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. 2022 Jul 7;12:11489. doi: 10.1038/s41598-022-15624-6

Figure 3.

Figure 3

Substrate regeneration system with LuxABCDE-Fre. (A) The schematic of the LuxABCDE-Fre substrate regeneration system. LuxAB generates light with reduced flavin mononucleotide (FMNH2) and long-chain aldehyde; those substrates are converted into oxidized flavin mononucleotide (FMN) and corresponding long-chain acid. NAD(P)H-flavin reductase (Fre) reduces FMN back to FMNH2, and LuxCDE reduces the acid back to the corresponding aldehyde. (B) Luminescence measurement with the LuxABCDE-Fre system. LuxAB-Fre and LuxCDE were expressed in TXTL and mixed with 1 mM long-chain fatty acids (octanoic acid, decanoic acid, dodecanoic acid, or tetradecanoic acid) or caprylic aldehyde, followed by luminescence measurement. The reaction also contained FMN, NADPH, and ATP. Control represents a reaction using TXTL without enzyme expression. (C) Luminescence kinetics measurement with decanoic acid. LuxAB-Fre and LuxCDE were expressed in TXTL. For LuxABCDE-Fre reaction, the TXTL expressing LuxAB-Fre and LuxCDE were mixed with 1 mM decanoic acid (time = 0). For LuxAB-Fre reaction, the TXTL expressing LuxAB-Fre was used. For Control reaction, TXTL without enzyme expression was used. The reaction also contained FMN, NADPH, and ATP. The luminescence was measured after 0.5, 1, 6, and 8 h. The graphs show means with error bars that signify SEM (n = 3).