Abstract
BackgroundDirect cloning combined with heterologous expression of a secondary metabolite biosynthetic gene cluster has become a useful strategy for production improvement and pathway modification of potentially valuable natural products present at minute quantities in original isolates of actinomycetes. However, precise cloning and efficient overexpression of an entire biosynthetic gene cluster remains challenging due to the ineffectiveness of current genetic systems in manipulating large-sized gene clusters for heterologous as well as homologous expression.ResultsA versatile Escherichia coli-Streptomyces shuttle bacterial artificial chromosomal (BAC) conjugation vector, pSBAC, was used along with a cluster tandem integration approach to carry out homologous and heterologous overexpression of a large 80-kb polyketide biosynthetic pathway gene cluster of tautomycetin (TMC), which is a protein phosphatase PP1/PP2A inhibitor and T cell-specific immunosuppressant. Unique XbaI restriction sites were precisely inserted at both border regions of the TMC biosynthetic gene cluster within the chromosome of TMC-producing Streptomyces sp. CK4412, followed by site-specific recombination of pSBAC into the flanking region of the TMC gene cluster. The entire TMC gene cluster was then rescued as a single giant recombinant pSBAC by XbaI digestion of the chromosomal DNA as well as subsequent self-ligation. Next, the recombinant pSBAC construct containing the entire TMC cluster in E. coli was directly conjugated into model Streptomyces strains, resulting in rapid and enhanced TMC production. Moreover, introduction of the TMC cluster-containing pSBAC into wild-type Streptomyces sp. CK4412 as well as a recombinant S. coelicolor strain resulted in a chromosomal tandem repeat of the entire TMC cluster with 14-fold and 5.4-fold enhanced TMC productivities, respectively.ConclusionsThe 80-kb TMC biosynthetic gene cluster was isolated in a single integration vector, pSBAC. Introduction of TMC biosynthetic gene cluster in TMC non-producing strains has resulted in similar amount of TMC production yield. Moreover, over-expression of TMC biosynthetic gene cluster in original producing strain and recombinant S. coelicolor dramatically increased TMC production. Thus, this strategy can be employed to develop a custom overexpression scheme of entire metabolite pathway clusters present in actinomycetes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0325-2) contains supplementary material, which is available to authorized users.
Highlights
Direct cloning combined with heterologous expression of a secondary metabolite biosynthetic gene cluster has become a useful strategy for production improvement and pathway modification of potentially valuable natural products present at minute quantities in original isolates of actinomycetes
CK4412 as well as a recombinant S. coelicolor strain resulted in a chromosomal tandem repeat of the entire TMC cluster with 40-fold enhanced TMC productivities
After 5 days of culture, TMC production levels in TMC002 and TMC003 were about 1.3-fold (4.05 mg/L) and 1.26-fold (3.91 mg/L) higher than that in wild-type (3.1 mg/L), respectively (Fig. 3b). These results reveal that the pSBAC-driven heterologous expression of an entire TMC biosynthetic gene cluster resulted in rapid and enhanced TMC production
Summary
Direct cloning combined with heterologous expression of a secondary metabolite biosynthetic gene cluster has become a useful strategy for production improvement and pathway modification of potentially valuable natural products present at minute quantities in original isolates of actinomycetes. Nah et al Microb Cell Fact (2015) 14:140 within chromosomes, identification of the entire biosynthetic gene cluster is relatively straightforward Some of these biosynthetic genes are derived from non-culturable or not amenable to genetic manipulation microorganisms and do not express the target compounds [3]. To bypass such intrinsic limitations and achieve functional expression of uncharacterized potentially-valuable natural product biosynthetic pathways, a relatively well-characterized heterologous host should be utilized [4, 5]. It is more desirable to develop a general cloning strategy to quickly capture an entire biosynthetic gene cassette without depending on endogenous restriction sites in the cluster or a large DNA fragment ligation process
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