Development Of Genetic System For Rhodobacter Sphaeroides GY-2

Coenzyme Q10,a liposoluble antioxidant compound produced by biological fermentation of the natural high-yield strain of R.sphaeroides,has been widely used in medicine,food,and industry.Several methods including optimization of fermentation conditions and chemical mutagenesis have been applied to improve CoQ10 production in R.sphaeroides.In recent years,biosynthesis pathway of Coenzyme Q10 has gained prominence in in R.sphaeroides,it is very important to develop a new and reusable genetic system in R.sphaeroides which can be used to optimize CoQ10 synthetic pathway by metabolic engineering.In this study,a ddsa gene encoding decaprenyl diphosphate synthase which play a key important role in CoQ10 biosynthesis pathway was overexpressed in R.sphaeroides GY-2 using a broad-host-range plasmids pBBR1MCS-2.Unfortunately,the productivity of CoQ10 decreased due to growth burden caused by foreign plasmid.Therefore,a new genetic system in R.sphaeroides GY-2 with broad-host-range and temperature sensitive plasmid of pBBR1MCS-2M was developed.The result of the plasmid loss rate,REP protein secondary structure and plasmid copy number indicated that the a mutation of A to G at nucleotide number 73 resulting a codon change of lysine(Lys)to glutamic acid(Glu)at residue number 25 of REP protein causes the plasmid replication to be temperature sensitive.Moreover,the loss rate of the plasmid was approximate 100%in E.coli T1 under the condition of 42? and the plasmid loss rate was about 50%in R.sphaeroides GY-2 under the condition of 37?.The secondary structure REP protein have obvious change and the Tm value of Mrep protein is 4.2? lower than the Rep proteinby circular dichroism spectrum analysis.Furthermore,the copy number of pBBR1MCS-2M and pBBR1MCS-2 in E.coli T1 under the condition of 42? and 30?significantly decreased with the temperature sensitive plasmid copy number lower than the original plasmid,indicating that the temperature sensitive plasmid of pBBR1MCS-2M was successfully constructed.Both the copy number of pBBR1MCS-2M and pBBR1MCS-2 increased in R.sphaeroides GY-2 under the condition of 30? and 42?,indicating that the plasmids may be randomly integrated to the chromosome of R.sphaeroides GY-2.upp gene,encoding URTP(Uracil phosphoribosyl transferase),can be used in gene markerless deletion and replacement.The mutant strain R.sphaeroide GY-2 ?upp was successfully constructed by combining a temperature sensitive plasmid of pBBR1MCS-2M with a counterselectable marker through the markerless gene replacement method.At the same time,R.Sphaeroides GY-2 ?upp was transformed with the complementary plasmid of pBBR1MCS2-tac-upp.The growth experiments indicated that no apparent differences in cell growth were detected both in R.Sphaeroides GY-2?upp and wild-type R.Sphaeroides GY-2.5-FU resistance and kanamycin antibiotics sensitivity experiment showed that the strains of R.Sphaeroides GY-2 ?upp grow well in the sistrom's medium supplemented with 7?M 5-FU and are sensitive to kanamycin antibiotics,indicating excision of the temperature sensitive plasmid of pBBR1MCS-2M from the chromosome in the condition of 37?.However,both the strains of R.Sphaeroides GY-2 and R.Sphaeroides GY-2 ?upp accompanied the plasmid of pBBR1MCS2-tac-upp were lethal in the sistrom's medium supplemented with a final concentration of 2?M 5-FU.This method established the foundation for improving coenzyme Q10 production by the continuous related gene deletion in R.Sphaeroides GY-2.