Customized metabolite-responsive gene regulators find applications as molecular reporters and as novel gene control tools in synthetic biology and metabolic engineering. In this study the AraC regulatory protein of E. coli was engineered to control gene expression in response to mevalonate and to triacetic acid lactone (TAL; 4-Hydroxy-6-methyl-2-pyrone).  Residue positions in the AraC binding pocket were first randomized and mutants were expressed in E. coli cells containing a pBAD-GFP reporter construct. AraC variants having desired regulatory properties in the presence and absence of the effector of interest were isolated via FACS.  Our customized mevalonate reporter system was used to screen for improved mevalonate production in E. coli harboring a heterologous mevalonate synthesis operon by randomizing gene ribosomal binding sites. Colony screening yielded an RBS mutant resulting in ~10-fold improved mevalonate production.  Similarly, the TAL reporter was used to screen for improved TAL synthesis in E. coli expressing the Gerbera hybrida 2-pyrone synthase (2-PS). Random mutagenesis of the 2-PS gene led to improved TAL production and identification of substrate binding pocket residues tolerant to mutation.  Subsequent saturation mutagenesis at those positions and screening resulted in further enhanced TAL production.  Random chromosomal transposon insertions were also screened and yielded a mutant with ~15-fold improved TAL production.  Biochemical characterization of the 2-PS mutants and host chromosome modifications will be discussed.

Design and applications of customized regulatory proteins as molecular reporters in high throughput screening of novel biocatalysts

241st National Meeting (2011)

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