Application of serine integrases for secondary metabolite pathway assembly in Streptomyces

Abstract

Serine integrases have been shown to be efficient tools for metabolic pathway assembly. To further improve the flexibility and efficiency of pathway engineering via serine integrases, we explored how multiple orthogonally active serine integrases can be applied for use in vitro for the heterologous expression of complex biosynthesis pathways in Streptomyces spp., the major producers of useful bioactive natural products. The results show that multiple orthogonal serine integrases efficiently assemble the genes from a complex biosynthesis pathway in a single in vitro recombination reaction, potentially permitting a versatile combinatorial assembly approach. Furthermore, the assembly strategy also permitted the incorporation of a well-characterised promoter upstream of each gene for expression in a heterologous host. The results demonstrate how site-specific recombination based on orthogonal serine integrases can be applied in Streptomyces spp.

Keywords: Erythromycin; Pathway assembly; Serine integrase; Streptomyces.

Fig. 1

Plasmids used for integration in this study. In pHG2R2, pHG9A, and pHG22A, the eryB genes were assembled in the order of B45–B6–B3–B2–B7, and integrases used are TG1-Int7-Int9-Int4-Bxb1-SPBc, act1p was cloned at the upstream of each gene fragment (B45, B6, B3, B2 or B7). In pHG22A, the eryC genes were assembled in the order of C1–C23–C45–C6, the integrases used are TG1-Bxb1-Int9-Int7-SPBc, and act1p was cloned at the upstream of each gene fragment.

Fig. 2

The assembly of DNA fragments. (A) The construction of DNA fragments to be assembled. The eryB or eryC genes were cloned into pJET1.2/blunt, under the control of the promoter act1p. Next, primer pairs (F: forward primer; R: reverse primer) contained attB or attP sites (indicated using triangle) at the 3′ end were used to amplify the DNA fragment used in the assembly reaction. The primers only contain the sequence complemented with the sequences in the backbone vector flanking the target DNA fragment, rather than the specific eryB or eryC genes. So these primers are universal and could be used to amplify any gene inserted in the backbone vector in the same way. (B) The assembly strategy of pHG2R2. The length of each fragment is: pHG2B0 (10.2 kb), eryBII (1.4 kb), eryBIII (1.7 kb), eryBIV-BV (2.7 kb), eryBVI (1.9 kb) and eryBVII (1.0 kb); (C) The assembly reactions were carried out in four buffers, and the assembled plasmids were checked by digesting with BglII. Stars mark the clones have been assembled correctly. B: pHG2B0, M: NEB Fast DNA Ladder (from https://international.neb.com/products/n3238-fast-dna-ladder#Product%20Information). 0.8% agarose gel was used to check the digested bands.

Fig. 3

Assembly of eryC genes. (A) The assembly strategy of pHG22A. The length of each fragment is: pHG9A (19.3 kb), eryCI (1.5 kb), eryCII-CIII (2.7 kb), eryCIV-CV (3.1 kb) and eryCVI (1.1 kb); (B) The assembled plasmids were checked by digesting with XbaI. NEB Fast DNA Ladder was used to assess the size of digested fragments. From (A), XbaI could digest the correctly assembled plasmids into these fragments: Stars mark the clones have been assembled correctly. M: NEB Fast DNA Ladder. 0.8% agarose gel was used to check the digested bands.

Fig. 4

The biosynthetic pathway of erythromycin A.

Fig. 5

Production of EB in Streptomyces coelicolor M1152. (A) HPLC base peak chromatogram (BPC) showing the most intense peaks for each mass spectrometry scan in the extract from a S. coelicolor M1152:pBF20:pBF22:pBF24:pHG1 (M2) fermentation. (B) Extracted ion chromatogram (EIC) of the internal standard roxithromycin (m/z 837.53; peak 1) from the same extract as in panel A. (C) Extracted ion chromatogram of 6-dEB (m/z 409.2750) from the same extracted as in panel A, showing almost all 6-dEB has been catalysed by EryF. (D) Extracted ion chromatogram of EB (m/z 425.2700, peak 2) from the same extracted as in panel A.