Impacts On Mycolic Acid Deficiency In Corynebacterium Glutamicum ATCC13869

Mycolic acid(MA)is endemic component of CMN bacteria family,which stands for Corynebacteria—Mycobacteria—Nocardia.Corynebacterium glutamicum belongs to Corynebacteria,contains tons of MA in its cell wall.MA in Mycobacterium tuberculosis is intensively studied.However,it is still barely known about the functions on C.glutamicum MA.As a common industrial applied amino acid prototype producer,C.glutamicum is widely reconstructed by metabolic engineering to develop its production,and the study on MA could provide new orientation for it.In this study,a MA deficiency strain of C.glutamicum wild type ATCC13869 was constructed to investigate the impacts of non-MA on cells,and understand the functions of MA in C.glutamicum.The major conclusions are stated below:(1)Markless deletion of polyketide synthase coding gene BBD29_RS14045 in C.glutamicum ATCC13869 by using Cre/loxp system,the deletion mutant strain was renamed WG001;(2)Series of phenotypic experiments and morphological observations on WG001 demonstrated that many outstanding differences happened on many aspects in cells,such as growth,auto-aggregation,antibiotics resistence,electroporation efficiency,cell shape and cell division;(3)Flask fermentation of WG001 exhibited conspicuous elevations as over 20% on 10 amino acids measured by HPLC,especially for L-Aspartate as 1305%,L-Glutamate as 3467% and L-Threonine as 3728%,suggesting that MA could be a potential target for amino acid engineering;(4)Extraction of the MAs,fatty acids,phospholipids and cell wall glycosyls from ATCC13869 and WG001,with further analyses with TLC,ESI/MS and IC,WG001 cells were totally lack of MAs,longer and more unsaturated in fatty acids and phospholipids,contained less phosphatidylglycerol and galactose but more phosphatidylinositol and rhamnose.All these variations suggested that MA deficiency caused the alteration of cell wall and membrane;(5)With further transcriptome sequencing and analysis,comparing to ATCC13869,WG001 overall had 2692 genes transcriptionally regulated,which were relevant to many aspects on cell.Transcriptome data were identical and interactively supportive with the phenotypic changes shown by WG001.Otherwise,many uninvolved variations in experiment were revealed.All these signified that MA plays unparalleled roles on whole cell;(6)Polyketide synthase complemetary strain was constructed,of which growth,amino acid production,MA were all tendency to wild type but not completely.Selected genes arc and vgb,their recombination strains of WG001 were built,both growth were better but somewhat worse on amino acid production.