BACKGROUND The PAOX1-based expression system is the most widely used for producing recombinant proteins in the methylotrophic yeast Pichia pastoris (Komagataella phaffii). Despite relevant recent advances in regulation of the methanol utilization (MUT) pathway have been made, the role of specific growth rate (µ) in AOX1 regulation remains unknown, and therefore, its impact on protein production kinetics is still unclear. RESULTS The influence of heterologous gene dosage, and both, operational mode and strategy, on culture physiological state was studied by cultivating the two PAOX1-driven Candida rugosa lipase 1 (Crl1) producer clones. Specifically, a clone integrating a single expression cassette of CRL1 was compared with one containing three cassettes over broad dilution rate and µ ranges in both chemostat and fed-batch cultivations. Chemostat cultivations allowed to establish the impact of µ on the MUT-related MIT1 pool which leads to a bell-shaped relationship between µ and PAOX1-driven gene expression, influencing directly Crl1 production kinetics. Also, chemostat and fed-batch cultivations exposed the favorable effects of increasing the CRL1 gene dosage (up to 2.4 fold in qp) on Crl1 production with no significant detrimental effects on physiological capabilities. CONCLUSIONS PAOX1-driven gene expression and Crl1 production kinetics in P. pastoris were successfully correlated with µ. In fact, µ governs MUT-related MIT1 amount that triggers PAOX1-driven gene expression-heterologous genes included-, thus directly influencing the production kinetics of recombinant protein.

中文翻译：

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特定生长速率控制AOX1基因表达，影响具有不同目标基因剂量的巴斯德毕赤酵母（Komagataella phaffii）PAOX1驱动的重组生产菌株的生产动力学。

背景技术基于PAOX1的表达系统是在甲基营养酵母巴斯德毕赤酵母（Komagataella phaffii）中最广泛用于产生重组蛋白的系统。尽管最近在甲醇利用（MUT）途径的调控方面取得了相关进展，但仍不清楚比生长速率（µ）在AOX1调控中的作用，因此，其对蛋白质生产动力学的影响仍不清楚。结果通过培养两个PAOX1驱动的假丝酵母念珠菌脂肪酶1（Crl1）生产者克隆，研究了异源基因剂量以及操作模式和策略对培养生理状态的影响。具体而言，在化学恒温器和分批补料培养中，将整合了一个CRL1表达盒的克隆与一个包含三个表达盒的克隆进行了比较，该稀释盒具有较宽的稀释率和µ范围。恒化器的培养允许建立μ对MUT相关的MIT1库的影响，从而导致μ与PAOX1驱动的基因表达之间呈钟形关系，直接影响Crl1的产生动力学。同样，恒化器和分批补料培养也显示出增加CRL1基因剂量（qp最高可达2.4倍）对Crl1产生的有利影响，而对生理能力没有明显的不利影响。结论PAOX1驱动的基因表达和Crl1生产动力学在巴斯德毕赤酵母中已成功地与µ相关。实际上，μ控制着MUT相关的MIT1数量，该数量触发了PAOX1驱动的基因表达（包括异源基因），从而直接影响重组蛋白的生产动力学。