High-fidelity de novo synthesis of pathways using microchip-synthesized oligonucleotides and general molecular biology equipment

Engineering and evaluation of synthetic routes for generating valuable compounds require accurate and cost-effective de novo synthesis of genetic pathways. Here, we present an economical and streamlined de novo DNA synthesis approach for engineering a synthetic pathway with microchip-synthesized oligonucleotides (oligo). The process integrates entire oligo pool amplification, error-removal, and assembly of long DNA molecules. We utilized this method to construct a functional lycopene biosynthetic pathway (11.9 kb encoding 10 genes) in Escherichia coli using a highly error-prone microchip-synthesized oligo pool (479 oligos) without pre-purification, and the error-frequency was reduced from 14.25/kb to 0.53/kb. This low-equipment-dependent and cost-effective method can be widely applied for rapid synthesis of biosynthetic pathways in general molecular biology laboratories.


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Page Supplementary  Table S1 Primers and RBS sequences used in this study 3-5 Supplementary  Table S2 Primers used in lycopene gene synthesis 6 Supplementary  Table S3 Summary of oligonucleotides design of lycopene biosynthesis genes 7 Supplementary Table S4 Primers used in the construction of strain WW1 8 Supplementary Figure S1 Design of oligonucleotides of lycopene pathway genes for synthesis on microchips (mvaS oligonucleotides are described as an example).

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Supplementary Figure S2 Schematic representation of the construction of the WW1 strain harboring the genes of the lycopene biosynthetic pathway 10 Supplementary Figure S3 Production of lycopene via the engineered lycopene biosynthesis pathway 11 ATATAGTAAGTCGCATTGTTTATTCCTCCTATAAATCTACTGGCAAACACGCTTTTT (3'-5') a Underline: the restriction enzyme site b These RBS sequences were dsDNA, and prepared by annealing of the two complementary single strand oligos.
The sequences of primers were listed in Supplementary Table S1. a half-specific primer pair with one specific fragment primer and one F-primer. b full-specific primer pair with two specific fragment primers.  Figure S1. Design of oligonucleotides of lycopene biosynthetic pathway genes for synthesis on microchips (mvaS oligonucleotides are described as an example).

Construction of dxr-deleted Escherichia coli WW1 and expression of the synthetic lycopene pathway
The strategy to construct the WW1 strain is illustrated in Supplementary Figure S6.
Because Dxr-disrupted E. coli cannot grow without 2-C-methyl-D-erythritol (1), a copy of dxr in the pT-dxr plasmid was used to complement the deletion during the deficient strain construction. In brief, the Tc R gene sequence was PCR-amplified from pACYC184 with Tc-A-F and Tc-S-R primers (Supplementary Table S4) and the pET21c DNA fragment without the bla coding sequence was obtained by PCR using the pET21C-A-F and pET21C-S-R primers (Supplementary Table S4). These sequences were double-digested with AspI and SpeI and ligated with T4 DNA ligase to generate the plasmid pT-21c. The ORF of dxr was amplified from the genome of E. coli with the dxr-N-F and dxr-X-R primers (Supplementary Table S4) and cloned into pT-21c NheI-XhoI sites to generate the plasmid pT-dxr. pT-dxr was transformed into E. coli JM109 (DE3). Then the chromosomal dxr gene sequence was deleted based on the Red system (Supplementary Figure S6) as previously described (2). The dxr gene in pT-dxr was under the control of the T7 promoter and leaking expression of this gene was sufficient to support growth. After eliminating the chromosomal dxr gene sequence, plasmids pO1-O3 and pO2 were transformed into the constructed strain and transformants were screened on LB medium containing 100 µg/mL ampicillin and 50 µg/mL kanamycin (without tetracycline). Because operon 1 and operon 2 could complement the deficiency of the MEP pathway (blocked in the dxr mutant) with the MVA pathway, the pT-dxr-deleted strain could be screened on the medium without tetracycline and confirmed with the loss of Tet R . The resultant strain harboring the lycopene pathway genes is dxr deficient and named WW1.

PCR amplification of oligos
PCR of 50 µL contained 1 ng of template oligos, 100 pmol of primers, 5 µL of 2 mM dNTPs, 3 µL of 25 mM MgSO4, and 1 µL of KOD plus DNA polymerase in 1× reaction buffer. PCR parameters were set as follows: 94°C for 5 min, 30 cycles of 15 s at 94°C, 30 s at 50°C, 30 s at 68°C, and a final extension step of 10 min at 68°C. Amplified oligos were purified with the UNIQ-10 oligonucleotide kit (Sangon Biotech Co. (Shanghai, China)) according to the manufacturer's instructions.

PCR amplification of full-length operons
The 50 µL-PCR mixture contained 10 µL of 5 × reaction buffer, 4 µL of 2.5 mM dNTPs, 0.5 µL of PrimeSTAR HS DNA polymerase, 1 µL of Gibson assembly product, and 1 µL of each primer (10 µM). PCR parameters were: 98°C for 10 sec, 55°C for 5 sec, and 72°C for 4.5 min for 30 cycles, followed by 72°C for 5 min. The PCR mixture was isolated on 1% (w/v) agarose gels and extracted with the Axygen gel extraction kit according to the manufacturer's instructions.