The Metabolism Of Lactose And Galactose In Lactic Acid Bacteria And Its Potential Application

Yogurt is favored by consumers because of its high nutritional value,unique flavor and good health care effect.Lactose,a disaccharide composed of glucose and galactose connected by ?-1,4-glycosidic bonds,is the main carbon source in cow's milk.It is also the important energy source for the growth and reproduction of lactic acid bacteria during dairy fermentation.The traditional yogurt is fermented by the joint action of Streptococcus thermophilus and Lactobacillus delbrueckii subsp.bulgaricus.However,these two species can only use the glucose part of lactose,and release the galactose moiety into the extracellular circumstance.The unutilized lactose and accumulated galactose in fermented dairy will not only affect the quality of the product,but also cause a burden on human health,especially those with lactose intolerance and galactosemia.In the present study,the operons for lactose and galactose metabolism were identified from the genome of S.thermophilus.Although this species containes the entire genes for galactose metabolism pathway related enzymes,the promoter of the operon is inactivated.This is one of the important reasons why S.thermophilus does not use galactose.Therefore,the promoter of the galactose operon was optimized by constructing a random mutant promoter library.The ability to ferment galactose was improved by using the strongest promoter to express the galactose kinase gene galK and the galactose operon galKTE,respectively.In order to further decrease the galactose content in yogurt,Lb.plantarum,which has a strong ability to utilize lactose and galactose,was used to co-culture with yogurt starter strains.The total sugar content in yogurt was effectively reduced.Finally,an engineering strain of Lb.plantarum was constructed for the conversion of galactose into high value-added D-tagatose,which lays the foundation for the development of high-quality yogurt.The specific results are as follows:1.Structure and activity analysis of the lactose/galactose operon in Streptococcus thermophilusThe exploration of the reasons for the Gal phenotype in S.thermophilus can provide a new strategy for obtaining stable galactose fermentation strains.In the past,it was thought that S.thermophilus was short of enzymes for galactose metabolism.However,with the deepening of the genetic research of S.thermophilus and the development of whole genome sequencing technology,the galKTE operon encoding the enzymes required for the galactose metabolism Leloir pathway existed in all the published S.thermophilus genome.In the present study,44 S.thermophilus strains were tested for their galactose utilization ability,and 16%(7 strains)of them showed Gal+ phenotype.When growing in the lactose medium,S.thermophilus strains with either Gal+ or Gal-phenotype secrete galactose to the outside of the cell,leading to the accumulation of galactose.When the lactose content was reduced to about 1 g/L,the Gal+ phenotype and part of the Gal-phenotype S.thermophilus began to metabolize the pre-accumulated galactose at different speeds and degrees.However,the galactose could not be completely utilized until 24 h fermentation.Analysis of the gaIR-galK interval sequence revealed it played an important role in the Gal+phenotype of S.thermophilus,although the base sequence in this interval was not the only factor determining the Gal+phenotype.2.Construction of S.thermophilus strains with galactose utilization abilityThe S.thermophilus strains with galactose utilization ability play important roles in reduction the content of galactose in fermented dairy products such as yogurt.The low expression level of genes related to the Leloir pathway caused by insufficient gal promoter activity is the main reason that S.thermophilus is unable to metabolize galactose.In the present study,the gal promoter was optimized by constructing a random mutant promoter library.The promoter P59 with strong mutation was screened by using green fluorescent protein as the reporter and the activity of the promoter was increased 6.7-fold compared with the original promoter.Then promoter P59 was used to express the galactose kinase gene galK and the galactose operon galKTE,respectively.The results showed that the galactose utilization ability of the recombinant strains was improved.They could simultaneously metabolize lactose and galactose in the lactose medium,thereby reducing the accumulation of galactose in the medium.The obtained Gal+ S.thermophilus was used to prepare yogurt with L.bulgaricus and the result showed that the content of galactose in the yogurt was reduced.Furthermore,the original promoter of the gal operon was replaced by the mutant promoter on the genome of S.thermophilus with homologous recombination technology.However,the obtained strain was not only unable to utilize galactose,but also had reduced