Optimization of (2S)-naringenin production from L-tyrosine by engineering three modules.

<p>pBR322: origin of pETDuet-1; CDF: origin of pCDFDuet-1; p15A: origin of pACYCDuet-1; T7: <i>T7</i> promoter; Trc: <i>Trc</i> promoter. S1–S13 denotes strains 1–13 constructed in this study. Gray bars: <i>p</i>-coumaric acid (mg/L); white bars: (2S)-naringenin (mg/L).</p

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

<div><p>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 <i>Escherichia coli</i>. The fermentation system described here paves the way for the development of an economical process for microbial production of flavonoids.</p></div

DATEL: A Scarless and Sequence-Independent DNA Assembly Method Using Thermostable Exonucleases and Ligase

DNA assembly is a pivotal technique in synthetic biology. Here, we report a scarless and sequence-independent DNA assembly method using thermal exonucleases (<i>Taq</i> and <i>Pfu</i> DNA polymerases) and <i>Taq</i> DNA ligase (DATEL). Under the optimized conditions, DATEL allows rapid assembly of 2–10 DNA fragments (1–2 h) with high accuracy (between 74 and 100%). Owing to the simple operation system with denaturation-annealing-cleavage-ligation temperature cycles in one tube, DATEL is expected to be a desirable choice for both manual and automated high-throughput assembly of DNA fragments, which will greatly facilitate the rapid progress of synthetic biology and metabolic engineering

Nucleotide sequences of primers.

a<p>: Bold and underlined letters are restriction enzyme cut sites.</p

Assembling individual modules to enable de novo synthesis of (2S)-naringenin.

<p>S14–S16 denotes strains 14–16 constructed in this study. Gray bars: (2S)-naringenin (mg/L); dark gray bars: <i>p-</i>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 <i>lacZ</i> locus of <i>E. coli</i> BL21 under <i>T7</i> promoter.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Modular optimization of heterologous pathways for de novo synthesis of (2S)-naringenin.

<p>Schematics of the three modules: module one (TAL, 4CL), module two (CHS, CHI), and module three (<i>matB</i>, <i>matC</i>). <i>aroG</i>: the gene encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase. <i>tyrA</i>: the gene encoding chorismate mutase/prephenate dehydrogenase (CM/PDH). TAL: tyrosine ammonia lyase; 4CL: 4-coumarate:CoA ligase; CHS: chalcone synthase; CHI: chalcone isomerase. <i>matB</i>: the gene encoding <i>R. trifolii</i> malonate synthetase; <i>matC</i>: the gene encoding <i>R. trifolii</i> malonate carrier protein.</p

Effect of temperature on the activity and stability of eight five-site mutant enzymes.

<p>A: Effect of temperature on the activity of mutant enzymes. B: Effect of temperature on the stability of mutant enzymes. The inset presents the Arrhenius plot of the logarithm of the <i>k</i> values against the reciprocal of the absolute temperature (<i>T</i>). The values shown are activation energies calculated from the plot.</p

The effect of ribosome binding site design on L-AAD catalyst activity, cell growth and KIC production.

<p><b>a</b>: The comparison of biocatalyst activity with predicted translation rate. Gray bar: the biocatalyst activity and the BL21/pET28a-lad as the control (the yellow bar). ☆: predicted translation initiation rate. <b>b</b>: The effect of RBS mutants on cell growth. <b>c</b> and <b>d</b>: the KIC production and bioconversion rate of the 4 dominant RBS mutants and BL21/pET28a-lad. BL21/RBS3 (orange squares), BL21/RBS4 (green circles), BL21/RBS6 (black triangles), BL21/RBS8 (magenta triangles), BL21/pET28a-lad (violet rhombus), the additon of L-leucine (arrows).</p

Construction and Characterization of Broad-Spectrum Promoters for Synthetic Biology

Characterization of genetic circuits and biosynthetic pathways in different hosts always requires promoter substitution and redesigning. Here, a strong, broad-spectrum promoter, P_bs, for <i>Escherichia coli</i>, <i>Bacillus subtilis</i>, and <i>Saccharomyces cerevisiae</i> was constructed, and it was incorporated into the minimal <i>E. coli</i>–<i>B. subtilis</i>–<i>S. cerevisiae</i> shuttle plasmid pEBS (5.8 kb). By applying a random mutation strategy, three broad-spectrum promoters P_bs1, P_bs2, and P_bs3, with different strengths were generated and characterized. These broad-spectrum promoters will expand the synthetic biology toolbox for <i>E. coli</i>, <i>B. subtilis</i>, and <i>S. cerevisiae</i>