Studies On The Mechanisms In The Production Of Lactic Acid With High Optical Purity By Bacterial Fermentation

The optically pure L-lactic acid monomers can be used as precursors in the synthesis of polylactic acid(PLA),which is renewably biodegradable material and its demand is consistently on the rise.Bacillus coagulans is an excellently thermostable producer of optically pure L-lactic acid(the optical purities reach 99%).However,little is known about the mechanism of synthesis of the highly optically pure L-lactic acid produced by this strain.Three enzymes are responsible for lactic acid production in gene annotation of genome-NAD-dependent L-lactate dehydrogenase(L-nLDH1;encoded by ldhL1,L-LDH2;encoded by ldhL2),NAD-dependent D-lactate dehydrogenase(D-nLDH;encoded by ldhD)were systematically investigated in order to study the relationship between these enzymes and the optical purity of lactic acid.Sporolactobacillus inulinus(a D-lactic acid producer)were also examined in this study as comparative strains.The specific activities of key enzymes for lactic acid production in the two strains were characterized in vitro.DNA fragments were cloned,overexpressed and the purified enzymes were obtained the enzymatic properties were studied respectively.For Sporolactobacillus inulinus,only D-nLDH activity was detected.For Bacillus coagulans,although D-nLDHs were found to have catalytic activities,the catalytic efficiency of D-nLDH toward pyruvate was lower than that of L-nLDH.Furthermore,the Km of D-nLDH was lower than that of L-nLDH,indicating that L-nLDH had a higher affinity for pyruvate than D-nLDH.Exponentially growing cells were collected and Native-PAGE was used before active staining of nLDHs.Only L-nLDH activities were detected in B.coagulans,which was in agreement with only D-nLDH activities were detected in Sporolactobacillus inulinus.To examine the transcription levels of the genes encoding nLDHs at the different phases,RealTime-PCR assays were performed.For B.coagulans,IdhL1 transcription levels were higher than those of ldhD and ldhL2 gene in different growth phases.For the other stain in this study only ldhD transcription levels were detected.Therefore,the high catalytic efficiency of L-nLDH toward pyruvate and the high transcription ratios of ldhLl is the key of production of highly optically pure L-lactic acid by B.coagulans.Furthermore,a novel markerless gene deletion system was for B.coagulans in this studyto analyze the change of the metabolic pathways by ldh gene deletion.Deletion of the IdhL2 gene showed no difference of fermentation profile with the wild type.Deletion of the IdhL2 gene,the strains B.coagulans?ldhL1?ldhL2 produced acetic acid as the main product,together with ethanol and other organic acids,indicating that ldhLl deletion led to alteration of the primary fermentation product from L-lactic acid to other by-products.Complementation of ldhLl gene in the gene knockout strain back to the fermentation profiles with the wild strain.This study first demonstrated that ldhL1 affected the metabolic pathways of B.coagulans.And ldhL1 is crucial for high optically pure L-lactic acid metabolism in B.coagulans.