Biocatalytic Synthesis Of Ump And Orotic Acid Ribose Transfer Enzyme Characteristics

Nucleotides are central metabolites in the complex metabolic network of cell. They are the precursors of DNA and RNA,and carriers of reductive power as well as playing an essential role in the energy transduction and in the energy conservation of cell.They also play important functions in the coordination of metabolic processes and control.Pyrimidine nucleotides are wildly used in the agriculture,food industry and medical industry.They are directly involved in carbon and lipid metebolism providing energy-rich precursors for biosynthesis of oligosaccharides, which have been the focus of much research because of their roles in biochemical recognition processes.Uridine 5'-monophosphate(UMP)is an important nucleotide which can be easily converted to other pyrimidine nucleotides.It is an essencial material in production of oligosaccharides and some anti-cancer drugs. However,the synthesis of UMP usually needs a long time and results in a low yield. The separation and isolation of product are also generally difficult and expensive to perform.Biocatalytic process for nucleotides production use microorganism as enzyme source to convert relative precursor into nucleotides.Compared with other nucleotide production methods,this way can shorten the productive time,improve the yield and make the separation and isolation of product easilier.Furthermore, complex nucleotides,sugar nucleotide and even some oligosaccharides can be synthesized by this method using coupling metabolically engineered bacteria.This is important for the industry of nucleotide,medicine,glycochemistry and glycobiology.In this dissertation,two methods to assay nucleotides were established. Thin-layer chromatography(TLC)was used to assay orotate and UMP in the biocatalytic reaction mixture.While by using HPLC,several different nucleotides and bases could be separated and quantified.This part laid a foundation for the subsequent research of UMP production by biocatalytic process. Attempts were made with success to develop a two-step biocatalytic process for UMP production from orotic acid by Corynebacterium ammoniagenes ATCC 6872; the strain was first cultivated in a high salt mineral medium,and then cells were harvested and used as the catalyst in the UMP production reaction.Effects of cultivation and reaction conditions on UMP production were investigated.The cells exhibited the highest biocatalytic ability when cultivated in a medium containing corn steep liquor at pH 7.0 for 15 h in the exponential phase of growth.To optimize the reaction,both "one-factor-at-a-time" method and statistical method were performed. By "one-factor-at-a-time" optimization,orotic acid,glucose,phosphate ion (equimolar KH₂PO₄ and K₂HPO₄),MgCl₂,Triton X-100 were shown to be the optimum components for the biocatalytic reaction.Phosphate ion and C. ammoniagenes cell were furthermore demonstrated as the most important main effects on UMP production by Plackett-Burman design,indicating that 5-phosphoribosyl-1-pyrophosphate(PRPP)synthesis was the rate-limiting step for pyrimidine nucleotides production.Optimization by a central composition design (CCD)was then performed,and up to 32 mM(10.4 gl^-1)UMP was accumulated in 24 h from 38.5 mM(6 gl^-1)orotic acid.The yield was three-fold higher than the original UMP yield before optimization.Orotate phosphoribosyltransferase(OPRTase)catalyzes the reaction between orotate and the ribose-5-phosphate donor 5-phosphoribosyl-1-pyrophosphate(PRPP) to form orotidine-5-phosphate(OMP)and pyrophosphate.This is the first step in the biocatalytic process of UMP production.A 623-bp fragment was cloned by nested PCR and colony blotting method from C.ammoniagenes ATCC 6872,which was used in the biocatalytic production of UMP.Sequence analysis revealed that the fragment (p26)was consisted with a 555-nt-long coding region identical to the pyrE gene from C.glutamicum ATCC 13032,and a 63-nt region upstream the initiation trinucleotide, ATG The amino acid sequence of the cloned OPRTase,as deduced from the nucleotide sequence was compared with the reported OPRTase sequences from other organisms.Although the OPRTases from Escherichia coli and Salmonella typhimurium,which had been studied in detail,showed only 28% similarity with the cloned enzyme,a PRPP binding motif,a solvent-exposed catalytic loop,and some essential residues(Arg 99,Lys 100,and Lys 103)are well assigned in the cloned enzyme.However,it is noteworthy that several amino acid residues in active site of the S.typhimurium OPRTase can not be assigned in the C.ammoniagenes sequence by the alignment.Some of these residues are displaced by other residues with similar side chain or with similar polarity.But sequence