1. Establishing an innovative carbohydrate metabolic pathway for efficient production of 2-keto-l-gulonic acid in <italic>Ketogulonicigenium robustum</italic> initiated by intronic promoters.
- Author
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Wang, Cai-Yun, Li, Ye, Gao, Zi-Wei, Liu, Li-Cheng, Zhang, Meng-Yue, Zhang, Tian-Yuan, Wu, Chun-Fu, and Zhang, Yi-Xuan
- Subjects
CARBOHYDRATE metabolism ,VITAMIN C ,BACILLUS megaterium ,RIBOSOMAL RNA ,GENE expression ,CELL communication - Abstract
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
Ketogulonicigenium vulgare andBacillus megaterium . 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,Ketogulonicigenium robustum SPU_B003 was reported for its capability to grow well independently and to produce more 2-KGA than that ofK. vulgare in a mono-culture system. The complete genome ofK. robustum SPU_B003 was sequenced, and the metabolic characteristics were analyzed. Compared to the four reportedK. vulgare genomes,K. robustum 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 (orf_01408 promoter) and P2 (orf_02221 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 intoK. robustum SPU_B003 by expressing heterologous phosphoketolase (xfp ) and phosphotransacetylase (pta ) initiated by internal promoters. After gene optimization, the recombinant strainK. robustum /pBBR-P1_xfp2502 -P2_pta2145 enhanced acetyl-CoA approximately 2.4-fold and increased 2-KGA production by 22.27% compared to the control strainK. robustum /pBBR1MCS-2. Accordingly, the transcriptional level of the 6-phosphogluconate dehydrogenase (pgd ) and pyruvate dehydrogenase genes (pdh ) decreased by 24.33 ± 6.67 and 8.67 ± 5.51%, respectively. The key genes responsible for 2-KGA biosynthesis, sorbose dehydrogenase gene (sdh ) and sorbosone dehydrogenase gene (sndh ), were up-regulated to different degrees in the recombinant strain. Conclusions: The genome-based functional analysis ofK. robustum 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]- Published
- 2018
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