초록
<P>As <I>Pichia pastoris</I> (syn. <I>Komagataella</I> sp.) yeast can secrete pure recombinant proteins at high rates, it is a desirable production system. The function of a novel synthetic variant of the AOX1 promoter was characterised comprehensively using a strain secreting <I>Candida antarctica</I> lipase B (CALB) as a model. A new time-saving approach was introduced to determine, in only one experiment, the hitherto unknown relationship between specific product formation rate (<I>q</I><SUB>p</SUB>) and specific growth rate (<I>μ</I>). Tight control of recombinant protein formation was possible in the absence of methanol, while using glycerol as a sole carbon/energy source. CALB was not synthesised during batch cultivation in excess glycerol (>10 g l<SUP>−1</SUP>) and at a growth rate close to <I>μ</I><SUB>max</SUB> (0.15 h<SUP>−1</SUP>). Between 0.017 and 0.115 h<SUP>−1</SUP> in glycerol-limited fedbatch cultures, basal levels of <I>q</I><SUB>p</SUB> > 0.4 mg g<SUP>−1</SUP> h<SUP>−1</SUP> CALB were reached, independent of the <I>μ</I> at which the culture grew. At <I>μ</I> > 0.04 h<SUP>−1</SUP>, an elevated <I>q</I><SUB>p</SUB> occurred temporarily during the first 20 h after changing to fedbatch mode and decreased thereafter to basal. In order to accelerate the determination of the <I>q</I><SUB>p</SUB>(<I>μ</I>) relationship (kinetics of product formation), the entire <I>μ</I> range was covered in a single fedbatch experiment. By linearly increasing and decreasing glycerol addition rates, <I>μ</I> values were repeatedly shifted from 0.004 to 0.074 h<SUP>−1</SUP> and vice versa. Changes in <I>q</I><SUB>p</SUB> were related to changes in <I>μ</I>. A rough estimation of <I>μ</I> range suitable for production was possible in a single fedbatch, thus significantly reducing the experimental input over previous approaches comprising several experiments.</P>