Mechanism For Conjugated Linoleic Acid Transformation By Bifidobacteria

Conjugated linoleic acid?CLA?has been identified to exert heal-promoting effect due to its anti-oxidation,anti-inflammation,anti-cancer.Recently,the source of dietary CLA was mainly from dairy and meat products.However,the amount of CLA among these products was very low,which could not meet our daily needs.Special microbes have been reported to produce CLA and in the future,microbial CLA generation might be one of the most attractive methods due to its simple composition of the products,as well as characteristics of short period.Bifidobacteria possessing CLA-producing ability has been widely to be beneficial to the health.Even though several studies have reported that bifidobacteria possessed CLA-producing ability,the whole transformation pathway,functional gene and also the regulating mechanism were not still unknown.In order to systemically understand more about the precise transformation pathway and also the regulating mechanism of bifidobacterial CLA production,the transcriptomics,gene heterogenous expression and gene knock-out has been employed,which aimed at providing theoretical and technical foundation for the industrialization of CLA-producing bifidobacteria.This study firstly screened the generation of CLA and10-hydroxy-cis-12-octadecenoic acid?10-HOE?of 169 Bifidobacterium strains and analyzed the producing characteristics at the level of bifidobacterial species.In this study,the CLA conversion rate of Bifidobacterium breve?B.breve?CCFM683 was identified to be the highest.The precise CLA-producing pathway from linoleic acid?LA?was confirmed through the method of stable isotope labelling with ¹³C-labelled LA as the substrate.And then the key gene involved in bifidobacterial CLA production has been discovered in combination of the transcriptomics,weighted gene co-expression network analysis?WGCNA?,heterologous expression.Furthermore,the precise action form of this enzyme in helping strains respond to the stress of free LA would be also discussed.Finally,the regulating gene as well as the regulating mechanism in process of CLA production would be also investigated.The main results were as follows:?1?This article identified the characteristics of CLA-and 10-HOE production by different Bifidobacterium species and also elucidated the general transformation pathway of LA into CLA and 10-HOE.This study firstly screened the conversion rate of CLA and 10-HOE among the 169 bifidobacterial strains,and results showed that B.breve were identified to be high CLA-producers,in which B.breve CCFM683revealed the highest CLA-producer with generation ratio as 90.3%,followed by B.pseudocatenulatum,B.longum,B.dentium.However,no strains,belonging to B.adolescentis,B.bifidum,B.animalis,B.angulatum,have been screened to generate CLA.In addition,10-HOE was found to be generated by nearly all the selected bifidobacterial strains,in which B.pseudocatenulatum CCFM749 revealed the highest conversion rate.Furthermore,B.breve CCFM683 could produce CLA with either LA or 10-HOE as the substrate.However,in this case,10-HOE must be firstly transferred into LA and the generated LA could be then converted into CLA.?2?This article discovered the gene encoding linoleic acid isomerase among B.breve CCFM683.This study firstly employed stable isotope labelled-linoleic acid(¹³C-LA)as the substrate and the final products labelled with ¹³C would be analyzed through GC-MS.Results showed that no other intermediates existed in the process of transformation of LA into CLA,suggesting that this process was a one-step reaction.Transcriptomic analysis revealed that free LA would induce significant gene transcription variation?p<0.05?.The two genes with no annotated function?CCFM683GM001103 and CCFM683GM1531?have been predicted to be candidate genes encoding linoleic acid isomerase among bifidobacteria via WGCNA.And finally,the results revealed that ony the gene with ID as CCFM683GM001103,was responsible for CLA generation,named as Bifidobacterium breve isomerase?BBI?.BBI contains nine trans-membrane domains,the function of which was firtly reported.In addition,this isomerase was the single enzyme with known amino acid sequence,in recent time,that was responsible for generating cis9,trans11-CLA through one step.In addition,BBI could only employ free LA as the substrate.Additionally,this enzyme preferred to employing the compounds?such as LA,linolenic acid?with more than two double bonds as the substrates and could not use the compounds?such as oleic acid and stearic acid?with the double bonds less than two.In recent time,the conversion rate for different substrates was as follows:?-linolenic acid?ALA?>LA>?-linolenic acid?GLA?.?3?BBI and MCRA would exert important role in helping B.breve CCFM683respond to the stress of free LA.Results showed that knock-out of bbi gene would result in the decreased adaptability of B.breve CCFM683 to the stress of free LA.Additionally,knock-down of mcra gene could also lead to its decreased adaptability to the stress of free LA.These results suggested that these two proteins would be very pivotal in protecting the strain from the damage of the free linoleic acid.Furthermore,the combination of differential centrifugation and western blot demonstrated that BBI existed in the cytoplasmic membrane,and MCRA was in the cytoplasm.Further study about the intra-cellular and extra-cellular variation of LA,CLA and 10-HOE suggested that LA might be possibly converted into CLA directly by BBI extracellularly and MCRA would only employ the little permeated LA as the substrate to generate 10-HOE.?4?Clg R,a HTH?Helix-Turn-Helix?-type transcription regulator,could directly regulate the transcription of bbi and mcra.Overexpression of clgr gene would increase the transcription of bbi,mcra and also the adaptability to free LA was also increased;knock-down of clgr gene would decrease the transcription of bbi,mcra and also the adaptability to free LA was also decreased.Electrophoretic mobility shift assay?EMSA?revealed that Clg R could be specially bound to the upstream of bbi and mcra.These results suggested that Clg R would be responsible for the transcription of bbi and mcra.Further study showed that the fourth alpha-helix?from N-terminal to C-terminal?would be the functional region bound with the upstream of bbi and mcra.And also the precise interacting point for the upstream of bbi with Clg R was?G?7?--G---GCG-----TAG--CG--C?32??,while the point for upstream of mcra with Clg R was?G?7?--G------GCACA------TAT—CG?34??.These results suggested that Clg R could regulating the response of B.breve CCFM683 to the free LA through controlling the transcripton of bbi and mcra.?5?Distribution of bbi and mcra gene has been related with the generation of CLA and 10-HOE by bifidobacteria,respectively.Bioinformatic analysis demonstrated that those CLA-producing bifidobacteria,including B.breve,B.longum,B.pseudocatenulatum,B.dentium,contained bbi gene,while bbi gene was not detected among those non-CLA producers,such as B.bifidum,B.adolescentis,B.animalis,B.angulatum.However,several strains within bbi gene could not produce CLA.Similarly,those which could generate 10-HOE possess the mcra gene,and however,the bifidobacterial strains with mcra gene,could possibly not produce10-HOE.