Genomic Analysis Of The Interspecies Interactions In Vitamin C Fermentation And Enhancement Of2-keto-L-gulonic Acid Production

Clarifying the interaction between Ketogulonigenium vulgare and its companionBacillus sp. and obtaining a consortium with enhanced2-keto-L-gulonic acid (2-KLG)production are of great importance to vitamin C industry. In this study, the interactionwithin the consortium was systematically analyzed on genome scale. L-sorboseconversion pathway and cofactor pyrroloquinoline quinone (PQQ) synthesis pathwaywere constructed into a series of modules, and the optimal combination betweenmodules or adaptive evolution within the chassis were investigated to increase theproduction of2-KLG.Genome sequencing of the laboratory strain K. vulgare HB602and the industrialcompanion strain Bacillus endophyticus Hbe603were performed by next-generationDNA sequencing technologies. The K. vulgare genome encoded a large number ofproteins involved in degrading, assimilating and metabolizing proteins, peptides oramino acids, which might be provided by the companion. Various transcriptionalregulators, chemotaxis regulators and the flagella synthesis system were predicted tobe associated with cellular response.5copies of sorbose dehydrogenase and2copiesof sorbosone dehydrogenase were present in the genome, and multi-copy of these keyenzymes in the L-sorbose pathway was responsible for efficient substrate conversion.The B. endophyticus genome contained a set of genes involved in spore formation,while lacked several spore coat synthesis genes and the rap-phr system coding genes.It indicated that B. endophyticus was able to form spore with specific spore coatstructure, which was initiated by a certain regulation mechanism. Comparisonbetween the genome scale metabolic networks of K. vulgare and companion B.megaterium showed that K. vulgare contained less reactions in metabolism ofcarbohydrate, lipid, cofactors and vitamins, indicating the poor growth of K. vulgare.The companion bacterium was necessary for K. vulgare to complement its metabolicdefect in galactose and butanoate metabolism, lipid degradation, glutamate synthesis,methionine cycle, valine/leucine/isoleucine degradation, urea cycle, tryptophanmetabolism and metabolism of cofactors and vitamins.Sorbose/sorbosone dehydrogenase genes (sdh and sndh) and synthesis genes(pqqABCDEN) of the adjoint cofactor PQQ was constructed into3L-sorbose conversion modules and3PQQ synthesis modules respectively.9combinations ofthese two types of modules were constructed to investigate the adaptation betweenmodules, and the combinational module of ss-pqqABCDEN exhibited optimaladaptation by a20%increase in the production of2-KLG (79.1±0.6g/L) than that ofthe parental K. vulgare HB602(65.9±0.4g/L) in co-cultures. The enhancement of2-KLG production in engineered industrial strain K. vulgare HKv604was achieved byco-cultured adaptive evolution of50transfers. Combinational modules ofsdh-pqqABCDEN and ss-pqqABCDEN demonstrated improved adaptation to theindustrial chassis consortium, which produced higher concentration of2-KLG(79.47±0.00g/L and80.48±0.55g/L) than they performed before adaptation.