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.

The more than 50,000 isoprenoids found in nature are all derived from the 5-carbon diphosphates isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Natively, IPP and DMAPP are generated by the mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways, which have been engineered to produce compounds with numerous applications. However, as these pathways are inherently constrained by carbon, energy inefficiencies, and their roles in native metabolism, engineering for isoprenoid biosynthesis at high flux, titer, and yield remains a challenge. To overcome these limitations, here we develop an alternative synthetic pathway termed the isoprenoid alcohol (IPA) pathway that centers around the synthesis and subsequent phosphorylation of IPAs. We first established a lower IPA pathway for the conversion of IPAs to isoprenoid pyrophosphate intermediates that enabled the production of greater than 2 g/L geraniol from prenol as well as limonene, farnesol, diaponeurosporene, and lycopene. We then designed upper IPA pathways for the generation of (iso)prenol from central carbon metabolites with the development of a route to prenol enabling its synthesis at more than 2 g/L. Using prenol as the linking intermediate further facilitated an integrated IPA pathway that resulted in the production of nearly 0.6 g/L total monoterpenoids from glycerol as the sole carbon source. The IPA pathway provides an alternative route to isoprenoids that is more energy efficient than native pathways and can serve as a platform for targeting a repertoire of isoprenoid compounds with application as high-value pharmaceuticals, commodity chemicals, and fuels.

No abstract

No abstract