Ruiz-Molina Natalia, Parsons Juliana, Schroeder Sina, Posten Clemens, Reski Ralf, Decker Eva L
Plant Biotechnology, Faculty of Biology, University of Freiburg, Freiburg, Germany.
Institute of Process Engineering in Life Sciences III Bioprocess Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
Front Bioeng Biotechnol. 2022 Feb 17;10:837965. doi: 10.3389/fbioe.2022.837965. eCollection 2022.
The moss Physcomitrella is an interesting production host for recombinant biopharmaceuticals. Here we produced MFHR1, a synthetic complement regulator which has been proposed for the treatment of diseases associated to the complement system as part of human innate immunity. We studied the impact of different operation modes for the production process in 5 L stirred-tank photobioreactors. The total amount of recombinant protein was doubled by using fed-batch or batch compared to semi-continuous operation, although the maximum specific productivity (mg MFHR1/g FW) increased just by 35%. We proposed an unstructured kinetic model which fits accurately with the experimental data in batch and semi-continuous operation under autotrophic conditions with 2% CO enrichment. The model is able to predict recombinant protein production, nitrate uptake and biomass growth, which is useful for process control and optimization. We investigated strategies to further increase MFHR1 production. While mixotrophic and heterotrophic conditions decreased the MFHR1-specific productivity compared to autotrophic conditions, addition of the phytohormone auxin (NAA, 10 µM) to the medium enhanced it by 470% in shaken flasks and up to 230% and 260%, in batch and fed-batch bioreactors, respectively. Supporting this finding, the auxin-synthesis inhibitor L-kynurenine (100 µM) decreased MFHR1 production significantly by 110% and 580% at day 7 and 18, respectively. Expression analysis revealed that the MFHR1 transgene, driven by the Physcomitrella () promoter, was upregulated 16 h after NAA addition and remained enhanced over the whole process, whereas the auxin-responsive gene was upregulated within the first 2 hours, indicating that the effect of auxin on promoter-driven expression is indirect. Furthermore, the day of NAA supplementation was crucial, leading to an up to 8-fold increase of MFHR1-specific productivity (0.82 mg MFHR1/g fresh weight, 150 mg accumulated over 7 days) compared to the productivity reported previously. Our findings are likely to be applicable to other plant-based expression systems to increase biopharmaceutical production and yields.
苔藓小立碗藓是生产重组生物制药的一种有趣的宿主。在此，我们生产了MFHR1，这是一种合成补体调节因子，作为人类固有免疫的一部分，已被提议用于治疗与补体系统相关的疾病。我们研究了5升搅拌罐式光生物反应器中不同操作模式对生产过程的影响。与半连续操作相比，采用补料分批或分批操作时，重组蛋白的总量增加了一倍，尽管最大比生产率（mg MFHR1/g鲜重）仅提高了35%。我们提出了一个非结构化动力学模型，该模型能准确拟合在2% CO富集的自养条件下分批和半连续操作的实验数据。该模型能够预测重组蛋白的产量、硝酸盐吸收和生物量增长，这对过程控制和优化很有用。我们研究了进一步提高MFHR1产量的策略。与自养条件相比，混合营养和异养条件降低了MFHR1的比生产率，而在培养基中添加植物激素生长素（NAA，10 µM），在摇瓶中可使其提高470%，在分批和补料分批生物反应器中分别提高多达230%和260%。支持这一发现的是，生长素合成抑制剂L - 犬尿氨酸（100 µM）在第7天和第18天分别使MFHR1产量显著降低110%和580%。表达分析表明，由小立碗藓（）启动子驱动的MFHR1转基因在添加NAA后16小时上调，并在整个过程中保持增强，而生长素响应基因在最初2小时内上调，这表明生长素对启动子驱动表达的影响是间接的。此外，添加NAA的日期至关重要，与之前报道的生产率相比，MFHR1比生产率提高了多达8倍（0.82 mg MFHR1/g鲜重，7天内积累150 mg）。我们的发现可能适用于其他基于植物的表达系统，以提高生物制药的产量。
