Construction Of Lactiplantibacillus Plantarum Food-grade Expression System Based On The Key Enzyme In Lactose Metabolism

Lactiplantibacillus plantarum is one of the most important and widely studied lactic acid bacteria,which has been used in many fields.Moreover,Lp.plantarum is widely present in various ecological niches such as dairy products,fruits and vegetables,meat,wine,animal gastrointestinal tract,and human urogenital tract.This ubiquitous specificity demonstrates the fantastic adaptability of Lp.plantarum and the diversity of its metabolic pathways.In addition,Lp.plantarum is a microorganism with a documented history of food use,with a qualified presumption of safety status from the European Food Safety Authority(EFSA)and GRAS strain from the U.S.Food and Drug Administration.In recent years,Lp.plantarum strains modified using molecular biology have shown advantages in pharmaceuticals and other areas,such as the ability to assist in the production of some high-value bioactive substances(specific antigens,proteins for particular purposes),which are essential in the fields of recombinant enzyme production,industrial food fermentation,genetic and metabolic engineering,where food-grade expression vectors are one of the commonly used methods.β-galactosidase(EC 3.2.1.23)can catalyze the hydrolysis of lactose and plays a vital role in lactose metabolism.The metabolic background of Lp.plantarum is straightforward and can use lactose as a carbon source for good growth.In this study,we investigated the feasibility of using lacM with small molecular weight as a screening marker and studied a food-grade system for Lp.plantarum,focusing on some areas as follows.(1)Transcriptome results and analysis of Lp.plantarum in a lactose environment.In this chapter,transcriptome sequencing was used to reveal the expression of different genes in Lp.plantarum LP01 when it was cultured with glucose and lactose medium,respectively.That is,203 genes were significantly up-regulated and 36 were significantly down-regulated when Lp.plantarum LP01 metabolized lactose compared to glucose.Moreover,the genomic loci of Lp.plantarum LP01 closely related to lactose metabolism,showed high expression after lactose induction,with the genes encoding β-galactosidase Lac A,Lac L,and Lac M being up-regulated by 9.86,9.12,and 9.53 fold,respectively.This provides a theoretical basis for exploring the growth and metabolic mechanism of Lp.plantarum in lactose medium.(2)Lp.plantarum LP01 has two genes encoding β-galactosidases,lacA,and lacLM.In this chapter,we investigate the specific roles of some key enzymes in the lactose metabolic pathway of Lp.plantarum LP01.First,the key genes in the lactose metabolic pathway in Lp.plantarum were identified by transcriptome analysis of the genes differentially expressed in Lp.plantarum when grown in glucose and lactose,respectively.Then,mutant strains of Lp.plantarum ΔlacA,Lp.plantarum ΔlacLM and Lp.plantarumΔlacAlacLM were constructed in this chapter of the study,and it was found that the knockout strain Lp.plantarum ΔlacA could still grow in MRS lactose medium during the knockout strain Lp.plantarum ΔlacLM could grow in an MRS lactose medium.Lp.plantarum ΔlacLM and Lp.plantarum ΔlacAlacLM could not grow in the MRS lactose medium,indicating that the gene lacLM plays a key role in the lactose metabolic pathway.Furthermore,by observing the effects of different carbon sources on the mutant strains,it could be found that β-galactosidase Lac LM was more important in lactose and cottonseed utilization.At the same time,β-galactosidase Lac A was more important in utilizing oligo galactose.These results suggest that the two β-galactosidases,encoded by the genes lacA and lacLM,respectively,have different effects on the metabolism of different sugars and synergistic effects on specific substrates.(3)Construction of food-grade expression vector in Lp.plantarum using the lacM gene as a screening marker.Based on the effect of the backfill strain,the food-grade vector p MG36 m was obtained by removing the antibiotic resistance gene using the lacM gene as a screening marker.Genetic stability experiments showed that the plasmid presence rate of the transformant Lp.plantarum ΔlacAlacM/p MG36 m was still close to100% after 98 generations.Subsequently,the expression vector p MG36m-gfp was constructed using the green fluorescent protein gene(gfp)as a reporter gene,and fluorescence detection and microscopic observation showed that the green fluorescent protein could be successfully expressed in Lp.plantarum.The fluorescence intensity of the transformant Lp.plantarum ΔlacAlacM/p MG36m-gfp was measured up to 380061.8RFU,and the stability of the expression was examined and found that the fluorescence value decreased to a certain extent and then remained stable as the transformant Lp.plantarum ΔlacAlacM/p MG36m-gfp was passed on.(4)To construct food-grade double plasmid expression system of Lp.plantarum.After reviewing the literature and transcriptome analysis,it was found that galactose kinase encoded by gal K in Lp.plantarum can catalyze the conversion of galactose to galactose 1-phosphate and then further produce UDP mono glucose and integrate into the glycolytic pathway,a key enzyme in the metabolic galactose pathway in Lp.plantarum.The gal K gene was knocked out from the knockout strain Lp in the present study.Plantarum ΔlacAlacLM to make the galactose gene unavailable,and the lacL and gal K genes were ligated to the plasmid p MSP3535,and the erythromycin resistance gene was removed to obtain.The red fluorescent protein gene m Cherry was ligated to the food grade expression vector p MSPlk to obtain the plasmid p MSPlk-m Cherry electrotransferred into Lp.plantarum ΔlacAlacLMgal K and screened for galactose as the only carbon source.Finally,the transformant Lp.plantarum ΔlacAlacLMgal K/p MSPlkm Cherry was electrotransferred into the food-grade plasmid p MG36m-gfp to obtain the transformant Lp.plantarum ΔlacAlacLMgal K/p MSPlk-m Cherry.Lp.plantarumΔlacAlacLMgal K/p MSPlk-m Cherry/p MG36m-gfp.The transformants Lp.plantarumΔlacAlacLMgal K/p MSPlk-m Cherry/p MG36m-gfp were tested to express both red and green fluorescence,demonstrating that the expression of red fluorescence with lacL,lacM,and gal K genes as marker genes to construct a food-grade dual plasmid expression system.The above results indicate that a food-grade dual plasmid expression system of Lp.plantarum with lacL and lacM genes as complementary screening markers and lactose as screening conditions has been successfully constructed,laying the foundation for applying Lp.plantarum.