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

Abstract

Increasingly complex metabolic pathways have been engineered by modifying natural pathways and establishing de novo pathways with enzymes from a variety of organisms. Here we apply retro-biosynthetic screening to a modular pathway design to identify a redox neutral, theoretically high yielding route to a branched C6 alcohol. Enzymes capable of converting natural E. coli metabolites into 4-methyl-pentanol (4MP) via coenzyme A (CoA)-dependent chemistry were taken from nine different organisms to form a ten-step de novo pathway. Selectivity for 4MP is enhanced through the use of key enzymes acting on acyl-CoA intermediates, a carboxylic acid reductase from Nocardia iowensis and an alcohol dehydrogenase from Leifsonia sp. strain S749. One implementation of the full pathway from glucose demonstrates selective carbon chain extension and acid reduction with 4MP constituting 81% (90±7 mg l À 1 ) of the observed alcohol products. The highest observed 4MP titre is 192±23 mg l À 1 . These results demonstrate the ability of modular pathway screening to facilitate de novo pathway engineering.

Microbial Engineering for Aldehyde Synthesis

“…Although microbial FFAs have been produced for decades, recent work has demonstrated the potential for obtaining advanced fuels or valuable chemicals as derivatives of FFAs (50)(51)(52)(53). Based on the broad substrate range and known activities of carboxylic acid reductases, their addition to these pathways can result in production of C 4 to C 18 aliphatic aldehydes (25,26). Microbial synthesis of other valuable aldehyde classes, such as terpenoid aldehydes, could potentially occur in E. coli using variations of previously engineered terpenoid pathways (54).…”

“…Later publications from Rosazza and colleagues demonstrated that Car Ni requires one-time activation by a phosphopantetheinyl transferase and that Car Ni has activity in vitro on a broader range of substrates that includes several citric acid cycle dicarboxylic acids (23,24). Motivated by the activity of Car Ni on diverse carboxylic acid substrates, we investigated its activity on straight-chain and branched-chain aliphatic acids ranging from C 2 to C 8 (25). A homolog of Car Ni from Mycobacterium marinum was also demonstrated to have activity on straight-chain aliphatic acids ranging from C 6 to C 18 (26).…”

Designer Microorganisms for Optimized Redox Cascade Reactions – Challenges and Future Perspectives

“…[22,25] Withint he context of metabolic pathwayi ncorporation, scientists in the Prather group used the Duet systemf or the biosynthesis of (R)-and (S)-3-hydroxybutyrate, [26] butanol [27] and the non-natural alcohols pentanol [28] and 4-methyl-1-pentanol. [29] Thel ast compound is formed via at en-step de novo pathway with enzymest aken from nine different microorganisms.I n the cases of non-naturala lcohol production, enzymes are groupedi nm odules that allowed rational combination of different modules to elevate product titers.…”

Metabolic Engineering for Advanced Biofuels Production and Recent Advances Toward Commercialization

“…Batch, 48 h Overexpression of LeuABCD pathway; Rational enzyme mutagenesis on leuA and kivD [28] E. coli 4-Methyl-pentanol 192 mg L À1 Batch, 48 h Retrosynthetic design for creation of de novo pathways [29] E. coli n-Pentanol 109 mg L À1 Batch, 48 h Retrosynthetic design for creation of >de novo pathways [30] Isoprenoids…”

“…Such an approach substantially increased the relative purity of products in E. coli strains harboring de novo pathways designed for 4-methyl-pentanol and n-pentanol. [29,30] …”