Abstract
Background2-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 and Bacillus 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.ResultsIn this study, Ketogulonicigenium robustum SPU_B003 was reported for its capability to grow well independently and to produce more 2-KGA than that of K. vulgare in a mono-culture system. The complete genome of K. robustum SPU_B003 was sequenced, and the metabolic characteristics were analyzed. Compared to the four reported K. 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 into K. robustum SPU_B003 by expressing heterologous phosphoketolase (xfp) and phosphotransacetylase (pta) initiated by internal promoters. After gene optimization, the recombinant strain K. 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 strain K. 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.ConclusionsThe genome-based functional analysis of K. 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.
Highlights
To investigate the effect of oversupply of acetyl-CoA on cell growth and 2-Keto-l-gulonic acid (2-KGA) production, a higher efficiency acetyl-CoA biosynthetic pathway with lower carbon loss was constructed by heterologously expressing phosphoketolase and phosphotransacetylase in K. robustum SPU_B003
Because the XFP-PTA pathway is helpful in promoting the tricarboxylic acid (TCA) cycle and generating α-ketoglutarate, the precursor of glutamate, this route is presumably conducive to the biosynthesis of pyrroloquinoline quinone (PQQ), and affects the PQQ-regulated electron transfer in the l-sorbose oxidation process, affecting the transcription of sdh and sndh
To further enhance 2-KGA production, an innovative acetyl-CoA biosynthetic pathway was constructed by expressing phosphoketolase and phosphotransacetylase initiated by a species-specific promoter
Summary
Ketogulonicigenium robustum SPU_B003 was reported for its capability to grow well indepen‐ dently and to produce more 2-KGA than that of K. vulgare in a mono-culture system. Compared to the four reported K. vulgare genomes, K. robustum SPU_B003 contained more tRNAs, rRNAs, NAD and NADP biosynthetic genes, as well as regulation- and cell signaling-related genes. To efficiently produce 2-KGA with decreased C O2 release, an innovative acetyl-CoA biosynthetic pathway (XFP-PTA pathway) was introduced into K. robustum SPU_B003 by expressing heter‐ ologous phosphoketolase (xfp) and phosphotransacetylase (pta) initiated by internal promoters. After gene optimiza‐ tion, the recombinant strain K. 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 strain K. robustum/pBBR1MCS-2. 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
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