Abstract
BackgroundIn an effort to identify alternate recombinant gene expression systems in Pseudomonas fluorescens, we identified genes encoding two native metabolic pathways that were inducible with inexpensive compounds: the anthranilate operon (antABC) and the benzoate operon (benABCD).ResultsThe antABC and benABCD operons were identified by homology to the Acinetobacter sp. anthranilate operon and Pseudomonas putida benzoate operon, and were confirmed to be regulated by anthranilate or benzoate, respectively. Fusions of the putative promoter regions to the E. coli lacZ gene were constructed to confirm inducible gene expression. Each operon was found to be controlled by an AraC family transcriptional activator, located immediately upstream of the first structural gene in each respective operon (antR or benR).ConclusionWe have found the anthranilate and benzoate promoters to be useful for tightly controlling recombinant gene expression at both small (< 1 L) and large (20 L) fermentation scales.
Highlights
In an effort to identify alternate recombinant gene expression systems in Pseudomonas fluorescens, we identified genes encoding two native metabolic pathways that were inducible with inexpensive compounds: the anthranilate operon and the benzoate operon
P. fluorescens MB214 can utilize anthranilate and benzoate as sole carbon source To characterize the functional catabolic and regulatory potential of Pseudomonas fluorescens strain MB214 (Table 1), the strain was tested for the ability to utilize a variety of aromatic and aliphatic hydrocarbons or aromatic acids as sole sources of carbon for growth
Subsequent experiments conducted in M9 media with glucose, glycerol, citrate, or succinate as primary growth substrates indicated that benzoate and anthranilate catabolism in strain MB214 was inducible by addition of the individual aromatic acid substrates
Summary
In an effort to identify alternate recombinant gene expression systems in Pseudomonas fluorescens, we identified genes encoding two native metabolic pathways that were inducible with inexpensive compounds: the anthranilate operon (antABC) and the benzoate operon (benABCD). To facilitate control of gene expression for production of proteins in an organism like P. fluorescens, which has been developed as a robust recombinant protein expression system[1,2], it is desirable to have a collection of expression cassettes. These cassettes would contain a variety of promoters that are tightly regulated, of differing strengths, induced under different growth conditions, and/or by different chemicals. Salicylate, phenylacetate, phthalate, phenol, naphthalene, n-octane, ndecane, n-dodecane, n-hexadecane and toluene were not able to support grow of P. fluorescens
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