Tandem assembly of the epothilone biosynthetic gene cluster by in vitro site-specific recombination

Abstract

We describe a site-specific recombination-based tandem assembly (SSRTA) method for reconstruction of biological parts in synthetic biology. The system was catalyzed by Streptomyces phage φBT1 integrase, which belongs to the large serine recombinase subfamily. This one-step approach was efficient and accurate, and able to join multiple DNA molecules in vitro in a defined order. Thus, it could have applications in constructing metabolic pathways and genetic networks.

Figure 1. Site-specific recombination-based tandem assembly in vitro.

Multiple DNA modules were flanked by pairs of non-compatible recombination sites of the φBT1 integration system. A series of mutated attB sites were placed upstream of each module, and mutated attP sites were located downstream. After incubation of all the DNA modules with φBT1 integrase, tandemly assembled products are produced in a one-step reaction. Differently coloured arrows represented different pairs of recombination sites.

Figure 2. Strategy for assembling the epothilone biosynthetic gene cluster by SSRTA.

(a) Map of seven ‘entry vectors' used in this study. Arrows in pink, green, dark green, red, lime, orange and blue represent pairs of recombination sites numbered 0, 6, 13, 7, 12, 3, and 15, respectively. (b) Map of plasmid pZLE10. Details are available in the Online Methods and Supplementary Table 2. (c) Schematic of the tandem assembly of pZLE10-epoD. Five groups of recombination reactions occur simultaneously, between attB₀/attP₀, attB₆/attP₆, attB₁₃/attP₁₃, attB₃/attP₃ and attB₁₅/attP₁₅. (d) Schematic of pZLE10-epo assembly. Seven groups of recombination reactions take place, between attB₀/attP₀, attB₆/attP₆, attB₁₃/attP₁₃, attB₇/attP₇, attB₁₂/attP₁₂, attB₃/attP₃ and attB₁₅/attP₁₅.

Figure 3. Identification of the assembly products.

(a) Analysis of assembled pZLE10-epoD by pulsed-field gel electrophoresis. The sizes of the substrates were 6316 bp, 6610 bp, 7417 bp, 5178 bp and 7115 bp. The final assembly product was a 29-kb plasmid. (b) Identification of pZLE10-epoD by PCR of the linker regions. The predicted sizes were 322 bp (vm3), 1615 bp (m3m4), 177 bp (m4m5), 249 bp (m5m6) and 468 bp (m6v). (c) ApaI (lane 1), HindIII & NheI (lane 2) digests of pZLE10-epoD. (d) Analysis of assembled pZLE10-epo by pulsed-field gel electrophoresis. The sizes of substrates were 4447 bp, 5749 bp, 7019 bp, 24857 bp, 11568 bp, 8840 bp and 7115 bp and the final assembly product was a 62.4-kb plasmid. (e) Identification of pZLE10-epo by PCR of the linker regions. The predicted sizes were 467 bp (vm0), 420 bp (m0m1), 420 bp (m1m2), 386 bp (m2m3), 177 bp (m4m5), 249bp (m5m6), 277 bp (m6m7), 306 bp (m8m9) and 411 bp (m9v). (f) HindIII (lane 1) and NdeI (lane 2) digests of pZLE10-epo.