Introduction of the Menaquinone Biosynthetic Pathway into Rhodobacter sphaeroides and de Novo Synthesis of Menaquinone for Incorporation into Heterologously Expressed Integral Membrane Proteins.,ACS Synthetic Biology

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Introduction of the Menaquinone Biosynthetic Pathway into Rhodobacter sphaeroides and de Novo Synthesis of Menaquinone for Incorporation into Heterologously Expressed Integral Membrane Proteins.
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-04-21 , DOI: 10.1021/acssynbio.0c00066
Daniel Jun ₁ , Tomas Richardson-Sanchez ₁ , Amita Mahey ₁ , Michael E P Murphy ₁ , Rachel C Fernandez ₁ , J Thomas Beatty ₁

Affiliation

Quinones are redox-active molecules that transport electrons and protons in organelles and cell membranes during respiration and photosynthesis. In addition to the fundamental importance of these processes in supporting life, there has been considerable interest in exploiting their mechanisms for diverse applications ranging from medical advances to innovative biotechnologies. Such applications include novel treatments to target pathogenic bacterial infections and fabricating biohybrid solar cells as an alternative renewable energy source. Ubiquinone (UQ) is the predominant charge-transfer mediator in both respiration and photosynthesis. Other quinones, such as menaquinone (MK), are additional or alternative redox mediators, for example in bacterial photosynthesis of species such as Thermochromatium tepidum and Chloroflexus aurantiacus. Rhodobacter sphaeroides has been used extensively to study electron transfer processes, and recently as a platform to produce integral membrane proteins from other species. To expand the diversity of redox mediators in R. sphaeroides, nine Escherichia coli genes encoding the synthesis of MK from chorismate and polyprenyl diphosphate were assembled into a synthetic operon in a newly designed expression plasmid. We show that the menFDHBCE, menI, menA, and ubiE genes are sufficient for MK synthesis when expressed in R. sphaeroides cells, on the basis of high performance liquid chromatography and mass spectrometry. The T. tepidum and C. aurantiacus photosynthetic reaction centers produced in R. sphaeroides were found to contain MK. We also measured in vitro charge recombination kinetics of the T. tepidum reaction center to demonstrate that the MK is redox-active and incorporated into the QA pocket of this heterologously expressed reaction center.

中文翻译：

将甲基萘醌生物合成途径引入球形球形红细菌中，并从头开始合成甲基萘醌，以掺入异源表达的整体膜蛋白中。

醌是氧化还原活性分子，在呼吸和光合作用过程中在细胞器和细胞膜中运输电子和质子。除了这些过程在维持生命方面的根本重要性外，人们对利用它们的机制用于从医学进步到创新生物技术的各种应用也产生了极大的兴趣。这些应用包括针对病原细菌感染的新型治疗方法，以及将生物混合太阳能电池制造为可替代的可再生能源。泛醌（UQ）是呼吸和光合作用中主要的电荷转移介质。其他醌，例如甲萘醌（MK），是额外的或替代的氧化还原介体，例如在细菌的光合作用中，例如温热嗜热色杆菌和绿屈挠菌。球形红细菌被广泛用于研究电子转移过程，最近又成为从其他物种生产完整膜蛋白的平台。为了扩大球形红球菌中氧化还原介体的多样性，在新设计的表达质粒中，将九种编码由分支酸和聚异戊二烯基二磷酸合成MK的大肠杆菌基因组装成一个合成操纵子。我们显示，基于高效液相色谱法和质谱法，当在球形红球菌细胞中表达时，menFDHBCE，menI，menA和ubiE基因足以用于MK合成。发现在球形红球藻中产生的T. tepidum和C. aurantiacus光合反应中心含有MK。我们还测量了T的体外电荷重组动力学。

更新日期：2020-04-09

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