Study On The Influence Of The Pgs Bca Components’ Interaction On Poly-γ-glutamic Acid Synthesis

γ-PGA is a natural biopolymer with physiological functions such as biocompatibility,biodegradability and moisturizing.γ-PGA has important applications in the fields of biomedicine,agriculture and cosmetics.γ-PGA synthase(PgsBCA multiprotein complex)is the only enzyme responsible for the synthesis of γ-PGA,consisting of three component proteins,PgsB,PgsC and PgsA.In the early stage of our research group,the PgsBCA from Bacillus licheniformis was expressed heterologously with the industrial strain of Corynebacterium glutamicum F343 as the chassis cell,and the de novo synthesis of γ-PGA was successfully achieved with glucose as the substrate.However,the mechanism by which the PgsBCA multiprotein complex affects the synthesis of γ-PGA is still unclear,limiting the further improvement of the synthesis ability of γ-PGA.This study focused on the influence of PgsBCA components’ interaction on γ-PGA synthesis,and firstly determined the localization of PgsB,PgsC and PgsA and their necessity for γ-PGA synthesis.On this basis,the regularity of the interaction of PgsB,PgsC and PgsA component on the production of γ-PGA was found by changing the expression intensity and source of PgsB,PgsC and PgsA respectively.This study provides a theoretical basis for further improving the production of γ-PGA by regulating the expression of PgsBCA multi-protein complex.The main research contents are as follows:(1)In order to study the location of PgsB,PgsC and PgsA,the subcellular localization and transmembrane region of PgsB,PgsC and PgsA were firstly analyzed by bioinformatics,and it was predicted that PgsB,PgsC and PgsA were all localized on the cell membrane.Then,C.glutamicum F343 was used as the host to express the fusion fluorescent protein,and the localization of the component was visualized by laser confocal.The membrane localization of PgsB,PgsC and PgsA was further determined.Finally,the 3D structures of PgsB,PgsC and PgsA were predicted using Alphafold2.Combining the information of the transmembrane helix,a model for the localization of components in C.glutamicum is proposed.(2)In order to explore the necessity of PgsB,PgsC and PgsA for the synthesis of γ-PGA,C.glutamicum F343,a high glutamate-producing species,was used as the host in this study.Single expression and combined expression were used to verify the γ-PGA synthesis ability of the strain.It was found that γ-PGA could be synthesized only when PgsB,PgsC and PgsA were simultaneously expressed,thus confirming that PgsB,PgsC and PgsA are indispensable for γ-PGA synthesis.(3)In order to explore the effect of the interaction of PgsB,PgsC and PgsA at different expression levels on the synthesis of γ-PGA,operon engineering was used to regulate PgsB,PgsC and PgsA at high,medium and low levels,respectively.It was found that the synthesis ofγ-PGA was favorable when PgsB and PgsC were expressed at high levels and PgsA was expressed at medium levels.Moreover,the expression level of PgsC had the greatest effect onγ-PGA synthesis.(4)In order to explore the effect of the interaction of different sources of PgsB,PgsC and PgsA components on the synthesis of γ-PGA,the sources of PgsB,PgsC and PgsA were replaced respectively on the basis of expressing PgsBCA derived from B.licheniformis ATCC9945 a in C.glutamicum F343.It was found that replacing the source of PgsB alone is beneficial to γ-PGA synthesis,replacing the source of PgsC alone will make the titer of γ-PGA drop sharply,and replacing the source of PgsA has a little effect on the titer of γ-PGA.