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Tuning Gene Expression by Phosphate in the Methanogenic Archaeon Methanococcus maripaludis
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2021-10-19 , DOI: 10.1021/acssynbio.1c00322 Taiwo S Akinyemi 1 , Nana Shao 1 , Zhe Lyu 2 , Ian J Drake 3 , Yuchen Liu 3 , William B Whitman 1
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2021-10-19 , DOI: 10.1021/acssynbio.1c00322 Taiwo S Akinyemi 1 , Nana Shao 1 , Zhe Lyu 2 , Ian J Drake 3 , Yuchen Liu 3 , William B Whitman 1
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Methanococcus maripaludis is a rapidly growing, hydrogenotrophic, and genetically tractable methanogen with unique capabilities to convert formate and CO2 to CH4. The existence of genome-scale metabolic models and an established, robust system for both large-scale and continuous cultivation make it amenable for industrial applications. However, the lack of molecular tools for differential gene expression has hindered its application as a microbial cell factory to produce biocatalysts and biochemicals. In this study, a library of differentially regulated promoters was designed and characterized based on the pst promoter, which responds to the inorganic phosphate concentration in the growth medium. Gene expression increases by 4- to 6-fold when the medium phosphate drops to growth-limiting concentrations. Hence, this regulated system decouples growth from heterologous gene expression without the need for adding an inducer. The minimal pst promoter is identified and contains a conserved AT-rich region, a factor B recognition element, and a TATA box for phosphate-dependent regulation. Rational changes to the factor B recognition element and start codon had no significant impact on expression; however, changes to the transcription start site and the 5′ untranslated region resulted in the differential protein production with regulation remaining intact. Compared to a previous expression system based upon the histone promoter, this regulated expression system resulted in significant improvements in the expression of a key methanogenic enzyme complex, methyl-coenzyme M reductase, and the potentially toxic arginine methyltransferase MmpX.
中文翻译：
通过磷酸盐调节产甲烷古生菌 Methanococcus maripaludis 的基因表达
Methanococcus maripaludis是一种快速生长的氢营养型产甲烷菌，具有将甲酸盐和 CO 2转化为 CH 4的独特能力。基因组规模代谢模型的存在以及用于大规模和连续培养的成熟稳健系统使其适合工业应用。然而，缺乏用于差异基因表达的分子工具阻碍了其作为微生物细胞工厂生产生物催化剂和生物化学品的应用。在本研究中，基于pst设计和表征了一个差异调节启动子库。促进剂，它响应生长培养基中的无机磷酸盐浓度。当培养基磷酸盐降至生长限制浓度时，基因表达增加 4 至 6 倍。因此，这种调节系统将生长与异源基因表达分离，而无需添加诱导剂。最小的pst启动子被鉴定并包含一个保守的富含AT的区域、一个因子B识别元件和一个用于磷酸盐依赖性调节的TATA盒。B因子识别元件和起始密码子的合理变化对表达没有显着影响；然而，转录起始位点和 5' 非翻译区的变化导致蛋白质产生差异，而调节保持完整。与以前基于组蛋白启动子的表达系统相比，这种受调控的表达系统显着改善了关键产甲烷酶复合物、甲基辅酶 M 还原酶和潜在有毒的精氨酸甲基转移酶 MmpX 的表达。
更新日期：2021-11-19
中文翻译：
通过磷酸盐调节产甲烷古生菌 Methanococcus maripaludis 的基因表达
Methanococcus maripaludis是一种快速生长的氢营养型产甲烷菌，具有将甲酸盐和 CO 2转化为 CH 4的独特能力。基因组规模代谢模型的存在以及用于大规模和连续培养的成熟稳健系统使其适合工业应用。然而，缺乏用于差异基因表达的分子工具阻碍了其作为微生物细胞工厂生产生物催化剂和生物化学品的应用。在本研究中，基于pst设计和表征了一个差异调节启动子库。促进剂，它响应生长培养基中的无机磷酸盐浓度。当培养基磷酸盐降至生长限制浓度时，基因表达增加 4 至 6 倍。因此，这种调节系统将生长与异源基因表达分离，而无需添加诱导剂。最小的pst启动子被鉴定并包含一个保守的富含AT的区域、一个因子B识别元件和一个用于磷酸盐依赖性调节的TATA盒。B因子识别元件和起始密码子的合理变化对表达没有显着影响；然而，转录起始位点和 5' 非翻译区的变化导致蛋白质产生差异，而调节保持完整。与以前基于组蛋白启动子的表达系统相比，这种受调控的表达系统显着改善了关键产甲烷酶复合物、甲基辅酶 M 还原酶和潜在有毒的精氨酸甲基转移酶 MmpX 的表达。
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