caa a22 4500 605505861 CHVBK 20210128100624.0 cr unu---uuuuu 210128e20150701xx s 000 0 eng 10.1007/s00253-015-6500-x doi (NATIONALLICENCE)springer-10.1007/s00253-015-6500-x The glyoxylate shunt is essential for CO2-requiring oligotrophic growth of Rhodococcus erythropolis N9T-4 [Elektronische Daten] [Takanori Yano, Nobuyuki Yoshida, Fujio Yu, Miki Wakamatsu, Hiroshi Takagi] 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. Springer-Verlag Berlin Heidelberg, 2015 Random mutagenesis nationallicence Carbon dioxide nationallicence Glyoxylate shunt nationallicence TCA cycle nationallicence Oligotroph nationallicence Rhodococcus erythropolis N9T-4 nationallicence Yano Takanori Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, 630-0192, Ikoma, Nara, Japan aut Yoshida Nobuyuki Department of Applied Chemistry and Biochemical Engineering, Graduate school of Engineering, Shizuoka University, 3-5-1 Johoku, Naka-ku, 432-8561, Hamamatsu, Japan aut Yu Fujio Yokohama Research Labolatories, Mitsubishi Rayon Co., Ltd., 10-1, Daikoku-cho, Tsurumi-ku, 230-0053, Yokohama, Japan aut Wakamatsu Miki Yokohama Research Labolatories, Mitsubishi Rayon Co., Ltd., 10-1, Daikoku-cho, Tsurumi-ku, 230-0053, Yokohama, Japan aut Takagi Hiroshi Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, 630-0192, Ikoma, Nara, Japan aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/13(2015-07-01), 5627-5637 0175-7598 99:13<5627 2015 99 253 https://doi.org/10.1007/s00253-015-6500-x text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6500-x text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Yano Takanori Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, 630-0192, Ikoma, Nara, Japan aut NATIONALLICENCE 700 1- Yoshida Nobuyuki Department of Applied Chemistry and Biochemical Engineering, Graduate school of Engineering, Shizuoka University, 3-5-1 Johoku, Naka-ku, 432-8561, Hamamatsu, Japan aut NATIONALLICENCE 700 1- Yu Fujio Yokohama Research Labolatories, Mitsubishi Rayon Co., Ltd., 10-1, Daikoku-cho, Tsurumi-ku, 230-0053, Yokohama, Japan aut NATIONALLICENCE 700 1- Wakamatsu Miki Yokohama Research Labolatories, Mitsubishi Rayon Co., Ltd., 10-1, Daikoku-cho, Tsurumi-ku, 230-0053, Yokohama, Japan aut NATIONALLICENCE 700 1- Takagi Hiroshi Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, 630-0192, Ikoma, Nara, Japan aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/13(2015-07-01), 5627-5637 0175-7598 99:13<5627 2015 99 253