growth ability in lactose medium.RT-PCR results showed that the galR transcription level increased and the gal operon transcription level decreased in the mutant strain compared with the wild strain.It was speculated that the transcription regulator GalR inhibited the transcription of the galKTEM operon,which resulted in the strain could not use galactose.The results also suggested that editing promoters on the genome should also consider the effects on other nearby genes.3.Low-sugar yogurt making by the co-cultivation of Lactobacillus plantarum WCFS1 with yogurt starter culturesIn order to further reduce the lactose/galactose content in yogurt,this part explored the influence of Lb.plantarum co-cultured with traditional yogurt starters on the sugar content in yogurt.In this study,we found that Lb.plantarum WCFS1 could effectively metabolize both lactose and galactose.Comparative genomic analysis demonstrated the constant presence of a chromosome-encoded Leloir pathway for galactose metabolism in Lb.plantarum species,and the gal operon was driven by a strong constitutive promoter in Lb.plantarum WCFS1,displaying great potential in low-sugar yogurt making.To test this hypothesis,Lb.plantarum WCFS1 was co-cultured with S.thermophilus or Lb.bulgaricus in lactose-based medium.As results,lactose was consumed up completely,and galactose was also metabolized efficiently.For yogurt making,co-cultivation of Lb.plantarum WCFS1 with traditional starters reduced the total sugars content from 18.40%to 26.00%,and the viable cell counts increased 1.6-fold compared with the traditional fermentation processes.Besides,the sensory analysis indicated that the yogurt fermented with yogurt starter cultures and Lb.plantarum WCFS1 was acceptable to consumers in appearance,texture,and flavor.Therefore,this study emphasized the potential to manufacture low-sugar yogurt by the co-cultivation of Lb.plantarum with yogurt starter cultures.4.Lactose/galactose-inducible expression system of Lactobacillus plantarum and its applicationThe lactose-inducible expression system was widely used in bacteria due to the safety of its inducers.However,the limited driving strength of this kind of promoters in lactic acid bacteria restricted their application and development.Here,the?-galactosidase activity in Lactobacillus plantarum WCFS1 was detected in lactose or galactose medium,but not in glucose medium.Sequence analysis showed that two?-galactosidase encoding genes lac A and lacLM were identified in the genome of Lb.plantarum WCFS1.Using green fluorescent protein as a reporter,we found that the promoter PlacA of lacA was lactose-induced,while promoter PlacLM of lacLM was lactose/galactose-induced.Furthermore,by optimizing the sequences of-35,-10 regions and ribosome binding sites of these two promoters,the maximum strength of the promoter PlacA derivative increased 10.4-fold by lactose induction,while PiacLM derivative increased 12.7-fold by lactose induction and 9.0-fold by galactose induction compared with their original promoters,respectively.When the optimized promoters were used to drive the ?-galactosidase gene from Streptococcus thermophilus SDMCC050242,the highest ?-galactosidase activity was 45.72±0.44 U/mL under PlacLM-35-10 control.The strength of these promoters was also confirmed by western-blot analysis.Thereby,this study provided a set of novel lactose/galactose-inducible promoters with high expression efficiency and thus expanded the toolbox to express recombinant protein in Lb.plantarum.5.Biotechnological production of D-tagatose from lactose using metabolically engineered Lactobacillus plantarumD-Tagatose,a unique and rare hexoketose monosaccharide,has been receiving interest as a non-caloric sweetener substitute for sucrose.In this study,we describe a novel way to produce D-tagatose from lactose by a metabolic engineered Lactobacillus plantarum.This was achieved by introducing L-arabinose isomerase AraA from L.casei SDMCC050286 into the Lb.plantarum WCFSl/?galK-PlacLM-35-10,in which the galactokinase gene galK was deficient and the original promoter of the ?-galactosidase gene lacLM was replaced by a strong lactose/galactose-inducible promoter PlacLM-35-10.This provided us with a D-tagatose production strain,in which P-galactosidase activity was increased 5.4-folds,galactose metabolism pathway was blocked and L-arabinose isomerase was heterologously expressed.The optimal conditions for D-tagatose production by resting cells were investigated.The resulted cells exhibited maximum D-tagatose producing activity at 65? and pH 7.5.Under the optimum conditions,the resting cells could hydrolyze lactose completely in 125 g/L lactose for 4 h,and the conversion rate of D-tagatose from lactose was achieved 33%at 56 h.This study verified the feasibility of single-step production of D-tagatose from lactose,thereby laying down the foundation for industrial usage of lactose.