Abstract
BackgroundThe Actinomycete Actinosynnema pretiosum ssp. auranticum has commercial importance due to its production of ansamitocin P-3 (AP-3), a potent antitumor agent. One way to increase AP-3 production would be to constitutively express selected genes so as to relieve bottlenecks in the biosynthetic pathway; however, an integrative expression vector for A. pretiosum is lacking. The aim of this study was to construct a vector for heterologous gene expression in A. pretiosum.ResultsA series of integrative expression vectors have been made with the following features: the IS117 transposase from Streptomyces coelicolor, the constitutive ermE* promoter from Saccharopolyspora erythraea, different ribosome-binding site (RBS) sequences and xylE as a translational reporter. Positive E. coli clones and A. pretiosum transconjugants were assayed by catechol. pAP42, containing an E. coli consensus RBS, and pAP43, containing an asm19 RBS, gave strong and moderate gene expression, respectively. In addition, an operon construct capable of multi-gene expression was created. Plasmid integration sites in transconjugants were investigated and four different sites were observed. Although the most common integration site was within a putative ORF with sequence similarity to NADH-flavin reductase, AP-3 levels and cell growth of transconjugants were unaffected.ConclusionA set of integrative vectors for constitutive gene expression in A. pretiosum has been constructed. Gene translation is easily determined by colorimetric assay on an agar plate. The vectors are suitable for studies relating to AP-3 biosynthesis as they do not affect AP-3 production.
Highlights
The Actinomycete Actinosynnema pretiosum ssp. auranticum has commercial importance due to its production of ansamitocin P-3 (AP-3), a potent antitumor agent
Plasmid containing IS117 with unmodified ends did not result in A. pretiosum transconjugants
Consensus sequences of up to 19 nucleotides flanking attM have been identified in the genomes of S. lividans [14] and M. smegmatis [16], suggesting a role in alignment of the integration site
Summary
The Actinomycete Actinosynnema pretiosum ssp. auranticum has commercial importance due to its production of ansamitocin P-3 (AP-3), a potent antitumor agent. Auranticum has commercial importance due to its production of ansamitocin P-3 (AP-3), a potent antitumor agent. One way to increase AP-3 production would be to constitutively express selected genes so as to relieve bottlenecks in the biosynthetic pathway; an integrative expression vector for A. pretiosum is lacking. Actinosynnema pretiosum is a commercially important organism due to its ability to produce ansamitocin P-3 (AP-3), a potent anti-tumor agent [1,2]. In immuno-toxin conjugates [3] Several of these conjugates are currently in late-phase clinical trials as therapeutic agents against solid tumors [4]. There is interest in generating strains of A. pretiosum that produce greater concentrations of AP-3 to meet increasing industrial (page number not for citation purposes). Deletion of a putative transcriptional repressor, asm, has been reported to increase AP-3 yield [7]
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