Integrated Proteomic and Metabolomic Analysis of an Artificial Microbial Community for Two-Step Production of Vitamin C

Abstract

An artificial microbial community consisted of Ketogulonicigenium vulgare and Bacillus megaterium has been used in industry to produce 2-keto-gulonic acid (2-KGA), the precursor of vitamin C. During the mix culture fermentation process, sporulation and cell lysis of B. megaterium can be observed. In order to investigate how these phenomena correlate with 2-KGA production, and to explore how two species interact with each other during the fermentation process, an integrated time-series proteomic and metabolomic analysis was applied to the system. The study quantitatively identified approximate 100 metabolites and 258 proteins. Principal Component Analysis of all the metabolites identified showed that glutamic acid, 5-oxo-proline, L-sorbose, 2-KGA, 2, 6-dipicolinic acid and tyrosine were potential biomarkers to distinguish the different time-series samples. Interestingly, most of these metabolites were closely correlated with the sporulation process of B. megaterium. Together with several sporulation-relevant proteins identified, the results pointed to the possibility that Bacillus sporulation process might be important part of the microbial interaction. After sporulation, cell lysis of B. megaterium was observed in the co-culture system. The proteomic results showed that proteins combating against intracellular reactive oxygen stress (ROS), and proteins involved in pentose phosphate pathway, L-sorbose pathway, tricarboxylic acid cycle and amino acids metabolism were up-regulated when the cell lysis of B. megaterium occurred. The cell lysis might supply purine substrates needed for K. vulgare growth. These discoveries showed B. megaterium provided key elements necessary for K. vulgare to grow better and produce more 2-KGA. The study represents the first attempt to decipher 2-KGA-producing microbial communities using quantitative systems biology analysis.

Comparative Proteomic Analysis of Experimental Evolution of the Bacillus cereus-Ketogulonicigenium vulgare Co-Culture

“…According to our previous study, the purine biosynthesis was weak in K. vulgare, but it could be enhanced by the co-culturing with B. megaterium [15]. From the proteomic study of experimental evolution in this study, we found the biosynthesis of purine and pyrimidine was improved in the evolved K. vulgare in the co-culture.…”

Combinational expression of sorbose/sorbosone dehydrogenases and cofactor pyrroloquinoline quinone increases 2-keto-l-gulonic acid production in Ketogulonigenium vulgare–Bacillus cereus consortium

“…Recent studies demonstrated that Bacillus sp. could release metabolites in amino acid biosynthesis (Liu et al, 2011a;Zhou et al, 2011) and purine during cell lysis upon sporulation Ma et al, 2011;Zhang et al, 2010), which can be used to compensate the deficient metabolism of K. vulgare (Zou et al, 2012). In this study, our results showed that the blank plasmid did not impair the growth and 2-KLG production in the consortium of K. vulgare-B.…”

A plate method for rapid screening of Ketogulonicigenium vulgare mutants for enhanced 2-keto- l -gulonic acid production

“…However, the need to use a mixed fermentation system for 2-KLG production by K. vulgare and B. megaterium makes traditional screening methods for K. vulgare mutants capable of 2-KLG overproduction laborious and complicated, 7 leading to a much lower screening efficiency.…”

“…K. vulgare contains a complete enzyme system for converting l -sorbose to 2-KLG, 4 but its growth is poor in the monoculture, resulting in a very low yield of 2-KLG 5 . A companion strain, Bacillus megaterium , is generally considered to generate and release, in the fermentation broth, the metabolites that stimulate K. vulgare propagation and 2-KLG accumulation 6, 7, 8, 9, 10. These metabolites contain active proteins, 6 amino acids, 8 antioxidants, 10 and other active substrates 7, 9.…”

“…Thus, easy and efficient screening methods are desired for the screening and isolation of 2-KLG-overproducing mutants of K. vulgare . Since mixed fermentation involves co-cultivation of two bacterial strains, the associated screening process is relatively laborious and complicated 7 . Currently, only one method is available for the screening of K. vulgare mutants, i.e., the 2-KLG determination method 13 .…”