Establishing an innovative carbohydrate metabolic pathway for efficient production of 2-keto-l-gulonic acid in <italic>Ketogulonicigenium robustum</italic> initiated by intronic promoters.

Background: 2-Keto-l-gulonic acid (2-KGA), the precursor of vitamin C, is currently produced by two-step fermentation. In the second step, l-sorbose is transformed into 2-KGA by the symbiosis system composed of <italic>Ketogulonicigenium vulgare</italic> and <italic>Bacillus megaterium</italic>. Due to the different nutrient requirements and the uncertain ratio of the two strains, the symbiosis system significantly limits strain improvement and fermentation optimization. Results: In this study, <italic>Ketogulonicigenium robustum</italic> SPU_B003 was reported for its capability to grow well independently and to produce more 2-KGA than that of <italic>K. vulgare</italic> in a mono-culture system. The complete genome of <italic>K. robustum</italic> SPU_B003 was sequenced, and the metabolic characteristics were analyzed. Compared to the four reported <italic>K. vulgare</italic> genomes, <italic>K. robustum</italic> SPU_B003 contained more tRNAs, rRNAs, NAD and NADP biosynthetic genes, as well as regulation- and cell signaling-related genes. Moreover, the amino acid biosynthesis pathways were more complete. Two species-specific internal promoters, P1 (<italic>orf_01408</italic> promoter) and P2 (<italic>orf_02221</italic> promoter), were predicted and validated by detecting their initiation activity. To efficiently produce 2-KGA with decreased CO₂ release, an innovative acetyl-CoA biosynthetic pathway (XFP-PTA pathway) was introduced into <italic>K. robustum</italic> SPU_B003 by expressing heterologous phosphoketolase (<italic>xfp</italic>) and phosphotransacetylase (<italic>pta</italic>) initiated by internal promoters. After gene optimization, the recombinant strain <italic>K. robustum</italic>/pBBR-P1_<italic>xfp2502</italic>-P2_<italic>pta2145</italic> enhanced acetyl-CoA approximately 2.4-fold and increased 2-KGA production by 22.27% compared to the control strain <italic>K. robustum</italic>/pBBR1MCS-2. Accordingly, the transcriptional level of the 6-phosphogluconate dehydrogenase (<italic>pgd</italic>) and pyruvate dehydrogenase genes (<italic>pdh</italic>) decreased by 24.33 ± 6.67 and 8.67 ± 5.51%, respectively. The key genes responsible for 2-KGA biosynthesis, sorbose dehydrogenase gene (<italic>sdh</italic>) and sorbosone dehydrogenase gene (<italic>sndh</italic>), were up-regulated to different degrees in the recombinant strain. Conclusions: The genome-based functional analysis of <italic>K. robustum</italic> SPU_B003 provided a new understanding of the specific metabolic characteristics. The new XFP-PTA pathway was an efficient route to enhance acetyl-CoA levels and to therefore promote 2-KGA production. [ABSTRACT FROM AUTHOR]