Abstract
Scaling-up of high-cell-density fermentation (HCDF) of Pichia pastoris from the lab or pilot scale to the demonstration scale possesses great significance because the latter is the final technological hurdle in the decision to go commercial. However, related investigations have rarely been reported. In this paper, we study the scaling-up processes of a recombinant P. pastoris from the pilot (10 to 100-L) to the demonstration (1,000-L) scales, which can be used to convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol by the β-xylosidase for semi-synthesis of Taxol. We demonstrated that a pure oxygen supplement can be omitted from the HCDF if the super atmospheric pressure was increased from 0.05 to 0.10 ± 0.05 MPa, and we developed a new methanol feeding biomass-stat strategy (0.035 mL/g/h) with 1% dissolved oxygen and 100 g/L initial induction biomass (dry cell weight). The scaling-up was reproducible, and the best results were obtained from the 1,000-L scale, featuring a shorter induction time and the highest enzyme activities and productions, respectively. The specific growth and specific production rates were also determined. This study lays a solid foundation for the commercial preparation of 10-deacetyltaxol through the recombinant yeast. It also provides a successful paradigm for scaling-up HCDF of P. pastoris to the demonstration scale.
Highlights
Malhamensis[10], Arxula adeninivorans[11], Yarrowia lipolytica[13,14,15], Kluyveromyces marxianus[16,17], Saccharomyces cerevisiae[18,19] and P. pastoris[20,21,22,23]
Since the oxygen solubility of the liquid medium can be raised by increasing the total air pressure in the cultivation system, the desired dissolved oxygen (DO) can be maintained by keeping the jar air pressure higher in the absence of a pure oxygen supplement
Since the enzyme was produced in non-secreted form, all of the cells were harnessed as the biocatalyst to remove xylosyl residual from 7-β-xyloxyl-10-deacetyltaxol (XDT) in order to form 10-deacetyltaxol (DT), which is the semi-synthetic precursor of Taxol
Summary
Malhamensis[10], Arxula adeninivorans[11], Yarrowia lipolytica[13,14,15], Kluyveromyces marxianus[16,17], Saccharomyces cerevisiae[18,19] and P. pastoris[20,21,22,23]. Charoenrat et al.[21] reported that increasing air pressure from 1.2 to 1.9 bar increased the oxygen uptake rate (OUR) of P. pastoris by 59%, and the heterologous β-glucosidase yield increased by 50% in the HCDF of the 10-L scale with ~120 g/L DCW. This may be the largest scale on which the strategy of increasing jar air pressure for the HCDF of P. pastoris has been adopted. The glycoside hydrolase LXYL-P1-2 (GenBank accession: AET31459) was cloned from the fungus Lentinula edodes[28] This enzyme can remove the xylosyl group from 7-β-xylosyl-taxanes, which are the by-products of the anti-tumor drug Taxol isolated from yew plants (Taxus species), forming the corresponding 7-β-hydroxyl-taxanes. We scaled up HCDF from the pilot scales of 10-L and 100-L to the demonstration/commercial scale of 1,000-L
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