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Applied Microbiology and Biotechnology

, Volume 99, Issue 13, pp 5627–5637 | Cite as

The glyoxylate shunt is essential for CO2-requiring oligotrophic growth of Rhodococcus erythropolis N9T-4

  • Takanori Yano
  • Nobuyuki Yoshida
  • Fujio Yu
  • Miki Wakamatsu
  • Hiroshi Takagi
Applied microbial and cell physiology

Abstract

Rhodococcus erythropolis N9T-4 shows extremely oligotrophic growth requiring atmospheric CO2 and forms its colonies on an inorganic basal medium (BM) without any additional carbon source. Screening of a random mutation library constructed by a unique genome deletion method that we established indicated that the aceA, aceB, and pckG genes encoding isocitrate lyase, malate synthase, and phosphoenolpyruvate carboxykinase, respectively, were requisite for survival on BM plates. The aceA- and aceB deletion mutants and the pckG deletion mutant grew well on BM plates containing L-malate and D-glucose, respectively, suggesting that the glyoxylate (GO) shunt and gluconeogenesis are essential for the oligotrophic growth of N9T-4. Interestingly, most of the enzyme activities in the TCA cycle were observed in the cell-free extract of N9T-4, with perhaps the most important exception being α-ketoglutarate dehydrogenase (KGDH) activity. Instead of the KGDH activity, we detected a remarkable level of α-ketoglutarate decarboxylase (KGD) activity, which is the activity exhibited by the E1 component of the KGDH complex in Mycobacterium tuberculosis. The recombinant KGD of N9T-4 catalyzed the decarboxylation of α-ketoglutarate to form succinic semialdehyde (SSA) in a time-dependent manner. Since N9T-4 also showed a detectable SSA dehydrogenase activity, we concluded that N9T-4 possesses a variant TCA cycle, which uses SSA rather than succinyl-CoA. These results suggest that oligotrophic N9T-4 cells utilize the GO shunt to avoid the loss of carbons as CO2 and to conserve CoA units in the TCA cycle.

Keywords

Random mutagenesis Carbon dioxide Glyoxylate shunt TCA cycle Oligotroph Rhodococcus erythropolis N9T-4 

Notes

Acknowledgments

The authors greatly appreciate Maki Kaneoka for her technical assistance for construction and screening of the random mutation library. This work was partly supported by the Sasakawa Scientific Research Grant from The Japan Science Society to T.Y. and Global COE Program in NAIST from the Ministry of Education, Science, Culture, Sports and Technology of Japan.

Supplementary material

253_2015_6500_MOESM1_ESM.pdf (170 kb)
ESM 1 (PDF 169 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.Graduate School of Biological SciencesNara Institute of Science and TechnologyIkomaJapan
  2. 2.Department of Applied Chemistry and Biochemical Engineering, Graduate school of EngineeringShizuoka UniversityHamamatsuJapan
  3. 3.Yokohama Research LabolatoriesMitsubishi Rayon Co., Ltd.YokohamaJapan

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