Luna-Flores Carlos H, Weng Yilun, Wang Alexander, Chen Xiaojing, Peng Bingyin, Zhao Chun-Xia, Navone Laura, von Hellens Juhani, Speight Robert E
Faculty of Science, Queensland University of Technology (QUT), Brisbane, Queensland, Australia.
Australian Institute for Bioengineering and Nanotechnology, The University of Queensland (UQ), Brisbane, Queensland, Australia.
Biotechnol Bioeng. 2023 Nov;120(11):3276-3287. doi: 10.1002/bit.28510. Epub 2023 Jul 25.
Pichia pastoris (Komagataella phaffii) is a fast-growing methylotrophic yeast with the ability to assimilate several carbon sources such as methanol, glucose, or glycerol. It has been shown to have outstanding secretion capability with a variety of heterologous proteins. In previous studies, we engineered P. pastoris to co-express Escherichia coli AppA phytase and the HAC1 transcriptional activator using a bidirectional promoter. Phytase production was characterized in shake flasks and did not reflect industrial conditions. In the present study, phytase expression was explored and optimized using instrumented fermenters in continuous and fed-batch modes. First, the production of phytase was investigated under glucose de-repression in continuous culture at three dilution factors, 0.5 d , 1 d , and 1.5 d . The fermenter parameters of these cultures were used to inform a kinetic model in batch and fed-batch modes for growth and phytase production. The kinetic model developed aided to design the glucose-feeding profile of a fed-batch culture. Kinetic model simulations under glucose de-repression and fed-batch conditions identified optimal phytase productivity at the specific growth rate of 0.041 h . Validation of the model simulation with experimental data confirmed the feasibility of the model to predict phytase production in our newly engineered strain. Methanol was used only to induce the expression of phytase at high cell densities. Our results showed that high phytase production required two stages, the first stage used glucose under de-repression conditions to generate biomass while expressing phytase, and stage two used methanol to induce phytase expression. The production of phytase was improved 3.5-fold by methanol induction compared to the expression with glucose alone under de-repression conditions to a final phytase activity of 12.65 MU/L. This final volumetric phytase production represented an approximate 36-fold change compared to the flask fermentations. Finally, the phytase protein produced was assayed to confirm its molecular weight, and pH and temperature profiles. This study highlights the importance of optimizing protein production in P. pastoris when using novel promoters and presents a general approach to performing bioprocess optimization in this important production host.
巴斯德毕赤酵母（Komagataella phaffii）是一种生长迅速的甲基营养型酵母，能够利用多种碳源，如甲醇、葡萄糖或甘油。它已被证明具有分泌多种异源蛋白的出色能力。在之前的研究中，我们利用双向启动子对巴斯德毕赤酵母进行工程改造，使其共表达大肠杆菌AppA植酸酶和HAC1转录激活因子。在摇瓶中对植酸酶的产生进行了表征，但未反映工业条件。在本研究中，使用仪器化发酵罐以连续和补料分批模式对植酸酶的表达进行了探索和优化。首先，在连续培养中，在三个稀释因子（0.5 d、1 d和1.5 d）下研究了葡萄糖去阻遏条件下植酸酶的产生。这些培养物的发酵罐参数被用于建立分批和补料分批模式下生长和植酸酶产生的动力学模型。所建立的动力学模型有助于设计补料分批培养的葡萄糖补料曲线。在葡萄糖去阻遏和补料分批条件下的动力学模型模拟确定了在比生长速率为0.041 h时的最佳植酸酶生产力。用实验数据对模型模拟进行验证，证实了该模型预测我们新工程菌株中植酸酶产生的可行性。甲醇仅用于在高细胞密度下诱导植酸酶的表达。我们的结果表明，高植酸酶产量需要两个阶段，第一阶段在去阻遏条件下使用葡萄糖来产生生物量并同时表达植酸酶，第二阶段使用甲醇来诱导植酸酶表达。与在去阻遏条件下单独用葡萄糖表达相比，通过甲醇诱导植酸酶产量提高了3.5倍，最终植酸酶活性达到12.65 MU/L。与摇瓶发酵相比，最终的植酸酶体积产量代表了约36倍的变化。最后，对产生的植酸酶蛋白进行了分析，以确认其分子量以及pH和温度谱。本研究强调了在使用新型启动子时优化巴斯德毕赤酵母中蛋白质生产的重要性，并提出了在这个重要生产宿主中进行生物过程优化的一般方法。
