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
{"title":"Hansenula polymorpha methanol metabolism genes enhance recombinant protein production in Komagataella phaffi.","authors":"Maryam Khalifeh Soltani, Sareh Arjmand, Seyed Omid Ranaei Siadat, Abdolreza Bagheri, Seyed Hassan Marashi","doi":"10.1186/s13568-024-01743-y","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":7537,"journal":{"name":"AMB Express","volume":"14 1","pages":"88"},"PeriodicalIF":3.5000,"publicationDate":"2024-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11296995/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"AMB Express","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1186/s13568-024-01743-y","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0

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.

Abstract Image

查看原文
分享 分享
微信好友 朋友圈 QQ好友 复制链接
本刊更多论文
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。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 去求助
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.
期刊最新文献
Optimized production and characterization of a thermostable cellulase from Streptomyces thermodiastaticus strain. Marine chitinase AfChi: green defense management against Colletotrichum gloeosporioides and anthracnose. Antibiotics and lectin C for diarrhea control intervention in piglets and influences. Correction: Improving carboxymethyl cellulose edible coating using ZnO nanoparticles from irradiated Alternaria tenuissima. Detection and molecular characterization of carbapenem-resistant gram-negative bacterial isolates.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
已复制链接
已复制链接
快去分享给好友吧!
我知道了
×
扫码分享
扫码分享
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1