Construction Of A High-Capacity Shuttle Vector Of Corynebacterium Glutamicum

Corynebacterium glutamicum is a major producer of amino acids which produces over6 million tons of amino acids per year and plays an important role in the food,medicine and health industry.Genome engineering has become an preferable approach for the improvement of C.glutamicum strains.However,large-scale genome engineering in C.glutamicum is limited due to a lack of vectors that enable delivery of large plasmid DNA into cells as autonomous replicons.In this study,we constructed of an E.coli-C.glutamicum shuttle vector pCGBACT1,which is able to accommodate large DNAs,transfer the inserted large DNA from E.coli to C.glutamicum via bacterial conjugation and exist as stable replicons in the C.glutamicum cells.The shuttle vector and the construction approach of which,could serve as a reference to tackle the challenges in the delivery of large DNAs into C.glutamicum cells.Main results are summarized as follows:(1)A 3.7-kb DNA fragment containing par A,par B,repA genes and two 22 bp-boxes was synthesized and cloned into pOK12.The constructed plasmid,pOK12CG1,was transformed into C.glutamicum ATCC 13032 and shown to stably replicate in the host cells,demonstrating that the 3.7-kb fragment was competent for replication and distribution of the plamids in C.glutamicum.An E.coli-C.glutamicum shuttle vector,pCGBACT1,was constructed by the fusing the 3.7-kb fragment,a backbone of the bacterial artificial chromosome(BAC)vector pBelo BAC11,and the ori T region of the RP4 plasmid.Further experiments showed that pCGBACT1 could be transferred from E.coli S17-1 to C.glutamicum 13032 through bacterial conjugation,and it is compatible with the Corynebacterial pBL1 and pGA1 series plasmids.(2)A high-compacity shuttle vector,pCGBACYT1,was constructed by inserting a DNA fragment containing a yeast CEN/ARS and a HIS3 gene into pCGBACT1.Large DNAs ranging from 10 kb to 60 kb were successfully assembled through yeast in vivo recombination by co-transforming linearized pCGBACYT1 and 2 to 12 fragments ranging from 4 to 6 kb.The plasmids bearing assembled large DNAs were isolated from yeast, transfered from E.coli S17-1 to C.glutamicum ATCC 13032 and verified existing as stable replicons.