Enabling natural product biosynthesis with novel synthetic biology tools

Microorganisms and plants have evolved to produce a myriad array of complex molecules known as natural products or secondary metabolites. Recent advances in molecular biology and genomics have revolutionized our ability to discover biosynthetic pathways that synthesize natural products. During my thesis research, I used various synthetic biology strategies to enable Escherichia coli, Saccharomyces cerevisiae and Streptomyces lividans to heterologously produce two value-added compounds: triacetic acid lactone and fosfomycin. Triacetic acid lactone is an precursor used to synthesize an important aromatic compound, 1,3,5-trihydroxylbenzene. Rational design of a 6-methylsalycylic acid synthase variant allowed the biosynthesis of triacetic acid lactone from a renewable feedstock D-glucose in S. cerevisiae. The maximal titer was subsequently optimized to 1.7 g/L by fed-batch fermentation.;Fosfomycin belongs to a group of compounds, called phosphonates, many of which have useful therapeutic properties. The fosfomycin gene cluster was cloned and heterologously expressed in S. lividans. Gene insertion and deletion were performed and the minimal cluster was determined to be composed of fom1-4, fomA-D and a transcriptional regulator fomR, based on which, a new biosynthetic mechanism for fosfomycin was proposed. In addition, it was found that, despite the significant structural differences among many of phosphonates, their biosynthetic routes contain an unexpected common intermediate, 2-hydroxyethylphosphonate, which is synthesized from phosphonoacetaldeyhyde by a distinct family of metal-dependent alcohol dehydrogenases. Detailed biochemical studies on these enzymes revealed that the reduction of phosphonoacetaldeyhyde to 2-hydroxyethylphosphonate may represent a common step in the biosynthesis of many phosphonate natural products, an observation that may lend insight into the evolution of phosphonate biosynthetic pathways and may prove highly useful in the mining of microbial genomes for novel phosphonate antibiotics and for suggesting the potential chemical structures of the products of these gene clusters.;In addition, I developed several novel synthetic biology tools for natural product research and development. The first tool is DNA assembler, which allows rapid construction of large recombinant DNAs in a single-step fashion through yeast in vivo homologous recombination. Further applications of DNA assembler to construct a highly versatile shuttle system and assemble an entire genome are in progress.