| China is a big country about dairy production and consumption.By the end of 2015,the sales of Chinese yogurt market have exceeded 80 billion RMB,and it has been increasing at a high growth rate per year.S.thermophilus is commonly used in the fermentation dairy products and has great market potential.However our country's industrial starter cultures still rely on west countries' enterprises.Metabolism of S.thermophilus,especially the study of carbohydrate metabolism,plays an important role in optimizing the composition of high density culture medium and fermentation parameters to obtain high quality starter cultures.In this paper,S.thermophilus TF96 was used as target stains and cultivated in the chemical synthetic medium.During the controled p H batch fermentation of S.thermophilus TF96,the key gene expression about carbohydrate metabolism pathways and the changes of intracelluar or extracelluar carbohydrate were studied by transcriptomics and metabonomics,which aimed to reveal the mechanism of carbohydrate metabolism and regulation from transcription and metabolite level of carbohydrate metabolism pathways' key genes.It will lay a theoretical foundation for the further research on high density culture.In this work,four different growth phase of S.thermophilus TF96 that includes final lag phase(T1),mid-exponential phase(T2),late-exponential phase(T3)and stationary growth phase(T4),were investigated by de novo transcriptome.In the results of assembly,2255 unigenes and Contig were assembled,average length is 1119 nt,N50 is 2231 nt and median length is 1119 nt.At the same time,the distribution of Unigenes' length was calculated,over 45% sequences were no more than 500 nt in length.All Unigene was annotated after assembly.There are 1470 unigenes were annotated to the KEGG database.In addition,A total of 260 genes were annotated to 15 carbohydrate metabolism pathways.During the growth of S.thermophilus TF96,a total of 166 different expression genes involved in carbohydrate metabolism pathways.Suger transport was probably a major factor to control the bacteria growth..In this work,lac S,fru B,man X/Y and mal X were different expressed gene,which encoding lactose,fructose,mannose and maltose transport,respectively.In general,expression level of encoding PTS system gene is fluctuating during growth,T1 and T2 were expression peak for the phosphotransferase system gene.And Pts I is a key gene in phosphotransferase system.When bacteria reached the stationary phase,the expression of Pts I was significantly increased 1.53 times.The contrast is the level of transcription of Pts I was continue todrop from T1 to T3.Gene encoding the lactose transport(lac S)was significantly down-regulated1.8-fold in the T2.Then when the strains at T3,transcription level of lac S increased significantly.It indicated that final lag phase and late-exponential phase were transport peak of lactose.Because of limitation of lactose concentration,gene encoding ?-galactosidase was repressed more than 2fold in the stationary phase.Galactokinase is the key protease for galactose metabolism in streptococcus thermophilus.The results suggest that the expression of galK was increased before the stationary phase.The expression of gal K was induced 1.77 times from T1 to T2.However,when the S.thermophilus TF96 grew at T4,the expression of gene encoding galactokinase was significantly decreased 2.78 folds compared to T3,Our study showed that S.thermophilus TF96 may have the ability to metabolise galactose from transcription during the growth.Glycolysis is an important pathway in cell energy production.The study showed that the genes involved in glycolysis,such as glk,pgi,fba A,gap A,gpm A,pyk and CcpA,expressed differently,which encoding glucokinase,glucose-6-phosphate isomerase,fructose-bisphosphate aldolase,glyceraldehyde 3-phosphate dehydrogenase,2,3-phosphoglycerate mutase,pyruvate kinase and carbohydrate catabolism proteins,respectively.Gene of glucokinase and pyruvate kinase are the key enzymes for glycolytic,the level of transcription of glk and pyk were down-regulated throughout the growth phase,especially significantly down regulation at T2.T1 is expression peak of gene for pfl,adh E and ack A.Inaddition,when S.thermophilus TF96 grew at T2,transcription level of these genes significantly decreased for 3.44,5.57,1.79 and 2.76-fold,respectively,and with low expression state in the following stages.Pyruvate Oxidase which is encoded by the E1.2.3.3 gene was up-regulated(1.96)in the T2.High expression of E1.2.3.3 might create a pool of acetyl phosphate,could be converted into acetate and producing ATP later in the exponential phase.Then the level of transcription of E1.2.3.3 recovered to final lag phase,when bacteria at stationary phases.At the same time,it was obvious induction with an up-regulation for 2.6 folds of the gene encoding the phosphate acetyltransferase.The results showed that lactic acid is main metabolite of pyruvic acid.The expression of gene encoding lactate dehydrogenase was increased from T1.Through the analysis of gene expression levels for different time periods,it could found that the genes about the pathway of pyruvic acid involved in fatty acid biosynthesis were increased at transcription level at T4.A total 118 genes were identified which involved in cell membrane/wall at transcription level.Among of these genes,there are 49 genes expressed differently.During the controled p H batch fermentation of S.thermophilus TF96,lag phase is a period with high expression of genes involved in cell wall and membrane.When strains at log phase,level expression of most genes had decreased.The Fts W gene encoding bacterial cell division membrane protein,which is also key gene for peptidoglycan synthesis.At T2,transcription level of Fts W was significantly increased for1.6 folds,but it was down-regulated 1.9 times compare to T3,when bacterial at T4.In our study,expression of genes which encoded muramidase,amidase and endopeptidasewere detected,and these genes involved in degradation of peptidoglycan.Different growth phase of S.thermophilus TF96,expression of peptidoglycan hydrolase's genes had a great difference.At the transcriptional level,oat A gene involved in the regulation of cell membrane to external stress,it was significant decreased during the cultivation.When strains at T3,oat A gene was repressed for1.3 fold,compared to T1.Research suggested that Oat A could resist the hydrolyzation of peptidoglycan hydrolase on cell wall.With the extension of cultivation,oat A gradually decreased in gene expression level,and cell autolysis was increased.During the batch fermentation of S.thermophilus TF96,some metabolite abundance had changed in fermentation liquor,such as lactic acid,acetic acid,ethanol and acetaldehyde and other small molecules of metabolic compounds.The abundance of acetaldehyde,ethanol and acetic acid had significant increased in extracelluar from T1.But,abundance of acetaldehyde and acetic acid were decreased from T3 to T4 in culture medium.To the contrary,intracelluar abundance of acetaldehyde and ethanol with no significant change.However,acetaldehyde concentration was increased significantly at T3,following decreased to 31.98 at T4,while ethanol abundance increased to 13.25 from T3 to T4.At the beginning stages,with the extension of cultivation,acetic acid abundance was increased from 4915.26 to 6198.78,but it reduced significantly at T3.The phenomenon demonstrated that acetate may be reused as a carbon source.And acetaldehyde was converted into ethanol by acetaldehyde dehydrogenase.In addition,abundance of intracellular carbohydrate associated with cell wall synthesis increased gradually with the extension of cultivation. |
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