Knock-out of the major regulator Flo8 in Komagataella phaffii results in unique host strain performance for methanol-free recombinant protein production

IF 4.5 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

New biotechnology Pub Date : 2024-10-09 DOI:10.1016/j.nbt.2024.10.001

Corinna Rebnegger , Mirelle Flores-Villegas , Viktoria Kowarz , Sonakshi De , Amandine Pusterla , Hauke Holm , Nuria Adelantado , Christoph Kiziak , Diethard Mattanovich , Brigitte Gasser

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Abstract

Flo8 is a main transcriptional regulator of flocculation and pseudohyphal growth in yeast. Disruption of FLO8 in the popular recombinant protein production host Komagataella phaffii (Pichia pastoris) prevents pseudohyphal growth and reduces cell-to-surface adherence, making the mutant an interesting platform for research and industry. However, knowledge of the physiological impact of the mutation remained scarce. In-depth analysis of transcriptome data from FLO8-deficient K. phaffii revealed that Flo8 affects genes involved in cell cycle, mating, respiration, and catabolite repression additionally to flocculation targets. One gene with considerably increased expression in flo8 was GTH1, encoding a high-affinity glucose transporter in K. phaffii. Its promoter (P_G1) was previously established as a strong, glucose-regulatable alternative to methanol-induced promoters. P_G1 and its improved derivatives P_G1_-₃, D-P_GS4 and D-P_GS5, proved to be promising candidates for controlling recombinant protein production in the FLO8-deficient background. In small-scale screenings, P_G1₃-controlled intracellular EGFP levels were 2.8-fold higher, and yields of different secreted recombinant proteins were up to 4.8-fold increased. The enhanced productivity of the flo8 mutant in combination with the P_G1 variants was transferrable to glucose-limited fed-batch processes and could largely be attributed to higher transcriptional activity of the promoter, leading to a much higher productivity per chromosomally integrated gene copy. K. phaffii flo8 has many advantageous characteristics, such as reduced surface growth and increased transcriptional strength of glucose-regulatable promoters. These features turn the flo8 strain into a valuable new base strain for various experimental designs and establish flo8 as an excellent strain background for methanol-free recombinant protein production processes.

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敲除 Komagataella phaffii 中的主要调控因子 Flo8 可使宿主菌株在无甲醇重组蛋白生产中表现出独特的性能。

Flo8 是酵母絮凝和假茎生长的主要转录调节因子。在常用的重组蛋白生产宿主 Komagataella phaffii（Pichia pastoris）中，FLO8 的破坏会阻止假鳞茎生长并降低细胞与表面的粘附性，从而使该突变体成为一个有趣的研究和工业平台。然而，有关该突变的生理影响的知识仍然很少。对FLO8缺陷型K. phaffii的转录组数据进行深入分析后发现，FLO8除了影响絮凝靶标外，还影响参与细胞周期、交配、呼吸和代谢抑制的基因。在 Flo8 中表达量显著增加的一个基因是 GTH1，它在 K. phaffii 中编码一种高亲和性葡萄糖转运体。其启动子（PG1）先前已被确定为可替代甲醇诱导启动子的强葡萄糖调节启动子。PG1 及其改进衍生物 PG1-3、D-PGS4 和 D-PGS5 被证明是在 FLO8 缺陷背景下控制重组蛋白生产的有希望的候选者。在小规模筛选中，由 PG1-3 控制的细胞内 EGFP 水平提高了 2.8 倍，不同分泌型重组蛋白的产量提高了 4.8 倍。flo8突变体与PG1变体结合后生产率的提高可以转移到葡萄糖限制的饲料批量生产过程中，这在很大程度上归因于启动子的转录活性更高，导致每个染色体整合基因拷贝的生产率大大提高。K. phaffii flo8 具有许多有利的特性，如减少表面生长和提高葡萄糖可调控启动子的转录强度。这些特点使 flo8 菌株成为各种实验设计中一种有价值的新基础菌株，并使 flo8 成为无甲醇重组蛋白生产过程中的优良菌株背景。

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来源期刊

New biotechnology 生物-生化研究方法

CiteScore

11.40

自引率

1.90%

发文量

审稿时长

1 months

期刊介绍： New Biotechnology is the official journal of the European Federation of Biotechnology (EFB) and is published bimonthly. It covers both the science of biotechnology and its surrounding political, business and financial milieu. The journal publishes peer-reviewed basic research papers, authoritative reviews, feature articles and opinions in all areas of biotechnology. It reflects the full diversity of current biotechnology science, particularly those advances in research and practice that open opportunities for exploitation of knowledge, commercially or otherwise, together with news, discussion and comment on broader issues of general interest and concern. The outlook is fully international. The scope of the journal includes the research, industrial and commercial aspects of biotechnology, in areas such as: Healthcare and Pharmaceuticals; Food and Agriculture; Biofuels; Genetic Engineering and Molecular Biology; Genomics and Synthetic Biology; Nanotechnology; Environment and Biodiversity; Biocatalysis; Bioremediation; Process engineering.