Improvement Of Pyruvate Production By Escherichia Coli Via Genomic Mediated Mutagenesis

Pyruvate is one of the most important intermediate products,and also a component of the human body.It can be slowly polymerize when heated,and also can participate in a variety of chemical reactions;in the process of bio-metabolism pyruvic acid has a connecting link role in sugar,fat and protein metabolism route,which provides the raw material for the tricarboxylic acid cycle.Because of the simple genetic background and easy laboratory experiments,the pyruvic acid pathway construction in Escherichia coli has a great advantage.We used the Tn5 transposon and dnaQ mutant genome replication engineering methods to assist randomize mutations in the genomic level of the two pyruvate-producing E.coli strains to find more genes that are beneficial for pyruvate-producing.We constructed two mutant library,and found an efficient method for screening pyruvate-producing strains.The mutants of improved pyruvate production have been found and then the location of insertion sites has been found out,too.We also analyzed the possible reasons for improved pyruvate production.In order to further build a new high pyruvate yield strain,this paper provides a theoretical basis,at the same time by analyzed the individual gene in-depth way,more information have been obtained.In the first method,Escherichia coli MG1655 was used as the starting strain to construct the KLPP,which was a pyruvate-producing strain.Then the KLPP mutant library was built,containing 7197 monoclones which constructed by using pUT Mini-Tn5 vector.With the rapid chromogenic reaction of dinitrophenylhydrazine and effective utilization of eliasa,7197 mutant strains were screened successfully.The results showed that the yield of pyruvic acid produced by the selected six strains was 38%,31%,19%,28%,44%,14% higher than that of KLPP.In order to verify the stability of pyruvate yield increase,the six strains fermentation yield of 5L fermentor was evaluated by batch fermentation.The K30 fermentation yield was 67 g / L after 48 h fermentation.Then we not only used whole genome resequencing to identify four genomic insert sites,but also identified the genetic loci that could affect pyruvate production,which laid the foundation for the subsequent transformation of the strain.In the second method,in order to further improve the production capacity of pyruvate-producing E.coli strains,we used the new technology of evolutionary engineering(the genomic replication project based on dnaQ mutant)which did not harm the host bacteria to build the engineering mutant library.In this paper,we used a pre-built laboratory strains of E.coli which producing pyruvate as the starting strain named A1(base on the KLPP,cat-sacB inserted into the end of PdhR gene).Further random mutations were performed using three different intensity plasmid called pKD46-dnaQ-B(high),pKD46-dnaQ-K(middle),pKD46-dnaQ-A(low)to construct a randomized library of mutants containing 9664 monoclones.Then,9664 mutant strains were screened by pyruvate-based dinitrophenylhydrazi-ne colorimetric method,and three mutant strains were improved by about 14% higher pyruvate than that of the original strain A1.Then 10 genes which could affect pyruvate producting were identified by whole genome resequencing,and the causes of high pyruvic acid yield were analyzed.By comparing the two methods of establishing the mutant library,we found that transposon single gene mutations have a significant effect on the increase of pyruvate yield,this finding not only provides the basis for pyruvate biosynthesis,but also provides a method for the biosynthesis of other substances...