Robust reporter system based on chalcone synthase rppA gene from Saccharopolyspora erythraea.
Industrial overproducing strains present unique hosts for expression of heterologous gene clusters encoding secondary metabolite biosynthesis. For this purpose, efficient gene expression tools and methods are needed. A robust and versatile reporter system based on the rppA gene from Saccharopolyspora erythraea is presented as the method of choice when studying gene expression in actinomycete hosts. The method is easily scalable to accommodate high-throughput procedure, and collected samples can be easily stored and re-tested when needed. The product of RppA is an inert 1,3,6,8-tetrahydroxynaphthalene which spontaneously oxidises to a dark-red quinone flaviolin providing a qualitative visual assessment of gene expression on an agar plate as well as a quantitative spectrophotometric measurement in liquid broth without the need for invasive procedures or external substrate addition. The applicability of the reporter system has been demonstrated by expressing the rppA gene under the control of the heterologous promoters actII-ORF4/PactI, ermE and its upregulated variant ermE*. The model streptomycete Streptomyces coelicolor, and three industrially important species, Streptomyces tsukubaensis (FK506), Streptomyces cinnamonensis (monensin) and Streptomyces rimosus (oxytetracycline) were used as hosts. The reporter system has shown its utility independently of cultivation conditions or composition of growth medium, from simple laboratory to complex industrial media. The simplicity and robustness of the system, demonstrated even in industrial settings, shows great potential for wider use in different microbial hosts and applications, and may thus represent a new generic and versatile tool useful to a wider scientific community.
Magdevska
V
V
Acies Bio d.o.o., Tehnološki Park 21, SI-1000 Ljubljana, Slovenia.
Gaber
R
R
Goranovič
D
D
Kuščer
E
E
Boakes
S
S
Durán Alonso
M B
MB
Santamaría
R I
RI
Raspor
P
P
Leadlay
P F
PF
Fujs
S
S
Petković
H
H
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D017930:Genes, Reporter; D009284:Naphthols / Q000378:metabolism; D009285:Naphthoquinones / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016930:Saccharopolyspora / Q000201:enzymology*; D013053:Spectrophotometry; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*
C121288:1,3,6,8-tetrahydroxynaphthalene; D009284:Naphthols; D009285:Naphthoquinones; D011994:Recombinant Proteins; C041659:flaviolin; D000217:Acyltransferases; C020141:flavanone synthetase