Construction And Analysis Of Genome-scale Metabolic Model Of Lactococcus Lactis NZ9000

With the advent of the post-gene era,the metabolic engineering transformation of industrial microorganisms is playing an increasingly important role in industrial production.The Genome-scale metabolic model(GSMM)integrates all known metabolic information in the organism,and provides a good platform for understanding the metabolic state of the organism globally and guiding metabolic engineering transformation rationally.In this study,Lactococcus lactis NZ9000 was used as the target strain,and its physiological characteristics and metabolic capacity were analyzed by constructing its unique GSMM.In order to provide a new tool for mastering the overall intracellular metabolism mechanism of L.lactis NZ9000 and guiding the rational transformation of the metabolic engineering of this model strain.The main research contents are as follows:(1)According to the latest published whole genome sequence of L.lactis NZ9000,the semi-automatic construction method combining genomic function annotation and comparative genomics was used to complete the construction of the genome scale metabolic network model iWK560 of L.lactis NZ9000 based on MATLAB platform.The model iWK560 contains 560 genes,668 metabolites,and 840 reactions.The gene coverage reached 23.89%,and 840 responses were distributed in the intracellular and extracellular compartments respectively.Among them,there were 638 reactions with clear gene association,accounting for 76%.(2)Verify the performance parameters of the model.Through the analysis of carbon source utilization capacity,it is found that L.lactis NZ9000 can use 16 carbon sources as substrates for growth,and the agreement rate between the model prediction and the experiment is 100%,indicating the accuracy of the model for carbon source substrate utilization prediction.The amino acid utilization ability verification found that the strain could not grow without Arginine,Histidine,Isoleucine,Leucine,Valine,Methionine and Serine,and the model prediction results were consistent with the experimental results,indicating that the model accuracy of amino acid metabolism calculation.Based on CDM medium,the physiological parameters of i WK560 were verified by batch fermentation experiments and chemostat experiment data at different dilution rates.The average deviation of the model simulation calculation was less than 5%,indicating that the model is accurate for the prediction of strain growth and product synthesis.The above analysis and verification show that iWK560 can accurately predict the growth and product production of the strain,reflect the true physiological state of the cell,and can be used as a guide tool for metabolic engineering transformation and fermentation optimization.(3)The model iWK560 was used to analyze the physiological metabolism function of L.lactis NZ9000.(i)Analyze the characteristics of the model and compare it with other lactic acid bacteria models;(ii)The gene knockout algorithm was used to predict the essential genes and reactions under three different media conditions(MS15,CDM,M17).In response,the number of essential genes was 88,62,and 54 respectively,and the number of essential responses were 189,137,and 135,respectively,and their metabolic subsystem distributions were analyzed;(iii)Multi-factor deletion based on the model and single factor addition to analyze the nutrient absorption of the strain and apply it to the optimization of the synthetic medium to increase the biomass by 100.19%;(iv)The central carbon metabolism network of the model is analyzed,and the utilization of carbon source substrates was analyzed.Studies have shown that monosaccharide carbon source substrates were more conducive to the absorption and growth of strains;(v)Using the FVA algorithm to analyze the ability of the strain as a cell factory to synthesize different products,the study shows that the wild-type strain is more suitable for the production of pyruvate-derived products,and acetyl-CoA and acetolactate derivatives also have greater potential.(4)With(S,S)-2,3-butanediol as the target product,metabolic pathway analysis and fermentation strategy optimization were carried out.(i)The metabolic pathway of S-BDO was analyzed and verified,and three metabolic nodes and eight metabolic transformation targets during S-BDO synthesis were obtained through metabolic flux analysis;(ii)The effects of vitamin and amino acid addition and pH changes on product synthesis and cell growth were studied.Among them,orotic acid,Asparagine,Aspartic acid,Glycine,Serine,and Threonine significantly promoted product production,and compared the production of S-BDO,cell growth is more sensitive to changes in pH,so it is more conducive to the production of S-BDO by properly maintaining a weakly acidic environment.