Effect Of Hemoglobin Transporter Deficiency On Heme Metabolism Regulation In Corynebacterium Glutamicum

Heme is a biological macromolecule that is commonly found in living organisms.It can be used as a cofacter to participate in a variety of metabolic activities.At the same time,it can also provide iron for the organisms.But excessive free heme has a toxic effect,C.glutamicum has a rather sophisticated and complex regulatory system to ensure the dynamic balance of heme.It was easy to accumulate excessive downstream products in the production of ALA by C.glutamicum.Then cell growth and respiratory metabolism were affected.Due to the important physicological functions of heme,heme synthesis pathway could not be blocked or knocked out,it could only be weakened in the production of ALA by recombinant bacteria.In C.glutamicum,heme is regulated by two component systems,HrrSA and ChrSA at the transcriptional level.HrrSA is mainly used to regulate the transcriptional level of heme synthesis pathway and heme metabolic pathway,ChrSA is only activated when the heme concentration is excessive.ChrSA activates the transcriptional expression of the heme transporter HrtBA gene,to transport excess heme into the extracellular.In this paper,firstly we constructed the mutant strain WT-H and WT-HH to study the effects of heme on intracellular accumulation,heme synthesis pathway and metabolic regulation pathway.Experiments showed that the amount of intracellular free heme was dynamically balanced when the hemoglobin transporter was absent.Strains accumulated a large amount of heme metabolites and secreted into extracellular.The transcription level of HrrSA and ChrSA,heme oxidase gene,heme-binding protein gene was up regulated,the gene transcription level of heme synthesis pathway was down regulated.This was due to the accumulation of free heme in the cell,the two component system maintained the dynamic balance of intracellular free heme by reducing the transcriptional level of the heme synthesis pathway and improving the transcriptional level of the heme metabolic pathway.By adding a degradation tag to the C-terminus of the C.glutamicum hemB gene,we could effectively reduce the activity of intracellular ALA dehydratase,and reduce the accumulation of PBG and porphyrins.The amount of free heme in the cell could still maintain a dynamic balance.Secondly,we overexpressed the ALA gene in C.glutamicum to study the effect of ALA on heme accumulation.The results showed that intracellular free heme maintained a dynamic balance when ALA was accumulated excessively.The deletion of heme transporter was beneficial to increase the accumulation of ALA.When the cells produced ALA,the color of the bacteria was deepened.And it was found that a large amount of porphyrins accumulated in the fermentation broth,which proved that the color of the bacteria was deepened by porphyrins.The transcriptional level of the two component system and the heme pathway were up regulated.This is due to ALA could promote the downstream metabolism,thus accumulating excessive intermediate metabolites.As the two component system gene transcriptional level up regulated,then it adjusted the excess intracellular free heme to achieve a dynamic balance.The addition of a degraded label to the end of the hemB gene could effectively increase the accumulation of ALA.In summary,when the heme transporter gene was deleted,the amount of intracellular free heme maintained a dynamic balance,and the cells accumulated a large amount of porphyrins.The strain mainly regulated the amount of intracellular heme at the transcriptional level through a two component system.On the one hand,it inhibited the transcription level of the heme synthesis pathway.On the other hand,it enhanced the transcription level of heme metabolism related genes.Reducing the accumulation of downstream metabolites could effectively increase the yield of ALA.To study the effect of heme metabolic regulation on the deletion of heme transporter could provide a theoretical basis for the later study of heme and ALA.