Metabolic Engineering Of Escherichia Coli And Rhodobcter Sphaeroides For The Production Of Coenzyme Q₁₀

Coenzyme Q₁₀(CoQ₁₀),also known as ubiquinone,is a lipid-soluble compound with high redox potential.With the increasing applications of coenzyme Q₁₀ in the food,supplement,commetic,and pharmaceutical industries,coenzyme Q₁₀ is growing in demand.In response to the global market competition and reduce the production cost,it is necessary to improve the yield of coenzyme Q₁₀ of the strain by strain engineering.In this study,the bottlenecks of coenzyme Q₁₀ biosynthesis in Escherichia coli were studied and the coenzyme Q₁₀ biosynthetic pathway was metaboic engineered.The genetic manipulation system for industrial strain of Rhodobacter sphaeroides GY-2 was developed.To integrate genes into the genome of Rhodobacter sphaeroides,temperature sensitive plasmids were obtained by random mutation of the replication region of broad host plasmid,by which the genes of Rhodobacter sphaeroides were knockout.The research of the metabolic engineering of microbes for the production of coenzyme Q₁₀ has important theoretical significance and application value.?1?The bottleneck of coenzyme Qi0 synthesis in Escherichia coli was studied.The knockout of pgi gene and the overexpression of ddsA,dxs,idi and ispDF genes were very important in the synthesis of coenzyme Q₁₀.The production of coenzyme Q₁₀ was further improve by knocking out the metabolic branch of coenzyme Q8,methylnaphthoquinone and demethyl naphthoquinone which alters the metabolic flow towards the IPP synthesis.The overexpression of ddsA,Pcak genes,and the addition of 2 mM pHBA and 5 mM Met eliminated the bottleneck of pHBA and SAM supply deficiencies.The synthesis of IPP to DPP was identified as bottleneck node by using double plasmid coexpression system.The highest yield of coenzyme Q₁₀?3.36 mg/g DCW?was achieved by coexpression of ddsA and quinone ring modified pathway genes in a single plasmid.?2?The key gene of coenzyme Q₁₀ biosynthesis was overexpressed in R.sphaeroides GY-2 using broad host range plasmid.The function of promoters from E.coli was analyzed in R.sphaeroides by using GFP as reporter.It was confirmed that the RNA polymerase of R.sphaeroidesi could recognize the promoter sequence of E.coli,among which the tac and 119 promoters were the strongest.The ddsA gene was overexpressed under the control of tac or 119 promoters in R.sphaeroides GY-2 and the results showed that the yield of R.sphaeroides GY-2 coenzyme Q1O increased by 13.0%?15.3%.Further more,the AcPcak from A.calcoaceticus,the KpPcak of K.pneumoniae,the CgPcak gene of C.glutamicum were individually expressed in R.sphaeroides GY-2 to enhance the uptake of pHBA.With the increase of pHBA concentration,the Pcak from C.glutamicum and K.pneumoniae appeared to have the highest transport efficiency.The RS-CgPcak and RS-KpPcak produced 17.45 and 18.06 mg/g DCW coenzyme Q₁₀ content,an increase of 36.8%?32.2%compared with industrial strains of Rhodobacter sphaeroides when 0.5 mM pHBA was added to the medium.?3?Modification and Screening of temperature sensitive plasmid pBBRIMCS2-Ts.The specific coenzyme Q₁₀ yields of R.sphaeroides were increased by overexpressing ddsA and Pcak genes in expression vector.However,the cell growth and the biomass were decreased due to the extra plasmid.It is necessary to develop plasmid integration system for R.sphaeroides.The rep gene and its promoter and terminator of broad range host pBBR1MCS2 was mutated by error prone PCR and 4 temperature sensitive plasdmids were screened.The plasmid could be lost at a rate of 99.99%in E.coli cultured at 42 ?,while at a rate of 78%?81%in R.sphaeroides cultured at 37 ?.Chimeric structure analysis and point mutation revealed that a codon change of Lys to Glu at residue number 25 of REP protein resulting from the mutation of T to A at nucleotide number 1866 causes the plasmid replication to be temperature sensitived.The wild type and mutated REP were expressed,purified and analysised by circular dichroism.The result showed that the a-helix secondary structure of the mutated protein was destroyed and the spatial structure was more unstable.The plasmid copy number of temperature sensitive plasmid?PCN?was determinated by real time PCR.The results showed that the temperature sensitive plasmid was susceptible to loss even at 30 ?without resistance.The mutated REP protein was unstable at high temperature which leads the loss of plasmid.?4?Metabolic engineering of R.sphaeroides for the production of coenzyme Q₁₀.The upp?uracil phosphoribosyl transferase?gene of R.sphaeroides GY-2 was disrupted by temperature sensitive plasmid and a new chassis cell R.sphaeroides GY-2 ?upp which was resistant to 5-FU?5-fluorouracil?was obtained.Compared with R.sphaeroides GY-2,the R.sphaeroides GY-2 ?upp showed no significant change in the growth,biomass,coenzyme Q₁₀ productivity and yield.The sensitivity to 5-FU was restored by upp gene complementation.The double gene knockout strain R.sphaeroides GY-2 ?uppAcm whose chorismate mutase gene was disrupted was further constructed using temperature sensitive plasmid.The coenzyme Q₁₀ productivity and yield of the resulting strain was 189 mg/L and 15.1 mg/g DCW,increased by 15%and 13%compared with wild industrial strain.In summary,the bottlenecks of coenzyme Q₁₀ synthesis in the model strain E.coli were studied and the key enzymes involved in the biosynthesis of coenzyme Q₁₀ were overexpressed by pladmids.The scarless disruption of R.sphaeroides system was developed.Metabolic engineering of E.coli and R.sphaeroides for the production of coenzyme Q₁₀ were achieved.