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

• Published in: ACS synthetic biology 2020-05, Vol.9 (5), p.1190-1200
• Main Authors: Jun, Daniel, Richardson-Sanchez, Tomas, Mahey, Amita, Murphy, Michael E. P, Fernandez, Rachel C, Beatty, J. Thomas
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - genetics; Chromatography, High Pressure Liquid; Electron Transport; Kinetics; Membrane Proteins - genetics; Metabolic Engineering - methods; Photobleaching; Photosynthetic Reaction Center Complex Proteins - genetics; Plasmids - genetics; Plasmids - metabolism; Rhodobacter sphaeroides - chemistry; Rhodobacter sphaeroides - genetics; Rhodobacter sphaeroides - metabolism; Spectrometry, Mass, Electrospray Ionization; Ubiquinone - analysis; Ubiquinone - metabolism; Vitamin K 2 - chemistry; Vitamin K 2 - metabolism
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.0c00066

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.