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Modeling of growth and energy metabolism of Pichia pastoris producing a fusion protein

机译：产生融合蛋白的毕赤酵母生长和能量代谢的模型

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A fusion protein composed of a cellulose binding domain from Neocallimastix patriciarum cellulase A and Candida antarctica lipase B (CBD-lipase) was produced by Pichia pastoris methanol utilization plus phe-notype in high cell-density cultures. The genes expressing CBD-lipase were fused to the alpha-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter. To control the repression and induction of AOX1 and oxygen demand at high cell density, a four-stage process was used. Batch growth on glycerol was used in the first step to provide biomass (28 g L~(-1)) while product formation was prevented due to repression of the AOX1. The second stage was exponential fed-batch growth on glycerol, which caused a slight increase of the enzyme alcohol oxidase activity due to derepression of the AOX1. This procedure resulted in smooth transition to exponential fed-batch growth on methanol, the third stage, in which the AOX1 was strongly induced. The fourth stage was constant fed-batch growth on methanol used to control the oxygen demand at the high cell density. A kinetic model was developed that could predict biomass growth and oxygen consumption in processes with and without oxygen-enriched air. With oxygen enrichment to 34% O_2 in the inlet air the methanol feed rate could be increased by 50% and this resulted in 14% higher final cell density (from 140 to 160 g L~(-1) cell dry weight). The increased methanol feed rate resulted in a proportionally increased specific rate of product secretion to the medium. After an initial decrease, the synthesis capacity of the cell was kept constant throughout the cultivation, which made the product concentration increase almost constantly during the process. The kinetic model also describes how the low maintenance demand of P. pastoris compared with E. coli enables this organism to grow to such high cell densities.
机译：在高细胞密度培养中，利用毕赤酵母甲醇利用加phe-notype产生了一种融合蛋白，该融合蛋白由来自新Callimastix patriciarum纤维素酶A和南极假丝酵母脂肪酶B（CBD-脂肪酶）的纤维素结合域组成。表达CBD脂肪酶的基因与酿酒酵母的α因子分泌信号序列融合，并置于乙醇氧化酶基因（AOX1）启动子的控制下。为了控制在高细胞密度下对AOX1和氧气需求的抑制和诱导，使用了四个阶段的过程。第一步是在甘油上分批生长，以提供生物质（28 g L〜（-1）），同时由于抑制AOX1而阻止了产物形成。第二阶段是甘油的指数补料分批生长，由于AOX1的抑制，导致酶醇氧化酶活性略有增加。此步骤导致在甲醇中平稳过渡到指数补料分批生长，这是强烈诱导AOX1的第三阶段。第四个阶段是甲醇连续补料分批生长，用于控制高细胞密度下的氧气需求。建立了动力学模型，该模型可以预测在有和没有富氧空气的过程中生物量的增长和氧气的消耗。随着氧气在进气中富集到34％O_2，甲醇的进料速率可以提高50％，这将使最终细胞密度提高14％（从140 g L〜（-1）细胞干重增加到160 g L〜（-1））。增加的甲醇进料速率导致产物向培养基中分泌的比重成比例地增加。在最初减少之后，细胞的合成能力在整个培养过程中保持恒定，这使得产物浓度在过程中几乎恒定地增加。动力学模型还描述了与大肠杆菌相比，巴斯德毕赤酵母的低维护需求如何使该生物体生长到如此高的细胞密度。

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