Research And Application Of The Genes (hpr And GlyA) Involved In The Serine Cycle Of Methylobacterium Sp MB200

Methylotrophic bacteria are a group of microorganisms that are able to use compounds containing one-carbon as well as multi-carbons as energy and carbon sources.These organisms can convert methanol to chemicals and materials biologically.A strain named MB200,can utilize methanol as the sole carbon and energy soure,was obtained from environment in our former work.This bacteria pocesses L-serine cycle to assimilate methanol.In order to enhance accumulating L-serine amd glyoxylate by this strain,the improved strains were constructed through increase the expressing level of two key genes (hpr,glyA)involved in L-serine cycle.glyA,a gene which encodes serinehydroxy methyltransferase(SHMT)in organisms,SHMT plays an improtant role as the first enzyme in the assimilation of C1 compounds through the transfer of formaldehyde to glycine,thus giving the main intermediate in the pathway,serine.A glyA gene was cloned from the genomic DNA of M.sp MB200,this gene encdes for the 434-amino-acid protein with a calculated molecular mass of 48230 Da.The amino-acid sequence of the enzyme showed identity to the sequence of the enzyme from M.extorquens AM1 (94%).The transfermed E.coli cells could express SHMT which has enzyme activity.With the suicide plasmid pK18mob,a mutant MB200gTB inactivated in glyA gene has been constructed through homologous recombination caused by a single crossover event.The experiments showed that this mutant can't utilize methanol as the sole carbon source and can't accumulate L-serine any more,no SHMT enzyme activity can be detected in the cells,but could grow on the medium with multi-carbon coumpounds.This gene was ligated into the vector pLAFR3 to obtained recombinant plasmid pLAFRg,which was transfered into M.sp.MB200 to generate MB202.The improved strain MB202 was found to exhibit the higher L-serine productivity than the wild strain MB200,11.4±0.6 mg/mL L-serine was produced from 20mg/mL glycine and 70mg/mL methanol in 2d when used a resting cell system of MB202,the yield was about 5-fold of that produced by MB200.After optmizing the fermentation conditions,an optimun conditions for resting cell system were:methanol 70 mg/mL;glycine 50 mg/mL;pH8.8;and 37 degrees C,under the under above conditions,the improved strain MB202 can produce 24.3±1.0 mg/mL L-serine.When use the conditions of 0.3 g/mL of cells(as wet matter),3%sodium alginate and 6% CaCL₂,the average output of L-serine produced by immobilized cells is 12.7±0.9 mg/mL per bacth and can be fermented for 5 bacthes.The results will facilitate enhancing the conversion rate of methanol to L-serine using M.sp strains.hpr encodes hydroxypyruvate reductase(HPR),an enzyme catalyzes the reversible conversion of hydroxypyruvate to D(-)glycerate,and D(-)glycerate is further metabolized to glyoxylate in the serine cycle in M.sp bacteria.A hpr gene was amplified from the total DNA of M.sp.MB200,sequenceing analysis showed that an ORF of 945bp was included in this fragment coding 315 amino acids with a molecular weight of approximately 37,000 Da,The purified HPR enzyme was performed to use NADPH as cofactor,the optimal PH for the reduction of hydroxypyruvate is about 5.5,and temprature has litter effect on it's activity.The mutant MB200hTB was construct by the suicide plasmid pK18mob ligated a part of glyA sequence.The result showed that MB200hTB had no HPR activity and lost its ability to grow on C-1 compounds but retained the ability to grow on C-2 compounds and multi-carbon compounds.The gene was ligated into the vector pLAFR3 to obtained the recombinant plasmid pLAFRh,which was transferred into M.sp.MB200 to generate an recombinant strain MB201. Homologous expression of hpr under the control of the lacZ promoter led to the enhanced glyoxylate accumulation in cultures of Methylobacterium sp MB201. The yield of glyoxylate reached 14.4±1.1 mg/mL,representing nearly a two-fold increase when compared with the wild-type strain.An important strategy to develop a synthesis method of commercial chemicals and materials by bacteria is to deregulate and increase the levels of gene expression,especially to increase the copy number of genes,by genetic engineering.In this report,a glyA gene with it's promoter was over-expressed in M.sp.MB200 lead to ehance the yield of L-serine,and a hpr gene was over-expressed under the control of lacZ promoter in the pLAFR₃ vector in M. sp.MB200 to produce glyoxylate.The results will facilitate researching and applicating M.sp.MB200 and it's genes to biosynthesize more products.