Bioretrosynthesis of Functionalized N‐Heterocycles from Glucose via One‐Pot Tandem Collaborations of Designed Microbes

Abstract The design of multistrain systems has markedly expanded the prospects of using long biosynthetic pathways to produce natural compounds. However, the cooperative use of artificially engineered microbes to synthesize xenobiotic chemicals from renewable carbohydrates is still in its infancy. Here, a microbial system is developed for the production of high‐added‐value N‐heterocycles directly from glucose. Based on a retrosynthetic analysis, eleven genes are selected, systematically modulated, and overexpressed in three Escherichia coli strains to construct an artificial pathway to produce 5‐methyl‐2‐pyrazinecarboxylic acid, a key intermediate in the production of the important pharmaceuticals Glipizide and Acipimox. Via one‐pot tandem collaborations, the designed microbes remarkably realize high‐level production of 5‐methyl‐2‐pyrazinecarboxylic acid (6.2 ± 0.1 g L−1) and its precursor 2,5‐dimethylpyrazine (7.9 ± 0.7 g L−1). This study is the first application of cooperative microbes for the total biosynthesis of functionalized N‐heterocycles and provides new insight into integrating bioretrosynthetic principles with synthetic biology to perform complex syntheses.


General information
Commercial reagents were used as received: L-threonine (

Strains, plasmids and cultures
The details of the strains and plasmids used in this study are given in Supplementary

Supplementary Table 3. Primers for genomic recombination
Luria-Bertani (LB) medium (per liter: 10 g tryptone, 5 g yeast extract and 10 g NaCl) was used for all molecular cloning experiments and small-scale cultivations. The cultures used for high-density fermentations are summarized in Supplementary Table 4. When needed, ampicillin, streptomycin and kanamycin were used at final concentrations of 50 mg/L.

Analytical methods
High-performance liquid chromatography (HPLC) was performed on a LC-2030C HT system (SHIMADZU, Japan) equipped with a C18AQ column (4.6 × 250 mm, 5 μm, the Nde I and Hind III sites via Gibson assembly method [1] . Plasmids pBAD-Bstdh, pBAD-ldh, pBAD-nox were constructed analogously. Gene Bstdh was amplified from genome of B. subtilis 168 with primers BsTDH-F and BsTDH-R. Gene ldh was amplified from B. subtilis 168 genome using the primers LDH-F and LDH-SalI-R. Gene nox was amplified from pRB1s-nox using primers NOX-F and NOX-SalI-R.

DNA manipulation and genome editing
To obtain strain M11, the deletion of the iclR, tdh, lysA, metA, kbl in E. coli BW25113 was performed using the CRISPR-Cas9 system. The cassettes of J23119-rhtC-rrnB and tac-Cgppc-rrnB were amplified from pS95s-rhtC and pSC2s-Cgppc, respectively. Then, cassettes were assembled with about 500 nt of the upstream/downstream region of target genes by overlap-extension PCR, resulting in the donor DNA fragments for gene integration. The integration was carried out using the CRISPR-Cas9 system as well.
The plasmid pRB1k used for gene expression was derived from expression vectors previously developed in our laboratory (unpublished) and have the following features: a promoter (araBAD), multiple cloning sites, rrnB terminator, origin of replication RSF1030, and kanamycin resistance genes. The plasmid pRBthrA*BC was constructed by introducing the thrA C1034T BC operon of E. coli into the Nco I and EcoR I sites of pRB1k by the Gibson assembly method [1] .

The fed-batch fermentation
a) The fed-batch fermentation of M11 A single colony of strain M11 from a fresh LB-agar plate was used to prepare a seed culture by inoculation of 50 mL LB broth in a 250 mL flask, followed by cultivation to The real-time cell-to-cell ratio of the two components in the co-culture was analyzed by resistance screening. Strain M11 contained a plasmid with kanamycin resistant gene, whereas strain M25 contained a plasmid with ampicillin resistant gene. To conduct resistance screening, the co-culture sample was diluted 10 5 -to 10 6 -fold before being spread onto LB agar plates with 50 mg/L kanamycin or 50 mg/L ampicillin. After 24 h cultivation at 37 °C, the phenotypic screening was conducted by counting colonies on each plate.
b) The sequential biocatalysis of strains M11 and M25 Fed-batch fermentation with strain M11 was conducted with the protocols similar to that described above. After 36 h of fermentation, the concentration of L-threonine reached 62.7 g/L. Then the nutrient feeding solution delivering was stopped and the process of Stage I was complete. The pH value was maintained at 8.0 by feeding 4 M NaOH for the biotransformation. The collected cells of strain M25 (12 g/L DCW) together with 2 mM NAD + were added to the culture to initiate the Stage II conversion (L-threonine to DMP). The concentrations of L-threonine and DMP were monitored with HPLC.
c) The sequential biocatalysis of strains M25 and M38 The biotransformations of M38 were conducted with the protocols similar to that