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MIT team develops new synthetic pathway and modular engineering toolkit for direct biosynthesis of odd-chain molecules for fuels and chemicals

3 November 2012

Tseng
Metabolic pathway construction for direct microbial synthesis of pentanol from glucose or glycerol. The pentanol biosynthetic pathway consists of three modules, each of which was validated separately and then assembled together. Tseng and Prather 2012. Click to enlarge.

Researchers at MIT have adapted the butanol pathway for the synthesis of odd-chain molecules and have also developed a complementary modular toolkit to facilitate pathway construction, characterization, and optimization in engineered Escherichia coli bacteria.

The modular nature of the pathway enables multi-entry and multi-exit biosynthesis of various odd-chain compounds at high efficiency. By varying combinations of the pathway and toolkit enzymes, they demonstrated controlled production of propionate, trans-2-pentenoate, valerate, and pentanol—compounds with applications that include biofuels, antibiotics, biopolymers, and aroma chemicals.

Tseng2
Applications of various fermentation products synthesized from recombinant Escherichia coli strains carrying different combinations of pentanol pathway and CoA-activation/removing toolkit enzymes. Tseng and Prather Supplementary Information. Click to enlarge.

In a paper published in the Proceedings of the National Academy of Sciences (PNAS), Hsien-Chung Tseng and Kristala L. J. Prather note that their bypass strategy was effective even without the presence of freely membrane-diffusible substrates. The approach could prove useful for optimizing other pathways that use CoA-derivatized intermediates, they suggested, including fatty acid β-oxidation and the mevalonate pathway for isoprenoid synthesis.

The interest in microbial synthesis of fuels and chemicals has increased substantially as efforts to transition toward a “bio-based” economy have gained momentum; however, the ability to more broadly use biological systems for chemical production is somewhat limited by the natural repertoire of biosynthetic pathways. To expand upon these options, it is useful to consider the means by which a limited number of functional conversions can lead to a broad array of structures.

In many natural pathways, a carbon skeleton is initially formed from which a handful of compounds with diverse chemical architecture are generated. These compounds can be further modified by several decorating enzymes to yield an enormous array of products that exert physiological functions. For example, a few prenyl diphosphates produced from two simple isoprene units through the central isoprenoid pathway can be modified into over 50,000 functionally diverse isoprenoids by terpene-modifying enzymes. Other examples include polyketide biosynthesis, where polyketide chains supplied from the iterative carbon elongation pathway can be derivatized into numerous bioactive compounds and the tricarboxylic acid cycle (TCA cycle) that supplies precursors for biosynthesis of amino acids.

In an attempt to mimic nature’s design for supplying structurally diverse compounds, we began with an engineered l-butanol pathway from Clostridium acetobutylicum, and incorporated elements from the synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [poly(3HB-co-3HV)] in Cupriavidus necator (formerly known as Ralstonia eutropha), and threonine in Escherichia coli to create a unique core pathway for the production of industrially relevant chemicals and fuels, particularly n-pentanol, a fuel alternative with high energy density and enhanced physical properties that would allow better integration into the current infrastructure.

—Tseng and Prather

Implementing a heterologous pathway that uses nonnatural substrates (in this case, to produce pentanol rather than butanol) presents two challenges, they noted.

  • The extent to which each of the enzymes will accept the five-carbon unnatural substrates is unknown.

  • Limitations in activity and pathway bottlenecks are confounded by the assembly of a unique combination of genes from multiple source organisms into a single heterologous host.

Their approach was to use a bypass strategy for pathway construction that is conceptually analogous to process control in chemical engineering, in which a stream split from the feed to a process unit is combined with the outlet stream from that process, thereby bypassing the unit in question.

They divided their entire pathway into three modules: precursor supply (module 1), top pathway (module 2), and bottom pathway (module 3). They tested each module individually with separate inlet and outlet carbon streams to confirm in vivo functionality and establish an operating range for the constituent enzymes.

Because all of the intermediates are present as CoA derivatives and are retained intracellularly, their bypass approach required the development of a CoA-addition/removal enzyme toolkit that would either hydrolyze metabolites within a module to provide a product stream or activate exogenously supplied free acids once inside the cell to provide the desired feed stream.

In the present pathway, further engineering of the internal redox metabolism, as well as improved substrate specificity and activity of pathway enzymes, should lead to economically viable and precisely controlled production of desired products.

—Tseng and Prather

Resources

  • Hsien-Chung Tseng and Kristala L. J. Prather (2012) Controlled biosynthesis of odd-chain fuels and chemicals via engineered modular metabolic pathways. PNAS 109 (44) 17925-17930 doi: 10.1073/pnas.1209002109

November 3, 2012 in Bio-hydrocarbons, Biotech, Synthetic Biology | Permalink | Comments (0) | TrackBack (0)

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