Pla Itzcoatl A, Damasceno Leonardo M, Vannelli Todd, Ritter Gerd, Batt Carl A, Shuler Michael L
120 Olin Hall, School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, USA.
Biotechnol Prog. 2006 May-Jun;22(3):881-8. doi: 10.1021/bp060012+.
Extracellular secretion of over 4 g x L(-1) of the A33 scFv antibody fragment was achieved in Pichia pastoris at the 10 L bioreactor scale using minimal medium and feedback control of the methanol concentration. Since methanol acts as both inducer and carbon source, its close regulation is a crucial factor in achieving optimal fermentation conditions. The antibody fragment production levels of both Mut+ and MutS phenotypes were compared in a bioreactor under closed-loop PID control of the methanol level. As expected, the MutS phenotype has a growth rate lower than that of the Mut+ (0.37 vs 1.05 d(-1)) when growing under methanol. However, protein productivity and cell yield on substrate are almost double that of the Mut+ (18.2 vs 9.3 mg A33 sc per gram of methanol). Induction at wet cell weight of 350 g x L(-1) for the MutS also has a positive effect on the final product concentration. Both Mut+ and MutS phenotypes reach a maximum biomass density around 450 g x L(-1) wet cell weight, independent of methanol concentration, reactor scale, or induction density. This reactor configuration allows for reproducible fermentation schemes with different Pichia pastoris phenotypes with AOX promoters, without prior knowledge of the culture growth parameters.
在10 L生物反应器规模下，使用基本培养基并对甲醇浓度进行反馈控制，在毕赤酵母中实现了超过4 g/L的A33单链抗体片段的细胞外分泌。由于甲醇既是诱导剂又是碳源，对其进行严格调控是实现最佳发酵条件的关键因素。在甲醇水平的闭环比例积分微分（PID）控制下，在生物反应器中比较了Mut+和MutS两种表型的抗体片段生产水平。正如预期的那样，在甲醇存在下生长时，MutS表型的生长速率低于Mut+（分别为0.37 d⁻¹和1.05 d⁻¹）。然而，底物上的蛋白质生产率和细胞产量几乎是Mut+的两倍（每克甲醇产生18.2 mg A33 sc，而Mut+为9.3 mg）。对于MutS，在湿细胞重量为350 g/L时进行诱导对最终产物浓度也有积极影响。Mut+和MutS两种表型都能达到约450 g/L湿细胞重量的最大生物量密度，这与甲醇浓度、反应器规模或诱导密度无关。这种反应器配置允许使用具有AOX启动子的不同毕赤酵母表型进行可重复的发酵方案，而无需事先了解培养生长参数。
