Biosynthesis of chiral 3-hydroxyvalerate from single propionate-unrelated carbon sources in metabolically engineered E. coli.

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Microbial Cell Factories (Impact Factor: 4.25). 01/2010; 9:96. DOI: 10.1186/1475-2859-9-96
Source: PubMed

ABSTRACT The ability to synthesize chiral building block molecules with high optical purity is of considerable importance to the fine chemical and pharmaceutical industries. Production of one such compound, 3-hydroxyvalerate (3HV), has previously been studied with respect to the in vivo or in vitro enzymatic depolymerization of biologically-derived co-polymers of poly(3-hydroxybutyrate-co-3-hydroxyvalerate). However, production of this biopolymeric precursor typically necessitates the supplementation of a secondary carbon source (e.g., propionate) into the culture medium. In addition, previous approaches for producing 3HV have not focused on its enantiopure synthesis, and thus suffer from increased costs for product purification.
Here, we report the selective biosynthesis of each 3HV stereoisomer from a single, renewable carbon source using synthetic metabolic pathways in recombinant strains of Escherichia coli. The product chirality was controlled by utilizing two reductases of opposing stereoselectivity. Improvement of the biosynthetic pathway activity and host background was carried out to elevate both the 3HV titers and 3HV/3HB ratios. Overall, shake-flask titers as high as 0.31 g/L and 0.50 g/L of (S)-3HV and (R)-3HV, respectively, were achieved in glucose-fed cultures, whereas glycerol-fed cultures yielded up to 0.19 g/L and 0.96 g/L of (S)-3HV and (R)-3HV, respectively.
Our work represents the first report of direct microbial production of enantiomerically pure 3HV from a single carbon source. Continued engineering of host strains and pathway enzymes will ultimately lead to more economical production of chiral 3HV.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Aromatic aldehydes are useful in numerous applications, especially as flavors, fragrances, and pharmaceutical precursors. However, microbial synthesis of aldehydes is hindered by rapid, endogenous, and redundant conversion of aldehydes to their corresponding alcohols. We report the construction of an Escherichia coli K-12 MG1655 strain with reduced aromatic aldehyde reduction (RARE) that serves as a platform for aromatic aldehyde biosynthesis. Six genes with reported activity on the model substrate benzaldehyde were rationally targeted for deletion: three genes that encode aldo-keto reductases and three genes that encode alcohol dehydrogenases. Upon expressing a recombinant carboxylic acid reductase in the RARE strain and supplying the strain with benzoate during growth, benzaldehyde remained in the culture after 24 hours, with less than 12% conversion of benzaldehyde to benzyl alcohol. Although individual overexpression results demonstrated that all six genes could contribute to benzaldehyde reduction in vivo, additional experiments featuring subset deletion strains revealed that two of the gene deletions were dispensable under the conditions tested. The engineered strain was next investigated for the production of vanillin from vanillate and succeeded in preventing formation of the byproduct vanillyl alcohol. A pathway for the biosynthesis of vanillin directly from glucose was introduced and resulted in a 55-fold improvement in vanillin titer when using the RARE strain versus the wild-type strain. Finally, synthesis of the chiral pharmaceutical intermediate L-phenylacetylcarbinol (L-PAC) was demonstrated from benzaldehyde and glucose upon expressing a recombinant mutant pyruvate decarboxylase in the RARE strain. Beyond allowing accumulation of aromatic aldehydes as end products in E. coli, the RARE strain expands the classes of chemicals that can be produced microbially via aldehyde intermediates.
    Journal of the American Chemical Society 07/2014; · 11.44 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The modification of Escherichia coli K-12 metabolism leading to threonine overproduction is the most studied system in synthetic biology that has been used to elaborate the majority of the currently known approaches to constructing microbial producers. They include optimization of biosynthesis through search for rate-limiting stages, modification of substrate and product transport, elimination of side metabolic pathways and degradation systems, reinforcement of the regeneration of coenzymes that are required for product biosynthesis, and exclusion of futile cycles and metabolic pathways with low energy efficiency. Extensive research in functional genomics made it possible to selectively remove the “unnecessary genes,” the functions of which are useless for producing a strain or adversely affect its properties. In total, using various approaches to designing threonine-producing strains, over 150 genome loci that affect more than 30% genes in E. coli were directly modified, thus providing interesting data for researchers in the field of microbial synthesis, as well as in related biological sciences. This review is dedicated to the assessment of genetic engineering modifications in E. coli metabolism (primarily, on the basis of modern patent literature) that ensure threonine overproduction.
    Applied Biochemistry and Microbiology 12/2013; 49(9). · 0.66 Impact Factor
  • [Show abstract] [Hide abstract]
    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(-1)) of the observed alcohol products. The highest observed 4MP titre is 192±23 mg l(-1). These results demonstrate the ability of modular pathway screening to facilitate de novo pathway engineering.
    Nature Communications 09/2014; 5:5031. · 10.74 Impact Factor

Full-text (3 Sources)

Available from
May 16, 2014