Study Of Conjugated Linoleic Acid Bioconversion In Lactic Acid Bacteria

Conjugated linoleic acid (CLA) refers to a group of positional and geometricisomers of linoleic acid (LA) with conjugated double bonds and has attractedincreased interest because of its biologically beneficial functions. In the past decades,a number of publications reported microbial CLA producers such as butyrvibrio,propionbacteria, lactobacilli and bifidobacteria. Compared to the linoleate isomerasefrom Propionbacterium ances, which has been full characterized and crystallized, theenzyme(s) for CLA bioconversion in lactic acid bacteria is still unknown.To understand the CLA production and the differences among lactic acid bacteria,a selection of strains were assessed for CLA production from free linoleic acid and thepossible intermediates were analyzed firstly. Some high CLA producers were screened.Then MCRAs from Lactobacillus plantarum ZS2058, Lb. plantarum ST-III,Bifidobacterium animalis subsp. lactis BB-12, Lb. acidophilus NCFM, Lb. rhamnosusLGG and Lb. casei Zhang, which were highly homologous to the putative linoleateisomerase in lactic acid bacteria, were studied. The whole genome of Lb. plantarumZS2058was sequenced and analysed. And the mechanism for CLA production wasconfirmed. According to those results, the triple-component linoleate isomerase in Lb.plantarum ST-III, a non-CLA producer, was identified. At last the presence oftriple-component linoleate isomerase was analysed by bioinformatics in all of thelactic acid bacteria genomes. Finally the transcription levels of each content oflinoleate isomerase in several strains, which had differernt CLA production ability,were analyzed to illustrate the reason for different CLA production abilities. Mainresults were described as follows.(1) After culturing with free LA in the media, fatty acid profiles of bothsupernatant and pellets from different derived and species of lactic acid bacteriastrains were assessed to screen CLA producers. All the assessed88strains couldmetabolize linoleic acid to10-HOE at different concentrations. Totally16high CLAproducers were screened, such as Lb. plantarum, Lb. acidophilus, Lb. bulgaricus, Lb.reuteri and some Bifidobacteria strains. The ability for CLA bioconversion wasshown strain dependent significantly in which Lb. plantarum ZS2058, Lb. plantarumCCFM47, Lb. plantarum CCFM232and Lb. bulgaricus CCFM4could convert over30%of free linoleic acid to c9, t11-CLA and t9, t11-CLA. Oleic acid was increasednotably as well as CLA in20strains including Lb. bulgaricus CCFM29, Lb.plantarum CCFM169, Lb. plantarum CCFM176.(2) Myosin cross reactive antigens from Lb. plantarum ZS2058, B. animalissubsp. lactis BB-12, Lb. plantarum ST-III, Lb. acidophilus NCFM, Lb. casei Zhangand Lb. rhamnosus GG showed highly homologous to putative linoleate isomerase.The six MCRAs were cloned, expressed and purified in E. coli. With linoleic acid and oleic acid as substrate separately, the correspondent hydroxyl derivatives wereproduced by the recombinant MCRAs, which means MCRA was fatty acid hydratasenor linoleate isomerase and might be involved in the CLA synthesis.(3) Lb. plantarum ZS2058accumulated some intermediates during CLAproduction including10-HOE,10-oxo-cis-12-octadecenoic acid,10-oxo-trans-11-octadecenoic and10-oxo-octadacenoic acid. The complete genomeof Lb. plantarum ZS2058was sequenced and annotated. The putative linoleateisomerase in Lb. plantarum ZS2058was a triple-component enzyme includinglinoleate/oleate hydratase (MCRA), short-chain hydrogenase/oxidoreductase (DH),acetoactate decarboxylase (DC), which might be the genetic determinants for CLAproduction. Those three genes were cloned and expressed in E. coli. The productscatalysed by those enzymes were determined and those results showed linoleatehydratase converted LA into10-HOE, DH catalysed10-HOE to10-oxo-cis-12-octadecenoic acid and DC converted10-oxo-cis-12-octadecenoic acidto10-oxo-trans-11-octadecenoic acid. With the three enzymes combination, CLA wasproduced. Based on the fatty acid profile both from Lb. plantarum ZS2058andrecombinant proteins converted, the synthesis for CLA was multi-steps: LA hydrationto10-HOE, followed by10-oxo-cis-12-octadecenoic acid and10-oxo-trans-11-octadecenoic acid, with CLA as the end product.(4) The genome comparison between Lb. plantarum ST-III, which can’tsynthesize CLA significantly, and Lb. plantarum ZS2058was made by bioinformaticsmethods. It showed that the triple-component linoleate isomerase in Lb. plantarumST-III was found as linoleate hydrase, short-chain dehydrogenase/oxidoreductase andacetoacetate decarboxylase. The three genes were cloned and expressed in E. coliseparately. The fatty acid contents after each recombinant catalysed were analysed.The results indicated that LA was converted to10-HOE by linoleate hydrase followedby conversion to10-oxo-cis-12-octadecenoic acid,10-oxo-trans-11-octadecenoic acidand ended with CLA. From both genes and protein functions, Lb. plantarum ST-IIImight have the similar CLA production capacity as Lb. plantarum ZS2058, but it didnot exhibit CLA production, which was not caused by activity loss oftriple-component linoleate isomerase.(5) The presence of linoleate isomerase in all of the available lactic acid bacteriagenomes was analysed through bioinformatics. The results demonstrated that thetriple-component linoleate isomerase differed in lactic acid bacteria, but most strainshad the three genes. To understand the reason for non-CLA producers, thetranscription of the genes involved in LA metabolism and CLA bioconversion inseveral CLA producers and non-CLA producers including Lb. plantarum ZS2058, Lb.plantarum CCFM261, Lb. plantarum ST-III, Lb. reuteri ATCC55739, Lb. reuteriCCFM14, Lb. casei Zhang and Lb. casei BD-II, were analyzed by real-time quantityPCR. The results showed that the different transcription levels of short-chain dehydrogenase/oxidoreductase and acetoacetate decarboxylase resulted in differentCLA production ability.