Modular Optimization of Heterologous Pathways for De Novo Synthesis of (2S)-Naringenin in Escherichia coli

Abstract

Due to increasing concerns about food safety and environmental issues, bio-based production of flavonoids from safe, inexpensive, and renewable substrates is increasingly attracting attention. Here, the complete biosynthetic pathway, consisting of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS), chorismate mutase/prephenate dehydrogenase (CM/PDH), tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), malonate synthetase, and malonate carrier protein, was constructed using pre-made modules to overproduce (2S)-naringenin from D-glucose. Modular pathway engineering strategies were applied to the production of the flavonoid precursor (2S)-naringenin from L-tyrosine to investigate the metabolic space for efficient conversion. Modular expression was combinatorially tuned by modifying plasmid gene copy numbers and promoter strengths to identify an optimally balanced pathway. Furthermore, a new modular pathway from D-glucose to L-tyrosine was assembled and re-optimized with the identified optimal modules to enable de novo synthesis of (2S)-naringenin. Once this metabolic balance was achieved, the optimum strain was capable of producing 100.64 mg/L (2S)-naringenin directly from D-glucose, which is the highest production titer from D-glucose in Escherichia coli. The fermentation system described here paves the way for the development of an economical process for microbial production of flavonoids.

Fine-Tuning of the Fatty Acid Pathway by Synthetic Antisense RNA for Enhanced (2 S )-Naringenin Production from l -Tyrosine in Escherichia coli

“…Based on our previous study (20), a control strain overproducing (2S)-naringenin from L-tyrosine was constructed. The plasmids pCDF-Trc-TAL-Trc-4CL, pET-CHS-CHI, and pACYCmatC-matB were transformed into E. coli BL21(DE3), yielding a production titer of 91 mg/liter.…”

“…KF765779) (2, 19) was amplified from pUC57-TAL (20) with the primer pair Pf_TAL(NcoI)/Pr_TAL(EcoRI) and then digested and inserted into the NcoI/EcoRI sites of pCDFD-Trc to construct pCDF-Trc-TAL. Codon usage-optimized 4CL from Petroselinum crispum (GenBank accession no.…”

“…Codon usage-optimized 4CL from Petroselinum crispum (GenBank accession no. KF765780) (8) was amplified from pUC57-4CL (20) with the primer pair Pf_4CL(NcoI)/Pr_4CL(HindIII) and then digested and inserted into the NcoI/HindIII sites of pCDFD-Trc to construct pCDFTrc-4CL. The pTrc-4CL region, including the trc promoter and 4CL, was amplified with the primer pair Pf_Ptrc4CL(EcoRI)/Pr_4CL(HindIII) and then digested and inserted into the EcoRI/HindIII sites of pCDF-Trc-TAL to construct pCDF-Trc-TAL-Trc-4CL.…”

“…Codon usage-optimized CHI from Medicago sativa (GenBank accession no. KF765782) (8) was excised from pUC57-CHI (20) and cloned into the NdeI/BlnI sites of pET-CHS to construct pET-CHS-CHI. Codon usageoptimized matB from Rhizobium trifolii (GenBank accession no.…”

“…Codon usage-optimized matC from Rhizobium trifolii (GenBank accession no. KF765784) (8) was excised from pUC57-matC (20) and inserted into the EcoRI/HindIII sites of pACYC-matB to construct pACYC-matC-matB. Sequences of these synthesized genes are shown in the supplemental material (see Fig.…”