Screening for enhanced triacetic acid lactone production by recombinant Escherichia coli expressing a designed triacetic acid lactone reporter.

Triacetic acid lactone (TAL) is a signature byproduct of polyketide synthases (PKSs) and a valuable synthetic precursor. We have developed an endogenous TAL reporter by engineering the Escherichia coli regulatory protein AraC to activate gene expression in response to TAL. The reporter enabled in vivo directed evolution of Gerbera hybrida 2-pyrone synthase activity in E. coli . Two rounds of mutagenesis and high-throughput screening yielded a variant conferring ~20-fold increased TAL production. The catalytic efficiency (kcat/Km) of the variant toward the substrate malonyl-CoA was improved 19-fold. This study broadens the utility of engineered AraC variants as customized molecular reporters. In addition, the TAL reporter can find applications in other basic PKS activity screens.

[1]  Hossein Fazelinia,et al.  AraC regulatory protein mutants with altered effector specificity. , 2008, Journal of the American Chemical Society.

[2]  J. Vederas,et al.  Complete Reconstitution of a Highly Reducing Iterative Polyketide Synthase , 2009, Science.

[3]  J. Agrawal.  Recent trends in high-energy materials , 1998 .

[4]  W. Groutas,et al.  Substituted 2-pyrones, 2-pyridones, and other congeners of elasnin as potential agents for the treatment of chronic obstructive lung diseases. , 1985, Journal of medicinal chemistry.

[5]  C. Khosla,et al.  Tolerance and specificity of recombinant 6-methylsalicyclic acid synthase. , 1999, Metabolic engineering.

[6]  David S. Goodsell,et al.  AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility , 2009, J. Comput. Chem..

[7]  Robert Schleif,et al.  AraC protein, regulation of the l-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action. , 2010, FEMS microbiology reviews.

[8]  Zachary L. Fowler,et al.  Genome-scale metabolic network modeling results in minimal interventions that cooperatively force carbon flux towards malonyl-CoA. , 2011, Metabolic engineering.

[9]  Huimin Zhao,et al.  Improving cellular malonyl-CoA level in Escherichia coli via metabolic engineering. , 2009, Metabolic engineering.

[10]  M. Koffas,et al.  Engineering Central Metabolic Pathways for High-Level Flavonoid Production in Escherichia coli , 2007, Applied and Environmental Microbiology.

[11]  R. Bentley,et al.  Biosynthesis of tropolones in Penicillium stipitatum. VII. The formation of polyketide lactones and other nontropolone compounds as a result of ethionine inhibition. , 1967, Journal of the American Chemical Society.

[12]  Patrick C Cirino,et al.  Protein and RNA engineering to customize microbial molecular reporting , 2012, Biotechnology journal.

[13]  J. Rabinowitz,et al.  Absolute Metabolite Concentrations and Implied Enzyme Active Site Occupancy in Escherichia coli , 2009, Nature chemical biology.

[14]  Christian P. Ridley,et al.  Synthesis and biological activity of novel pyranopyrones derived from engineered aromatic polyketides. , 2007, ACS chemical biology.

[15]  James A. Dumesic,et al.  Triacetic acid lactone as a potential biorenewable platform chemical , 2012 .

[16]  Gavin J. Williams,et al.  Promiscuity of a modular polyketide synthase towards natural and non-natural extender units. , 2013, Organic & biomolecular chemistry.

[17]  Patrick C Cirino,et al.  Design and application of a mevalonate-responsive regulatory protein. , 2011, Angewandte Chemie.

[18]  Huimin Zhao,et al.  Rational pathway engineering of type I fatty acid synthase allows the biosynthesis of triacetic acid lactone from D-glucose in vivo. , 2004, Journal of the American Chemical Society.

[19]  J. Cronan,et al.  Overproduction of Acetyl-CoA Carboxylase Activity Increases the Rate of Fatty Acid Biosynthesis in Escherichia coli * , 2000, The Journal of Biological Chemistry.

[20]  S. Belkin,et al.  Where microbiology meets microengineering: design and applications of reporter bacteria , 2010, Nature Reviews Microbiology.

[21]  R. Bentley,et al.  Biosynthesis of tropolones in Penicillium stipitatum. 8. The utilization of polyketide lactones for tropolone formation. , 1967, Journal of the American Chemical Society.

[22]  M. Xian,et al.  Improved phloroglucinol production by metabolically engineered Escherichia coli , 2011, Applied Microbiology and Biotechnology.

[23]  Y Takamura,et al.  Changes in the intracellular concentration of acetyl-CoA and malonyl-CoA in relation to the carbon and energy metabolism of Escherichia coli K12. , 1988, Journal of general microbiology.

[24]  Y. Helariutta,et al.  New pathway to polyketides in plants , 1998, Nature.

[25]  M. Austin,et al.  Structural control of polyketide formation in plant-specific polyketide synthases. , 2000, Chemistry & biology.

[26]  V. de Lorenzo,et al.  Transcriptional regulators à la carte: engineering new effector specificities in bacterial regulatory proteins. , 2006, Current opinion in biotechnology.

[27]  J. W. Frost,et al.  Deoxygenation of polyhydroxybenzenes: an alternative strategy for the benzene-free synthesis of aromatic chemicals. , 2002, Journal of the American Chemical Society.

[28]  Huimin Zhao,et al.  Microbial synthesis of triacetic acid lactone , 2006, Biotechnology and bioengineering.

[29]  J. M. Dickinson,et al.  2-pyrones possessing antimicrobial and cytotoxic activities. , 2004, Bioorganic & medicinal chemistry.

[30]  Stefano Forli,et al.  Virtual screening with AutoDock: theory and practice , 2010, Expert opinion on drug discovery.

[31]  Xuefeng Lu,et al.  Overproduction of free fatty acids in E. coli: implications for biodiesel production. , 2008, Metabolic engineering.