| This dissertation focuses on the elucidation of physiological chrateristics and interactionof an artificial microbial ecosystem (AME) consisting of Ketogulonicigenium vulgare andBacillus megaterium, which was used in the vitamin C industrial production. This can berealized by reconstruction of genome scale metabolic model (GSMM) of these two bacteriaon the basis of information of biochemical and omics studies (such as genomics, proteomics,metabolomics). With GSMMs, constraints-based methods can be used to investigate thephysiological features and ineractions with B. megaterium. The main results are as follows:1. The genome of K. vulgare WSH001was annotated by four approaches: RAST, KAAS,PRIAM, and a local sequence similarity search (BLASTp) of UniProtKB/Swiss-Protdatabase. Totally,834proteins were annotated and assigned with an EnzymeCommission (EC) number, which can be further used in the metabolic reconstruction.For transport proteins, KAAS annotation and a BLASTp with TCDB database wereperformed and476transportors were annotated. In addition, new annotated genesidentified during the RAST annotation were rechecked by a BLASTp ofUniProtKB/Swiss-Prot database and a comparative genomics analysis:186genes out of231new genes in K. vulgare and153genes out of219new genes in B. megaterium wereproved because of a high similarity or having homologous protein(s) in other strains ofits species.2. We constructed the GSMM of K. vulgare, iWZ663, on the basis of Model SEED, KAAS,literature mining, public databases, and experimental data. It consists of663genes,649metabolites and830reactions. The gene coverage of model iWZ663is21.4%. Themodel reactions were divided into14metabolic subsystems, among which Transportingsystem, Carbohydrate metabolism, and Amino acid metabolism occupy the largestproportions:16.5%,15.3%, and15.2%, respectively. Metabolites that associated withenergy generation and nitrogen metabolism have the most extensive connectivities iniWZ663. Annotation of L-sorbose metabolic pathway indicated that L-sorbose can notonly be converted into2-KLG or vitamin C, but also enter into the central carbonmetabolism for the production of energy and biomass precursors.3. Model iWZ663was comprehensive analyzed with Cobra toolbox on MATLAB. Essentialgene analysis was performed and116genes were predicted to be essential, and all theessential genes are located on chromosome. In addition,153reactions were predicted asessential reactions. Flux balance analysis (FBA) was carried out to investigate thereasons for the poor growth of K. vulgare, and three main reasons were included:(1) K.vulgare could not de novo biosynthesize asparigine, L-cysteine, L-methionine, biotin,nicotinate, thiamine diphosphate, and dihydrofolate (DHF);(2) the carbon flux mainlyenter into the ED pathway and only5.7%into the PP pathway, resulting a lower level ofreducing power (NADPH) and shortage of carbon backbones such as ribose;(3) thedefect in sulfate metabolism hampering the syntheses of L-cysteine, L-methionine,coenzyme A (CoA), and glutathione. Meanwhile, K. vulgare has aboundant peptide transportors and peptidase and was capable of assimilating L-asparagine, L-aspartic acid,L-glutamic acid, L-glutamine, glycine, L-alanine, L-proline, L-serine, and L-threonineinto TCA cycle or purine metabolism.4. The previous B. megaterium GSMM iMZ992was reifined by integrating the informationof RAST new annotations, metabolomics data, growth phenotype data on differentcarbon and nitrogen sources, and protein sublocation information. The new model namediMZ1055, comprised of1055genes,1137reactions, and1011metabolites. Then, the twomodels iMZ1055and iWZ663were reconciled (named iMZ1055a and iWZ663a,respectively) and compared. It was found that they share453reactions and548metabolites. Comparison of the two GSMMs suggested that B. megaterium has a morediversity metabolism, because:(1) iMZ1055a has15unique metabolic subsystems whileiWZ663a has only one;(2) iMZ1055a has some special metabolic functions in Histidinemetabolism, Valine, leucine and isoleucine biosynthesis, Alanine, aspartate andglutamate metabolism, and Riboflavin metabolism;(3) the percentage of essentialreactions in iWZ663a was more than two folds of that in iMZ1055a, and iMZ1055a has51non-essential shared reactions which were essential for. These51reactions mainlydistributed in Purine metabolism, Pyrimidine metabolism, Riboflavin metabolism,Pantothenate and CoA biosynthesis;(4) iMZ1055a could biosynthesize and transport out78metabolites, while K. vulgare only22metabolites. In addition, the two models havedifferent metabolic mechanisms in Fructose and mannose metabolism, Ubiquinone andother terpenoid-quinone biosynthesis, Sulfur metabolism, Benzoate degradation, andTransporting system.5. A two-species metabolic interaction model of AME in vitamin C production wasconstructed by integrating iWZ663a and iMZ1055a. The resulted model, namediWZ-KV-663-BM-1055, consists of1718genes,1583metabolites, and1910reactions.FBA and robustness analysis found both mutualism and competition exist between K.vulgare and B. megaterium. Futher, FVA and essential reactions analysis revealed thatwith the companion of B. megaterium: forty-two reactions that could not carry flux iniWZ663a were capable of carrying flux; thirty-three reactions that were essential iniWZ663a became non-essential in iWZ-KV-663-BM-1055; thirty-eight non-essentialreactions in B. megaterium could affect K. vulgare reach its maximum growth rate.Further, analysis of metablic interactions between K. vulgare and B. megaterium shows:(1) metabolism of K. vulgare and B. megterium were connected mainly by extracelluarmetabolites and reactions;(2) B. megaterium could secrets23to K. vulgare, amongwhich15metabolites can be biosynthesized by K. vulgare;(3) most of reactions inamino acids, vitamins and cofactors pathways has no flux, excepting several reactions innucleotide salvage pathway and the biosynthesis of leucine, isoleucine, valine, and proline;(4) predictions of metabolic interaction were affected by the accuracy oftransporter annotations in iWZ-KV-663-BM-1055. |
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