A Synthetic Recursive “+1” Pathway for Carbon Chain Elongation

Abstract

Nature uses four methods of carbon chain elongation for the production of 2-ketoacids, fatty acids, polyketides, and isoprenoids. Using a combination of quantum mechanical (QM) modeling, protein–substrate modeling, and protein and metabolic engineering, we have engineered the enzymes involved in leucine biosynthesis for use as a synthetic “+1” recursive metabolic pathway to extend the carbon chain of 2-ketoacids. This modified pathway preferentially selects longer-chain substrates for catalysis, as compared to the non-recursive natural pathway, and can recursively catalyze five elongation cycles to synthesize bulk chemicals, such as 1-heptanol, 1-octanol, and phenylpropanol directly from glucose. The “+1” chemistry is a valuable metabolic tool in addition to the “+5” chemistry and “+2” chemistry for the biosynthesis of isoprenoids, fatty acids, or polyketides.

Identification of Homophenylalanine Biosynthetic Genes from the Cyanobacterium Nostoc punctiforme PCC73102 and Application to Its Microbial Production by Escherichia coli

“…Although genes involved in the synthesis of dihomotyrosine have not been identified, it is possible that an hph-like gene is involved, which should catalyze two rounds of methylene carbon extension in a manner similar to that for dihomomethionine synthesis catalyzed by leu-like genes from the plant Arabidopsis thaliana (30,31). Discovery of largamide-producing enzymes and comparison of the crystal structures of enzymes for homoamino acid and dihomoamino acid synthesis will provide a good opportunity to study control of side chain elongation on homoamino acid and allow rational protein engineering to generate novel enzymes that synthesize unnatural homoamino acids, like the method described previously (32).…”

Retro-biosynthetic screening of a modular pathway design achieves selective route for microbial synthesis of 4-methyl-pentanol

“…Recent work has been focused on improving modified natural pathways by substituting new enzymes to improve kinetics, improve expression or utilize alternate cofactors [12][13][14] . In some cases, pathways have been repurposed to synthesize new products by capitalizing on the natural capacity of enzymes to accept closely related substrates or by engineering protein specificity [15][16][17] . The boldest designs have utilized previously undescribed pathways created by combining the natural chemistry of individual enzymes from multiple hosts [18][19][20] .…”

Engineering Escherichia coli for Microbial Production of Butanone

“…It should be noted that acetyl phosphate, the intermediate in the Pta-AckA pathway, serves as an important metabolite for global regulation of gene expression and other fundamental processes and inactivating this pathway can potentially induce metabolic complications (29). For instance, elimination of acetyl phosphate can lead to inadequate turnover of the sigma factor RpoS and overall reductions in growth and metabolite formation (30,31). Although this phenomenon is well known and its etiology is not fully understood, it could be associated with the depletion of free intracellular CoA moieties caused by low rates of acetyl-CoA turnover (26).…”