Figure 1.
Modular optimization of heterologous pathways for de novo synthesis of (2S)-naringenin.
Schematics of the three modules: module one (TAL, 4CL), module two (CHS, CHI), and module three (matB, matC). aroG: the gene encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase. tyrA: the gene encoding chorismate mutase/prephenate dehydrogenase (CM/PDH). TAL: tyrosine ammonia lyase; 4CL: 4-coumarate:CoA ligase; CHS: chalcone synthase; CHI: chalcone isomerase. matB: the gene encoding R. trifolii malonate synthetase; matC: the gene encoding R. trifolii malonate carrier protein.
Table 1.
Nucleotide sequences of primers.
Table 2.
Plasmids used in this study.
Figure 2.
Optimization of (2S)-naringenin production from L-tyrosine by engineering three modules.
pBR322: origin of pETDuet-1; CDF: origin of pCDFDuet-1; p15A: origin of pACYCDuet-1; T7: T7 promoter; Trc: Trc promoter. S1–S13 denotes strains 1–13 constructed in this study. Gray bars: p-coumaric acid (mg/L); white bars: (2S)-naringenin (mg/L).
Figure 3.
HPLC and LC-MS analysis of (2S)-naringenin and p-coumaric acid produced by engineered E. coli strains.
A–B: Partial HPLC chromatograms show engineered strains have a significantly increased titer of (2S)-naringenin and a dramatically decreased titer of p-coumaric acid compared to the initial strain. A: Partial HPLC chromatograms of the initial strain; B: partial HPLC chromatograms of the optimized strain constructed through module engineering. C–D: HPLC chromatograms of p-coumaric acid (C) and (2S)-naringenin (D) in our sample. E–F: Selected ion chromatograms of (2S)-naringenin (m/z 271.0587 [M–H]−) produced by E. coli. E: LC/ESI-MS chromatogram of our compound; F: MS/MS spectrum of our compound.
Figure 4.
Assembling individual modules to enable de novo synthesis of (2S)-naringenin.
S14–S16 denotes strains 14–16 constructed in this study. Gray bars: (2S)-naringenin (mg/L); dark gray bars: p-coumaric acid (mg/L). S14 means the new modular pathway from glucose to L-tyrosine was expressed at the plasmid of pRSFDuet-1; S15 means the new modular pathway from glucose to L-tyrosine was expressed at the plasmid of pCOLADuet-1; S16 means the new modular pathway from D-glucose to L-tyrosine was integrated into the lacZ locus of E. coli BL21 under T7 promoter.