Construction Of Escherichia Coli L-Lysine Producing Strain Based On Metabolic Engineering

L-Lysine plays an important role in the growth and development of humans and animals.Since L-Lysine can not be synthesized by transamination,it is one of the essential amino acids of humans and animals,which is known as the first limiting amino acid.L-Lysine is widely used in food processing,pharmaceutical preparations,feed additives and so on.The total annual demand of the world market for L-lysine is about 1.6 million tons currently,and the annual growth rate is about 7 %-8 %.Therefore it is very important to increase the yield of L-lysine.At present,L-lysine is mainly produced by direct fermentation.The fermentative strains are usually auxotrophic or deregulation of Corynebacterium glutamicums which are obtained by physical or chemical mutagenesis.These strains have the disadvantages of slow growth rate,low sugar consumption rate and poor tolerance to adverse environmental.In this paper,Escherichia coli(E.coli)engineering strains were constructed by using metabolic engineering technique.The production of L-lysine was carried out by fermentation.Compared to the Corynebacterium glutamicum whose background is not yet fully clear,gene manipulation of E.coli is simpler and further reform is conducive.At the same time the growth cycle of E.coli is short,the culture conditions are simple and the cost is low.E.coli engineering strain can be safely,efficiently and stably applied to the industrial production of L-lysine.In this paper,the engineering strains of overexpression and heterologous expression of the key enzymes of L-lysine biosynthesis pathway were constructed.The aspartate kinase gene(lysC)was cloned into vector p Trc99 A.The successful recombinant plasmid pTrc99A-lysC was constructed into E.coli ML103 to form engineering strain ZDZM11.The glucose was used as substrate for shake flask fermentation.The L-lysine yield reached 1.55 g/L.Compared with yield of the original strain E.coli ML103 was only 0.0036 g/L,which increased by 430 times.The dicarboxylic acid dehydrogenase gene(ddh)of Corynebacterium glutamicum was cloned into vector pACYCDuet-1.The successful recombinant plasmid pACYCDuet-1-ddh was constructed into E.coli ML103 to form engineering strain ZDZM12.The glucose was used as substrate for shake flask fermentation.The L-lysine yield reached 1.38 g/L.In this paper,fructose-1,6-bisphosphatase gene(fbp)was cloned into vector pTrc99 A in order to improve the efficiency of carbon source utilization during the fermentation proceess.The successful recombinant plasmid pTrc99A-fbp was constructed into E.coli ML103 to form engineering strain ZDZM13.The fructose was used as substrate for shake flask fermentation.The L-lysine yield reached 1.06 g/L.While the original strain E.coli ML103 produced almost no L-lysine.For the construction of polygene co-expressed engineering strains,in this paper,the dihydropyridine dicarboxylate synthase gene(dapA)was cloned into vector pTrc99 A for the construction of recombinant plasmid pTrc99A-dapA,and the dapA gene was cloned into the recombinant plasmid pTrc99A-lysC for the construction of recombinant plasmid pTrc99A-lysC-dapA.The recombinant plasmid pTrc99A-lysC-dapA was transferred to E.coli ML103 for construction of engineering strain ZDZM21.The L-lysine yield reached 2.33 g/L.Compared with the engineering strain ZDZM11,the yield increased by 50.32 %.The recombinant plasmids pTrc99A-lysC and pACYCDuet-1-ddh were transferred into E.coli ML103 to form engineering strain ZDZM22.The L-lysine yield reached 2.33 g/L.Compared with the engineering strain ZDZM11 and ZDZM12,the yield increased by 56.13 % and 74.98 %,separately.The recombinant plasmids pACYCDuet-1-ddh and pTrc99A-dapA were transferred into E.coli ML103 to form engineering strain ZDZM23.The L-lysine yield reached 2.58 g/L.Compared with the engineering strain ZDZM12,the yield increased by 86.55 %.The recombinant plasmids pTrc99A-lysC-dapA and pACYCDuet-1-ddh were transferred into E.coli ML103 to form engineering strain ZDZM24.The L-lysine yield reached 2.98 g/L.Compared with the engineering strain ZDZM22 and ZDZM23,the yield increased by 23.14 % and 15.5 %,separately.Compared with yield of the original strain E.coli ML103,which increased by 430 times.In this paper,the Corynebacterium glutamicum diaminopimelate dehydrogenase gene was introduced into the L-lysine production metabolic pathway of Escherichia coli while overexpression of the key enzyme gene of Escherichia coli at the same time.The efficiency of the formation of diaminopimelic acid catalyzed by hexahydropyridine dicarboxylic acid was improved.The synthesis of L-lysine was greatly promoted.While overexpressing the fructose-1,6-bisphosphatase gene of E.coli.E.coli engineering strain could use fructose to produce L-lysine.The scope of application of E.coli fermentation substrate was increased.Making L-lysine production by E.coli fermentation more economical.