Study On The Co-production Of Farnesol And Coenzyme Q₁₀ Via Metabolic Engineering Of Rhodobacter Sphaeroides

Farnesol?FOH?is a kind of acyclic sesquiterpene widely distributed in nature.It has various biological functions such as signal transduction,quorum sensing,cell proliferation,apoptosis and mycelial inhibition.It is also an important raw material for anti-inflammatory and anti-cancer drugs.Coenzyme Q₁₀(CoQ₁₀)molecule consists of a benzoquinone ring nucleus and a hydrophobic side chain of deca-isoprene,which is an essential cofactor for electron transport in the electron transport chain.CoQ₁₀0 has anti-lipid peroxidation and participates in cellular energy metabolism,and plays an important role in the treatment of various diseases and drug synthesis.In this study,Rhodobacter sphaeroides was used as a starting strain to transform R.sphaeroides by metabolic engineering to achieve efficient co-production of FOH and CoQ₁₀.The biosynthetic pathway of FOH and CoQ₁₀0 has an important common synthetic precursor which is Farnesyl pyrophosphate?FPP?.FPP is the basic unit of CoQ₁₀0 decamer isoprene synthesis,and FPP can also directly synthesize FOH by pyrophosphorylase or terpene synthase.FPP is synthesized in the prokaryotic cell by the 2-methylerythritol 4-phosphate pathway?MEP?.In this study,the CoQ₁₀0 high-yield strain R.sphaeroides GY-2 was used as the starting strain,and four genes were over expressed.The phosphorylase gene?phosphatidylglycerophosphatase B,pgpbR?derived from R.sphaeroides 2.4.1encodes alkaline phosphatase,phosphatidylglycerolase B?pgpbE?from E.coli T1,sesquiterpene synthase?atps?derived from Brachypodium distachyon and Terpene synthase?tps?derived from Zea mays.Overexpression of these four genes increased the FOH production of Rhodobacter sphaeroides by 25%-50%compared with the original strain GY-2.The overexpression of tps gene increased the yield of FOH by50%compared with the original strain.There was no significant change in the yield of CoQ₁₀0 in the modified strain.In order to further increase the synthesis of FOH and CoQ₁₀,we used RNA interference method to inhibit the transcription of phytoene synthase?psy?,a key enzyme in the lycopene synthesis pathway that is a competitive pathway for FOH and CoQ₁₀0 synthesis.Thus it is possible to concentrate the carbon flux on the synthesis of the desired product.The strain with the strongest inhibition of psy was named RS-RNAiPSY,and its yield of FOH was increased by 26%compared with the original strain,while the yield of CoQ₁₀0 was not changed significantly compared with the control.We overexpressed three FOH synthase genes using RS-RNAiPSY as the starting strain,named RS-RNAiPSY-pgpbE,RS-RNAiPSY-tps and RS-RNAiPSY-atps,and found that RS-RNAiPSY-tps strain has the highest FOH yield?35.04 mg/L?which was 135%higher than that of wild type.Subsequently,we also carried out experiments on a 5L fermenter.These experimental data indicate that the strategy used to achieve efficient co-production of FOH and CoQ₁₀0 is effective in this study.It can be expected that the industrialization of FOH and CoQ₁₀0 will be initially realized through further research on the RS-RNAiPSY-tps strain of Rhodobacter sphaeroides.