Integration Of Heterologous 4-HBA Transport Proteins In Rhodobacter Sphaeroides For Enhancement Of CoQ₁₀ Production

Coenzyme Q10(CoQ10)is formed by the conjugation of a benzoquinone head group and a side chain of ten repeated isoprene units,which plays important roles such as participating in aerobic respiration and functioning as an electron carrier in the electron transfer system.In addition,it is also well known as an antioxidant and has been found to be beneficial in 'treatment and prevention of mitochondrial diseases and arterial hypertension.So far,it has gained increasing focus by researchers,who aims to improve its production.The biosynthesis route of CoQ10 in prokaryotes is consisted of three units:Methylerythritol Phosphate(MEP)pathway,Shikimate pathway and Ubiquinone pathway.Many strategies for enhancing CoQ10 production have been developed in various microorganisms to promote the industrial application of CoQio.Peak is an integral membrane protein in the microbial metabolism to facilitate the transmembrane transport of pHBA,which is an important precusor of CoQ10 biosynthesis in Klebsiella pneumoniae and Pseudomonas putida.We have already screened and preserved a CoQ10 producing strain named Rhodobacter sphaeroides GY-2 before.Firstly,we extracted the genomic DNA from different organisms(Acinetobacter calcoaceticus,Klebsiella peneumoniae and Corynebacterium glutamicum)and obtained the peak gene fragment by PCR.After that,we ligated the gene fragment to the vector named pBBR1MCS2 and expressed the recombinant vector pBBR1MCS2-Pcak in Rhodobacter sphaeroides GY-2.The three membrane transport proteins were individually heterologously expressed in Rhodobacter sphaeroides GY-2 to enhance the uptake of extracellular pHBA.Then the CoQ10 productivity and pHBA consumption have been detected through High Performance Liquid Chromatography(HPLC).HPLC results show that when 0.25mM pHBA was added,the CoQ10 production of RS-tRFP-KpPcak reached 18.06mg/g DCW(20.82%higher than Rhodobacter sphaeroides GY-2).Finally,the recombinant strains RS-KpPcak,RS-AcPcak and RS-CgPcak were scaled up with addition of 13C-labled pHBA in a 5L reactor in order to obtain the distribution of the extracellular pHBA for CoQ10 biosynthesis using isotope analysis.