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An artificial microbial community consisted of Ketogulonicigenium vulgare and Bacillus cereus has been used in industry to produce 2-keto-gulonic acid, the precursor of vitamin C.In order to investigate how two species interact with each other in an evolutionary angle of view, we performed a 150-day experimental evolution of these two species by transferring the bacteria into new seed medium every 24 h.The evolution process improved the cooperation of these two species and promoted the 2-KGA yield by 16% compared to the ancestral bacteria.Aiming to explore the the mechanism, an iTRAQ-based quantitative proteomic analysis was performed to compare the evolved and ancestral bacteria.We compared the 0th, 50th, 100th and 150th mono-cultured K.vulgare, B.cereus and the mix-cultured consortia respectively in seed medium.In total, we identified approximately 175 proteins in mono-cultured K.vulgare, 406 proteins in mono-cultured B.cereus.By performing hierarchy cluster analysis of the protein data, we found that the sorbose/sorbosone dehydrogenase in K.vulgare responsible for 2-KGA production was up-regulated with the ongoing of the evolution process, while when the 0th, 50th, 100th and 150th generation of K.vulgare were cultured in fermentation medium, which was less fertile than the seed medium, the expression of SSDH were gradually down-regulated.Similar phenomena also occurred to proteins participating in pentose phosphate pathway, TCA cycle and ATP synthesis.This result suggested the evolution of the consortia was environment dependent, and the evolution direction was to adapt to the environment.The evolution of B.cereus was multidirectional.The most furious change happened at the 50th generation, and afterwards, the evolution was much more stable.Proteins involved in sporulation and exosporium forming had variable expressions, suggesting the sporulation and cell lysis of B.cereus changed with the evolution, and thus could affect material supply for the growth and 2-KGA production of K.vulgare.