| Lactic acid is an important hydroxycarboxylic acid which has been widely used in food,pharmaceutical,feed,pesticide,chemical,leather and textile industries et al.The compounds L(+)and D(-)lactic acid are the two stereo?isomers of lactic acid,and only L(+)-lactic acid can be metabolized by humans.In recent years,the most important application of lactic acid is in producing polylactic acid,an environmentally friendly biomaterial.Polylactic acid is a non-toxic and nonirritating biomaterial with high tensile strength,plasticity and formability,which is the potential substitute for the plastic from unrenewable fossil resources.Lactic acid can be produced either via chemical synthesis,enzymatic catalyzation or fermentation.The advantages of fermentative lactic acid production are the high optical purity of lactic acid,the use of the renewable resources as feedstock,low cost,mild reaction condition and low energy consumption.Fermentation becomes the dominant method for lactic acid production industry.Currently,there are researches on lactic acid producing from corn starch,sweet potato starch and potato starch.The lactic acid produced from cassava starch is rarely reported.In order to establish the fermentation process of lactic acid producing from cassava starch,the optimization of fermentation condition,high efficiency Lactobacillus rhamnosus and L-lactic acid synthesis mechanism in Lactobacillus rhamnosus were studied.1.Optimization of fermentation conditions in producing lactic acid from cassava starchThrough single factor experiments combined with Plackett-Burman design,the fermentation conditions and key factors in medium for lactic acid production from cassava starch were optimized and screened.The results showed that the seed age of Lactobacillus rhamnosus used in fermentation of lactic acid from cassava starch was 12 h.The optimal fermentation medium was yeast extract as nitrogen source with 0.4%of MgSO4·7H2O and 0.01%MnSO4,and adding 7%of CaCO3 as neutralizer.The opitmal fermentation condition was achieved when the inoculum was 6%.The optimum temperature and pH for lactic acid production were 37? and 6.3,respectively.By Plackett-Burman test,the yeast extract,K2HPO4 and MgSO4·7H2O were identified as the key factors for lactic acid production.The linear regression equation using lactic acid as response value was obtained.The effect of optimization was verified by ferementation experiments.The lactic acid yield and conversion rate was increased after optimization,which provided the reference for the industrilzation of lactic acid producing from cassava starch in the future.2.High efficiency Lactobacillus rhamnosus for L-lactic acid producing from cassava starchIn order to improve the optical purity and yield of L(+)-lactic acid,the knockout plasmid pUC-ldhD-Ter containing homologous sequence of D-lactate dehydrogenase from Lactobacillus rhamnosus was transformed into Lactobacillus rhamnosus JCM1553.Two recombinant stains were successfully constructed.The highest yield of L-lactic acid in Erlenmeyer flasks from 10%glucose at 37—,200 rpm by ldhD negative strain 1 and ldhD negative strain 2 was(80.07±0.48)g/L and(80.48±1.94)g/L after 40 h fermentation.The conversion rate was 80.36%and 81.04%.OD600 value of the two strains was 12.75±0.21 and 12.33 ± 0.08.The residual sugar content was(4.31 ±0.22)g/L and(4.77±0.74)g/L,respectively.When the glucose concentration reached 20%,the highest yield L-lactic acid of ldhD negative strain 1 and ldhD negative strain 2 was(166.07±2.75)g/L and(167.90±1.86)g/L g/L after 72 h with 83.83%and 84.51%conversion rate.OD600 value of the two strains was 14.52±0.31 and 14.05±0.68.The residual sugar content of two strains was(9.27±0.62)g/L and(9.39±0.36)g/L,respectively.The results showed no significant differences were observed between recombinant and wild type strains.The results also indicate that the knockout of D-lactate dehydrogenase cannot increase the optical purity and yield of lactic acid.The classical mutagenesis,as an alternative method,was employed to improve the L-lactic acid yield of Lactobacillus rhamnosus.A high yield mutant strain of L-lactic acid fermentation,defined as SCT-10-10-60,was selected by mutagenesis of Lactobacillus rha.mnosus JCMl553 strain with the combination of U-V and Co60 radiation.The result of L-lactic acid production from different generation transfer culture verified SCT-10-10-60 had the genetic stability on L-lactic acid fermentation.In the shake flask fermentation with glucose as substrate under the condition of 37?,200 rpm,the L-lactic acid concentration of mutant strain in broth was reached to maximum of 195.67 g/L at 60h of fermentation and the maximum ratio of glucose converting to lactic acid reached to 95.33%.The maximum L-lactic acid yield,the fermentation rate and the ratio of glucose converting to L-lactic acid of mutant strain were respectively 16.24%,50.13%and 17.81%higher than those of start strain.In the fermentation with enzymatic hydrant of cassava starch as substrate under the same condition,the L-lactic acid concentration of mutant strain was reached to 227.33 g/L of maximum at 84 h fermentation,with the maximum L-lactic acid yield and the fermentation rate of mutant strain both 36.95%higher than that of start strain(166.00 g/L,84 h).The maximum ratio of glucose converting to L-lactic acid reached to 88.20%,31.74%higher than that of start strain(66.95%).Since SCT-10-10-60 strain has more advantage features than JCM1553 and current industrial strains in L-lactic acid fermentation,it is thought as a potential strain for L-lactic acid production industry.3.Comparative genomics and transcriptome analysis of Lactobacillus rhamnosusMechanisms for high L-lactic acid production remain unclear in many bacteria.Lactobacillus rhamnosus SCT-10-10-60 was previously obtained from L.rhamnosus JCM1553 via mutagenesis and showed improved L-lactic acid production.In this study,the genomes of strains SCT-10-10-60 and JCM1553 were sequenced.Both genomes are a circular chromosome,2.99 Mb in length with the GC content of approximately 46.8%.Eight split genes were identified in strain SCT-10-10-60,including two LytR family transcriptional regulators,two Rex redox-sensing transcriptional repressors,and four ABC transporters.In total,60 significantly up-regulated genes(log2fold-change?2)and 39 significantly down-regulated genes(log2fold-change?-2)were identified by a transcriptome comparison between strains SCT-10-10-60 and JCM1553.KEGG pathway enrichment analysis revealed that "pyruvate metabolism" was significantly different(P<0.05)between the two strains.The split genes and the differentially expressed genes involved in the "pyruvate metabolism" pathway are probably responsible for the increased L-lactic acid production by SCT-10-10-60.The genome and transcriptome sequencing information and comparison of SCT-10-10-60 with JCM1553 provide insights in to the anabolism of L-lactic acid and a reference for improving L-lactic acid production using genetic engineering.In conclusion,the fermentation conditions and key factors in medium for lactic acid production from cassava starch were optimized and screened,and a Lactobacillus rhamnosus,SCT-10-10-60,with enhanced L-lactic acid production capacity was obtained.Through comparative genomics and transcriptome analysis of Lactobacillus rhamnosus JCM1553 and SCT-10-10-60,the mechanisms for high L-lactic acid production in Lactobacillus rhamnosus were studied.These studies provide the reference for the industrialization of L-lactic acid producing from cassava starch,which can further promote the development of L-lactic acid production industry in China.In addition,the insights of the anabolism and regulation mechanism in L-lactic acid production provide ideas for genetic modification of lactic acid bacteria. |
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