3-dehydroshikimate and catechol

Adipic acid is a high-value compound used primarily as a precursor for the synthesis of nylon, coatings, and plastics. Today it is produced mainly in chemical processes from petrochemicals like benzene. Because of the strong environmental impact of the production processes and the dependence on fossil resources, biotechnological production processes would provide an interesting alternative. Here we describe the first engineered Saccharomyces cerevisiae strain expressing a heterologous biosynthetic pathway converting the intermediate 3-dehydroshikimate of the aromatic amino acid biosynthesis pathway via protocatechuic acid and catechol into cis,cis-muconic acid, which can be chemically dehydrogenated to adipic acid. The pathway consists of three heterologous microbial enzymes, 3-dehydroshikimate dehydratase, protocatechuic acid decarboxylase composed of three different subunits, and catechol 1,2-dioxygenase. For each heterologous reaction step, we analyzed several potential candidates for their expression and activity in yeast to compose a functional cis,cis-muconic acid synthesis pathway. Carbon flow into the heterologous pathway was optimized by increasing the flux through selected steps of the common aromatic amino acid biosynthesis pathway and by blocking the conversion of 3-dehydroshikimate into shikimate. The recombinant yeast cells finally produced about 1.56 mg/liter cis,cis-muconic acid.

Topics: Biosynthetic Pathways; Biotransformation; Carbon; Catechols; Gene Expression; Hydroxybenzoates; Metabolic Engineering; Recombinant Proteins; Saccharomyces cerevisiae; Shikimic Acid; Sorbic Acid

The toxicity of aromatics frequently limits the yields of their microbial synthesis. For example, the 5% yield of catechol synthesized from glucose by Escherichia coli WN1/pWL1.290A under fermentor-controlled conditions reflects catechol's microbial toxicity. Use of in situ resin-based extraction to reduce catechol's concentration in culture medium and thereby its microbial toxicity during its synthesis from glucose by E. coli WN1/pWL1.290A led to a 7% yield of catechol. Interfacing microbial with chemical synthesis was then explored where glucose was microbially converted into a nontoxic intermediate followed by chemical conversion of this intermediate into catechol. Intermediates examined include 3-dehydroquinate, 3-dehydroshikimate, and protocatechuate. 3-Dehydroquinate and 3-dehydroshikimate synthesized, respectively, by E. coli QP1.1/pJY1.216A and E. coli KL3/pJY1.216A from glucose were extracted and then reacted in water heated at 290 degrees C to afford catechol in overall yields from glucose of 10% and 26%, respectively. The problematic extraction of these catechol precursors from culture medium was subsequently circumvented by high-yielding chemical dehydration of 3-dehydroquinate and 3-dehydroshikimate in culture medium followed by extraction of the resulting protocatechuate. After reaction of protocatechuate in water heated at 290 degrees C, the overall yields of catechol synthesized from glucose via chemical dehydration of 3-dehydroquinate and chemical dehydration of 3-dehydroshikimate were, respectively, 25% and 30%. Direct synthesis of protocatechuate from glucose using E. coli KL3/pWL2.46B followed by its extraction and chemical decarboxylation in water gave a 24% overall yield of catechol from glucose. In situ resin-based extraction of protocatechaute synthesized by E. coli KL3/pWL2.46B followed by chemical decarboxylation of this catechol percursor was then examined. This employment of both strategies for dealing with the microbial toxicity of aromatic products led to the highest overall yield with catechol synthesized in 43% overall yield from glucose.

Topics: Catechols; Escherichia coli; Fermentation; Glucose; Hydro-Lyases; Hydroxybenzoates; Quinic Acid; Shikimic Acid