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.
Highlights
Phage-encoded serine integrases catalyse site-specific integration of DNA into bacterial host chromosomes in a highly controllable and predictable way [1], making these proteins powerful tools for molecular genetics
This is evident in the study of Colloms et al, who developed the serine integrase recombinational assembly (SIRA) method that used φC31 to assemble up to 5 genes into a functional pigment biosynthetic pathway; the efficiency dropped from 87% for the 3-gene assembly to 18% for the 5-gene assembly [11]
A strain producing Erythronolide B (EB) was needed to be the starting strain, and the production of Mycarosylerythronolide B (MEB) or erythromycin D could be tested after the assembly of eryB or eryC genes, to confirm the assembled pathways are functional
Summary
Phage-encoded serine integrases catalyse site-specific integration of DNA into bacterial host chromosomes in a highly controllable and predictable way [1], making these proteins powerful tools for molecular genetics. Studies have shown that this 2 bp sequence can be changed to other sequences and recombination efficiency is unaffected, but only if both attP and attB have the same 2-bp sequence [12,13] This central 2-bp site specificity has permitted the use of a single integrase to assemble DNA fragments together in a predictable order in a single recombination reaction [11,14]. This is evident in the study of Colloms et al, who developed the serine integrase recombinational assembly (SIRA) method that used φC31 to assemble up to 5 genes into a functional pigment biosynthetic pathway; the efficiency dropped from 87% for the 3-gene assembly to 18% for the 5-gene assembly [11]. This is because integrase attempts and fails to complete recombination between sites with non-identical crossover sites but recombination only proceeds to completion in reactions where the central 2-bp site is identical in both attP and attB
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