Hansenula polymorpha methanol metabolism genes enhance recombinant protein production in Komagataella phaffi.

IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY AMB Express Pub Date : 2024-08-02 DOI:10.1186/s13568-024-01743-y
Maryam Khalifeh Soltani, Sareh Arjmand, Seyed Omid Ranaei Siadat, Abdolreza Bagheri, Seyed Hassan Marashi
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Abstract

Recombinant protein production in Komagataella phaffi (K. phaffi), a widely utilized host organism, can be optimized by enhancing the metabolic flux in the central carbon metabolism pathways. The methanol utilization pathway (MUT) during methanol-based growth plays a crucial role in providing precursors and energy for cell growth and development. This study investigated the impact of boosting the methanol dissimilation pathway, a branch of MUT that plays a vital role in detoxifying formaldehyde and providing energy in the form of NADH, in K. phaffi. This was achieved by integrating two orthologous genes from Hansenula polymorpha into the K. phaffi genome: formaldehyde dehydrogenase (HpFLD) and formate dehydrogenase (HpFMDH). The HpFLD and HpFMDH genes were isolated from the Hansenula polymorpha genome and inserted under the regulation of the pAOX1 promoter in the genome of recombinant K. phaffi that already contained a single copy of model protein genes (eGFP or EGII). The expression levels of these model proteins were assessed through protein activity assays and gene expression analysis. The findings revealed that while both orthologous genes positively influenced model protein production, HpFMDH exhibited a more pronounced upregulation in expression compared to HpFLD. Co-expression of both orthologous genes demonstrated synergistic effects, resulting in approximately a twofold increase in the levels of the model proteins detected. This study provides valuable insights into enhancing the production capacity of recombinant proteins in K. phaffi.

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Hansenula polymorpha 甲醇代谢基因可提高 Komagataella phaffi 的重组蛋白产量。
Komagataella phaffi(K. phaffi)是一种广泛使用的宿主生物,其重组蛋白质的生产可通过提高中心碳代谢途径的代谢通量来优化。在以甲醇为基础的生长过程中,甲醇利用途径(MUT)在为细胞生长和发育提供前体和能量方面起着至关重要的作用。甲醇分解途径是 MUT 的一个分支,在解毒甲醛和以 NADH 形式提供能量方面发挥着重要作用。这是通过将 Hansenula polymorpha 的两个同源基因整合到 K. phaffi 基因组中实现的:甲醛脱氢酶(HpFLD)和甲酸脱氢酶(HpFMDH)。HpFLD 和 HpFMDH 基因是从 Hansenula polymorpha 基因组中分离出来的,并在 pAOX1 启动子的调控下插入重组 K. phaffi 的基因组中。通过蛋白质活性测定和基因表达分析评估了这些模式蛋白的表达水平。研究结果表明,虽然两个直向基因都对模型蛋白的产生有积极影响,但与 HpFLD 相比,HpFMDH 的表达上调更为明显。两个同源基因的共同表达产生了协同效应,使检测到的模型蛋白水平提高了约两倍。这项研究为提高 K. phaffi 重组蛋白的生产能力提供了宝贵的见解。
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来源期刊
AMB Express
AMB Express BIOTECHNOLOGY & APPLIED MICROBIOLOGY-
CiteScore
7.20
自引率
2.70%
发文量
141
审稿时长
13 weeks
期刊介绍: AMB Express is a high quality journal that brings together research in the area of Applied and Industrial Microbiology with a particular interest in ''White Biotechnology'' and ''Red Biotechnology''. The emphasis is on processes employing microorganisms, eukaryotic cell cultures or enzymes for the biosynthesis, transformation and degradation of compounds. This includes fine and bulk chemicals, polymeric compounds and enzymes or other proteins. Downstream processes are also considered. Integrated processes combining biochemical and chemical processes are also published.
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