Fig. 4: Implementation of module 1 and module 2 of THETA in vivo.
From: Construction and modular implementation of the THETA cycle for synthetic CO2 fixation
a, A simplified scheme of the THETA cycle, consisting of three modules. b, The scheme for implementing module 1 in vivo. c, Heterologous expression of Frd (T. brucei) or overexpression of NadB (E. coli) rescued the growth of the acetyl-CoA and succinyl-CoA auxotroph strain SL1 on M9 with 1% glucose and 10 mM acetate without supplementing succinate. d, The scheme for implementing module 2 in vivo. A short reaction sequence (C4-acetyl-CoA shunt) is added to channel crotonyl-CoA, the output molecule of module 2, into acetyl-CoA. e, Expression of crotonyl-CoA ligase (DmdB1 from P. aeruginosa) and the C4-acetyl-CoA shunt (pTE3217) enabled the acetyl-CoA auxotroph strain SL2 convert crotonate to acetyl-CoA and grow without supplementing acetate. f, Expression of module 2 (pTE3236 or pTE3237) and the C4-acetyl-CoA shunt (pTE3217) enabled the acetyl-CoA auxotroph strain SL2 to convert succinate to acetyl-CoA and grow without supplementing acetate. The growth was achieved through short-term evolution on plates. sucD encodes Scr and 4hbd encodes Ssr. g, 13C labelling of proteinogenic leucine (Leu) and alanine (Ala) after cultivating the strains with [U-13C]succinate (SL2 on M9 with 1% glucose, 10 mM acetate and 10 mM [U-13C]succinate; 1736 and 1737 on M9 with 1% glucose and 10 mM [U-13C]succinate). The labelling pattern confirmed operation of module 2 in vivo, since leucine (derived from one acetyl-CoA and two pyruvate) was mainly labelled twice while alanine (derived from pyruvate only) was predominately non-labelled. The growth data represent the mean ± s.d. obtained in n = 3 independent experiments. For enzyme abbreviations, see Supplementary Table 2. Glx, glyoxylate; αKG, α-ketoglutarate; Ac-CoA, acetyl-CoA; Suc-CoA, succinyl-CoA; Acac-CoA, acetoacetyl-CoA; 3HB-CoA, 3-hydroxybutyryl-CoA; Cro-CoA, crotonyl-CoA; 4HB-CoA, 4-hydroxybutyryl-CoA.
