−</SUP>). Compared with VHb<SUP>−</SUP> cells, the expression levels of YlLIP2 in VHb-expressing cells when oxygen was not a limiting factor were improved 31.5% in shake-flask culture and 22% in a 10-L fermentor. Under non-limiting dissolved oxygen (DO) conditions, the maximum YlLIP2 activity of VHb<SUP>+</SUP> in a 10-L fermentor reached 33,000U/mL. Oxygen limitation had a more negative effect on YlLIP2 productivity in VHb<SUP>−</SUP> cells than in VHb<SUP>+</SUP> cells. The highest YlLIP2 activity of VHb<SUP>+</SUP> cells was approximately 1.84-fold higher than that of VHb<SUP>−</SUP> cells at lower DO levels. Moreover, the recombinant strain VHb<SUP>+</SUP> exhibited a higher specific oxygen uptake rate and achieved higher cell viability under oxygen limiting and non-limiting conditions compared with VHb<SUP>−</SUP> cells. Therefore, the above results suggest that intracellular expression of VHb in recombinant <I>P. pastoris</I> has the potential to improve cell growth and industrial enzyme production.</P>