Synthetic Pathway for Production of Five-Carbon Alcohols from Isopentenyl Diphosphate

Abstract

f Synthetic biological pathways could enhance the development of novel processes to produce chemicals from renewable resources. On the basis of models that describe the evolution of metabolic pathways and enzymes in nature, we developed a framework to rationally identify enzymes able to catalyze reactions on new substrates that overcomes one of the major bottlenecks in the assembly of a synthetic biological pathway. We verified the framework by implementing a pathway with two novel enzymatic reactions to convert isopentenyl diphosphate into 3-methyl-3-butenol, 3-methyl-2-butenol, and 3-methylbutanol. To overcome competition with native pathways that share the same substrate, we engineered two bifunctional enzymes that redirect metabolic flux toward the synthetic pathway. Taken together, our work demonstrates a new approach to the engineering of novel synthetic pathways in the cell. The chemical and transportation industries currently use limited nonrenewable resources to produce raw materials that could instead be synthesized from renewable resources using metabolic engineering (18). Natural biological pathways have traditionally been the source of industrially important chemicals produced using fermentation (43). Some of the regulatory mechanisms that limit production in the native hosts are circumvented by reconstructing pathways in heterologous hosts (23). To synthesize chemicals not produced by natural pathways, enzymes are overexpressed in novel combinations in order to construct synthetic biological pathways (29,49).One of the rate-limiting steps in the assembly of a synthetic pathway is the identification of enzymes that can catalyze new reactions of interest. Reliance on known enzymatic reactions limits the number of synthetic pathways that could be assembled, and the task of engineering an enzyme to catalyze a novel reaction remains a challenge. Strategies that incorporate directed evolution or metagenomic libraries require product-specific high-throughput screens or selections in order to identify enzymes able to catalyze a desired reaction (17). Rational and in silico protein engineering strategies generate smaller libraries to alleviate the need for high-throughput technologies but require a priori knowledge about the protein being engineered (6). Therefore, the effort to discover an enzyme that can catalyze a desired reaction is often hindered by the lack of high-throughput screening or selection and insufficient knowledge about how to rationally engineer a protein.We provide a new framework to identify proteins able to catalyze a desired reaction on a new substrate based on models that describe pathway and enzyme evolution. The patchwork evolution model suggests that a new biological pathway is assembled by recruiting enzymes from existing pathways (16). The ability to assemble new pathways could be facilitated by the high degree of promiscuity demonstrated by enzymes. The promiscuity could be a source of divergence during enzyme evolution, since members of the same superfamily typically share the ...

Phosphorylation Mechanism of Phosphomevalonate Kinase: Implications for Rational Engineering of Isoprenoid Biosynthetic Pathway Enzymes

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Metabolic Engineering for Advanced Biofuels Production and Recent Advances Toward Commercialization

“…Unsaturated C 5 alcohols derived from either IPP or DMAPP are precursors for desirable gasoline additives based on their energy densities and octane numbers relative to gasoline. [33,34] Downstream recursive condensation of the IPP/DMAPP building blocks form larger hydrocarbons, serving as more suitable fuel candidates for diesel and jet engines. [35] 3.1.…”

“…[36] Introducing an E. coli ortholog, nudB, increased isopentenol production to 8% of its theoretical yield. [34] Metabolic engineering efforts for isopentenol production have undergone a series of improved metabolic engineering strategies to increase titers and pathway efficiency. Product purity and yield of isopentenol could be improved when the pathway was overexpressed in the absence of idi, which isomerizes IPP to DMAPP.…”

Isoprenoid-Based Biofuels: Homologous Expression and Heterologous Expression in Prokaryotes

“…Various endogenous and homologous phosphatases and pyrophosphatases that can convert prenyl pyrophosphates into respective bio- fuel compounds have been explored in Escherichia coli (12,16,37,53,54).…”