analysis shows that there is a lack of an important conserved lysine(Lys 73 in Salmonella typhimurium OPRTase) in the C.ammoniagenes OPRTase.This lysine has been considered to contribute to the initiation of catalysis.The enzyme was overexpressed and purified from a recombinant E.coli.The molecular weight of the purified OPRTase was determined to be 45.4±1.5 kDa by gel filtration.Since the molecular mass of the enzyme was 21.3±0.6 kDa for the subunit,the native enzyme exists as a dimer.Divalent magnesium was necessary for the activity of the enzyme,and can be substituted by Mn²⁺and Co²⁺.The optimal pH for forward(phosphoribosyl transfer)reaction is 10.5-11.5,which is higher than that of other reported OPRTase,and the optimal pH for reverse(pyrophosphorolysis) reaction is 5.5-6.5.The K_m values for the four substrates were determined to be 33μM for orotate,64μM for PRPP,45μM for OMP,and 36μM for pyrophosphate, respectively.Compared with other reported OPRTases,the OPRTase from C.ammoniagenes showed some different properties;(1)The dissimilar pH dependence between forward and reverse reactions.(2)The higher optimal pH for the forward reaction. (3)The K_m value for OMP is much larger than those of other organisms.These differences may be due to the absence of Lys 73 present in the active site of other OPRTases and known to interact with OMP and PRPP.And the catalytic biochemistry of the enzyme may be different from the known mehanisms.De novo synthesis of pyrimidines involves utilization of energy-rich labile intermediates such as carbamoyl phosphate,PRPP,and nucleoside triphosphates. The enzymes involved in UMP biosynthesis were strictly controlled by exogenous pyrimidines.In our research,the activity of OPRTase in C.ammoniagenes ATCC 6872 was showed repressed in the presence of exogenous uracil as well.Then the 63 nucleotides upstream the coding region were cloned together with the pyrE gene from C.ammoniagenes ATCC 6872.The expression experiments revealed that in E.coli, the specific activities of overexpressed OPRTase were depressed with exogenous uracil when the 63 nucleotides upstream exist,while the specific activities remained constant under the same conditions when the upstream region was removed.These indicate that pyrE expression is regulated in response to pyrimidines and the 63-nt upstream region participates in the regulation.The pyrimidine-sensitive transcriptional attenuation may be the most important and common mechanism in bacteria,which contributes to the regulation of the expression of pyrimidine synthetic(pyr)genes.The feature common to the mechanism is a Rho-independent terminator structure named attenuator in mRNA,whose key features are inverted repeats and a string of Ts in the nontemplate strand.However, sequence analysis showed the 63 nucleotides region immediately upstream of C. ammoniagenes pyrE contained neither a string of Ts nor a region of inverted repeats as the attenuator does.Moreover,the subsequent expression experiments performed in C.ammoniagenes showed that the expression of p26 was not sensitive to uracil, indicating that the mode of pyrimidine-specific regulation of the 63-bp fragment doesn't exist in Coryneform bacteria,but only in E.coli.C.ammoniagenes is a Gram-positive non-pathogenic soil bacterium,which has strong ATP regeneration activity with sufficient PRPP supply,and has been widely used to produce UMP from orotic acid by either fermentative process or biocatalytic process.Functioning as the enzyme source,the cell is obviously a controlling factor in UMP production.OPRTase catalyzes the first step in the UMP production between orotate and the ribose-5-phosphate donor PRPP to form OMP.The expression of the enzyme has been shown to be strictly controlled by pyrimidines nucleotides.When the in vivo concentration of UMP is high,the expression of the enzyme will be repressed.This mechanism limits the yield of UMP.Therefore,we cloned a promoter IJ59,which could initiate transcription sufficiently without induction both in C.ammoniagenes ATCC6872 and E.coli.The promoter was ligated into a shuttle vector pXMJ19 with p26 forming a plasmid pXMJIE,which could overexpress OPRTase in both E.coli and C.ammoniagenes ATCC6872 at absence of IPTG.The expression experiments in E.coli showed that when cultivated for 12 h without induction,the specific activity of OPRTase from the E.coli harboring pXMJIE were over 5-fold higher than that from the E.coli without the plasmid. Although the expression of the plasmid in C.ammoniagenes is still under investigating,the promoter IJ59 has been reported to be more effient in Coryneform bacteria than in E.coli.Therefore,a recombinant C.ammoniagenes which can overexpress OPRTase would be constructed,and could be used to improve the UMP yield.