Green fluorescent protein as a reporter for the spatial and temporal expression of actIII in Streptomyces coelicolor
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ORIGINAL PAPER
Green fluorescent protein as a reporter for the spatial and temporal expression of actIII in Streptomyces coelicolor Fernando Santos‑Beneit1 · Jeff Errington1
Received: 13 February 2017 / Revised: 1 March 2017 / Accepted: 7 March 2017 © The Author(s) 2017. This article is an open access publication
Abstract Polyketides constitute a large group of structurally diverse natural products with useful biological activities. Insights into their biosynthetic mechanisms are crucial for developing new structures. One of the most studied model polyketide is the blue-pigmented antibiotic actinorhodin, produced by Streptomyces coelicolor. This aromatic polyketide is synthesized by minimal type II polyketide synthases and tailoring enzymes. The ActIII actinorhodin ketoreductase is a key tailoring enzyme in actinorhodin biosynthesis. Previous papers have reported contradictory findings for localization of the protein in the cytoplasmic fraction or associated with the cell wall. We have now used green fluorescent protein as a reporter to analyse the spatial and temporal expression of actIII (SCO5086) in S. coelicolor under actinorhodin producing and non-producing conditions. We provide evidence in support of ActIII being a cytosolic protein, with limited if any association with the membrane or cell wall. Keywords Polyketides · Actinorhodin · Streptomyces coelicolor · ActIII · ActA · SCO5086 · SCO5083
Introduction Polyketides are an important class of natural products with many different biological functions that can be produced by Communicated by Yusuf Akhter. * Fernando Santos‑Beneit fernando.santos‑[email protected] 1
Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Medical School, Newcastle University, Newcastle upon Tyne, UK
bacteria, fungi and plants (Hopwood 1997). The enzymes that synthesize these complex molecules are known as polyketide synthases. These proteins are analogous to the closely related fatty acid synthases which use the decarboxylation of α-carboxylated precursors to drive the synthesis of carbon chains (Keatinge-Clay et al. 2004). Actinorhodin provides a classic example of how these secondary metabolites are synthesized and regulated. This polyketide antibiotic is produced by the soil bacterium Streptomyces coelicolor and, as a blue isochromanequinone compound, provides a convenient model for studies of regulation (Rawlings 1999). The actinorhodin biosynthetic gene cluster (Act) has been extensively studied and the function of most genes characterized (Malpartida and Hopwood 1986; FernandezMoreno et al. 1994). The carbon backbone of actinorhodin is assembled through the cooperation of a ketosynthase/ chain length factor (KS-CLF; actI-ORF1 and actI-ORF2), an acyl carrier protein (ACP; actI-ORF3), and a malonylCoA:ACP transacylase (MAT) which is shared between polyketide and fatty acid synthesis (Carreras and Khosla 1998; Dreier et al. 1999). The resulting backbone is tailored into actinorhodin by the action of various enzymes, including ketoreductas
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