Structural Modifications Of Mycolate In Corynebacterium Glutamicum And Its Application

Corynebacterium glutamicum has a unique mycolic acids layer structure mainly composed of mycolic acids and mycolic acid glycolipids.Mycolic acid glycolipids mainly include trehalose monocorynomycolates,trehalose dicorynomycolates and glucose monocorynomycolate,which plays an important role in maintaining the structural integrity of cell membrane walls,but its synthesis and assembly require a large amount of carbon and energy sources.The main objective of this study was to modify the mycolic acid layer of C.glutamate to maintain the structural integrity of the cell wall and save carbon sources and energy.In this paper,a series of mutants of C.glutamicum with altered mycolic acid glycolipid structure were constructed by genetic engineering.On the one hand,the changes of cell wall structure and lipid composition of these mutants were studied by electron microscopy,liquid chromatography mass spectrometry and high performance liquid phase.On the other hand,the changes of intracellular metabolism and the related mechanism of amino acid production increase were analyzed by transcriptomics and real-time quantitative fluorescence PCR.The main research results of this study are as following:（1）By completely blocking mycolic acid layer synthesis,the effects of mycolic acid layer on the cell morphology,cell wall structure and L-glutamate synthesis of mutant were studied.The mutant strainΔpks13 was generated by knocking out the pks13 gene encoding polyketo synthase,which catalyzes the condensation of both mycolic acid chains,in C.glutamicum ATCC13869.Compared with ATCC13869,the growth ofΔpks13 was significantly affected.The lipid molecules ofΔpks13 were extracted and analyzed by liquid chromatography mass spectrometry.It was found thatΔpks13 mainly synthesized cardiolipin and could not synthesize any mycolic glycolipid.Scanning electron microscopy analysis showed thatΔpks13 cells became larger and cell debris fell off on the surface.Transmission electron microscopy analysis of ultrathin sections revealed complete loss of the outermost mycolic acid layer of theΔpks13cell wall.L-glutamate production ofΔpks13 was 9-fold higher than that of ATCC13869 after72-hour incubation in fermentation medium.Real-time quantitative PCR analysis showed that the transcription level of mscCG was up-regulated,while odh A and arg BCDFGHJ were down-regulated inΔpks13.These results indicated that complete inhibition of mycolic acid layer synthesis significantly affected the growth of C.glutamicum,but was beneficial to increase L-glutamate production.（2）The mutant of C.glutamicum was constructed by deleting the related genes,and the effects of mycolic acid redox state structure on cell morphology,molecular composition of mycolic acid layer lipids and L-glutamate synthesis were studied.To investigate the effects of mycolic acid redox structure on the layer structure and intracellular metabolism of C.glutamicum,we knocked out the mycolic acid reductase gene cmr A in C.glutamicum ATCC13869 and constructed a mutant strainΔcmr A.After extraction and purification of mycolic acid layer lipid molecules,liquid chromatography mass spectrometry analysis showed that ATCC13869 synthesized large amounts of oxidized hydroxy-glucose monocorynomycolate,whileΔcmr A synthesized large amounts of reduced keto-glucose monocorynomycolate.Scanning electron microscopy analysis showed thatΔcmr A cells were larger than ATCC13869cells and had cell wall debris.Tem analysis of ultrathin sections revealed that the outermost mycolic acid glycolipid layer of theΔcmr A cell wall was thinned.The L-glutamate production ofΔcmr A was 10.77-fold higher than that of ATCC13869 after 72 hours of incubation in fermentation medium.Transcriptome analysis showed that the changes in fatty acid composition inΔcmr A were up-regulated in response to the transcriptional levels of the protein-coding gene far R,and the mechanosensitive channel protein-coding genes mscCG and mscCG2.However,the transcript levels of odh A,a gene encodingα-ketoglutarate dehydrogenase that competed with glutamate synthesis for carbon flow,and arg BCDFGHJ,genes involved in arginine synthesis,were down-regulated.These results indicate that mycolic acid redox state structure directly affects cell morphology and molecular composition of mycolic acid layer lipids,and is beneficial to improve L-glutamate production.（3）The effect of trehalose on the molecular composition of mycolic acid layer was studied by blocking the trehalose biosynthesis in C.glutamicum.The mutant strainΔSYA was generated by knocking out treS,treY and ots A,three key genes in the trehalose biosynthesis pathway,in C.glutamicum ATCC13869.Scanning electron microscopy analysis showed thatΔSYA cells were easily connected to each other.Lipid molecules were extracted and subjected to liquid chromatography mass spectrometry analysis,and it was found that the lipid molecules synthesized byΔSYA had shorter fatty acid chains than those synthesized by ATCC13869.ΔSYA mainly synthesized glucomycolic acid in the fermentation medium,but it could still synthesize a small amount of trehalose bimycolic acid.However,ΔSYA did not synthesize any trehalose bimycolic acid in MM medium.This indicates thatΔSYA synthesized trehalose bimycolic acid using a small amount of trehalose in the fermentation medium.When the concentration of glucose in MM medium increased from 10 g/L to 40 g/L,the synthesis of glucose monocorynomycolate by ATCC13869 gradually increased.However,ΔSYA synthesized only a small amount of glucose monocorynomycolate at 40 g/L glucose concentration.These results suggested that C.glutamicum could efficiently utilize glucose to synthesize glucommycolic acid to maintain membrane integrity in the absence of trehalose.（4）Blocking the trehalose synthesis pathway was beneficial to increase the production of L-glutamate in C.glutamicum.L-glutamate production ofΔSYA was 12.5-fold higher than that of ATCC13869.When 50 g/L glucose was added to MM medium,L-glutamate production ofΔSYA was 7.25-fold higher than that of ATCC13869.Transcriptomics and quantitative real-time PCR analysis showed that far R and mscCG were up-regulated,while odh A and arg BCDFGHJ were down-regulated inΔSYA.To further increase L-glutamate production,the key alanine biosynthesis genes avt A and ala T were knocked out inΔSYA to obtain mutantsΔSYAavt A,ΔSYAala T,andΔSYAavt Aala T.Among them,ΔSYAala T had the highest production of L-glutamate,which increased by 11%compared withΔSYA.（5）The production of L-isoleucine in C.glutamicum WM001 was increased by modifying the structure of mycolic acid layer.In L-isoleucine producing strain WM001,the production of L-isoleucine was increased by 15%after knockout of treS,treY and ots A.The knockout of glycogen synthesis pathway related genes glg C,glg A and fatty acid synthesis related genes fas B inΔtreSYA did not increase L-isoleucine production further.The mutantsΔtreSYAA,ΔtreSYAB andΔtreSYAAB were obtained by knockout of myt A and myt B inΔtreSYA.It was found that L-isoleucine production inΔtreSYAA andΔtreSYAB increased further,while the growth ofΔtreSYAAB slowed down,resulting in decreased L-isoleucine production.The natural promoter of ilv A was replaced with the L-isoleucine-induced promoter P_{brn FE7} on chromosomeΔtreSYAB,and the mutant strain WL001 was obtained by inserting the gene lrp,which encodes the positive regulation of L-isoleucine synthesis and secretion.The fermentation results showed that the yield of L-isoleucine in WL001 was 36.1%higher than that in WM001,reaching 24.98 g/L.At the same time,the by-product L-lysine production decreased by 36.2%compared withΔtreSYAB.