3-dehydroshikimate and muconic-acid

The current shift from a fossil-resource based economy to a more sustainable, bio-based economy requires development of alternative production routes based on utilization of biomass for the many chemicals that are currently produced from petroleum. Muconic acid is an attractive platform chemical for the bio-based economy because it can be converted in chemicals with wide industrial applicability, such as adipic and terephthalic acid, and because its two double bonds offer great versatility for chemical modification.. We have constructed a yeast cell factory converting glucose and xylose into muconic acid without formation of ethanol. We consecutively eliminated feedback inhibition in the shikimate pathway, inserted the heterologous pathway for muconic acid biosynthesis from 3-dehydroshikimate (DHS) by co-expression of DHS dehydratase from P. anserina, protocatechuic acid (PCA) decarboxylase (PCAD) from K. pneumoniae and oxygen-consuming catechol 1,2-dioxygenase (CDO) from C. albicans, eliminated ethanol production by deletion of the three PDC genes and minimized PCA production by enhancing PCAD overexpression and production of its co-factor. The yeast pitching rate was increased to lower high biomass formation caused by the compulsory aerobic conditions. Maximal titers of 4 g/L, 4.5 g/L and 3.8 g/L muconic acid were reached with glucose, xylose, and a mixture, respectively. The use of an elevated initial sugar level, resulting in muconic acid titers above 2.5 g/L, caused stuck fermentations with incomplete utilization of the sugar. Application of polypropylene glycol 4000 (PPG) as solvent for in situ product removal during the fermentation shows that this is not due to toxicity by the muconic acid produced.. This work has developed an industrial yeast strain able to produce muconic acid from glucose and also with great efficiency from xylose, without any ethanol production, minimal production of PCA and reaching the highest titers in batch fermentation reported up to now. Utilization of higher sugar levels remained conspicuously incomplete. Since this was not due to product inhibition by muconic acid or to loss of viability, an unknown, possibly metabolic bottleneck apparently arises during muconic acid fermentation with high sugar levels and blocks further sugar utilization.

Topics: Carboxy-Lyases; Catechol 1,2-Dioxygenase; Cloning, Molecular; DNA, Fungal; Fermentation; Gene Expression Regulation, Fungal; Glucose; Hydro-Lyases; Hydroxybenzoates; Industrial Microbiology; Metabolic Engineering; Metabolic Networks and Pathways; Pyruvate Decarboxylase; Recombinant Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Shikimic Acid; Sorbic Acid; Xylose

Muconic acid is a platform chemical and an important intermediate in the degradation process of a series of aromatic compounds. Herein, a plasmid-free synthetic pathway in Pseudomonas chlororaphis HT66 is constructed for the enhanced biosynthesis of muconic acid by connecting endogenous ubiquinone biosynthesis pathway with protocatechuate degradation pathway using chromosomal integration. Instead of being plasmid and inducer dependent, the engineered strains could steadily produce the high muconic acid using glycerol as a carbon source. The engineered strain HT66-MA6 achieved a 3376 mg/L muconic acid production with a yield of 187.56 mg/g glycerol via the following strategies: (1) block muconic acid conversion and enhance muconic acid efflux pumping with phenazine biosynthesis cluster; (2) increase the muconic acid precursors supply through overexpressing the rate-limiting step, and (3) coexpress the "3-dehydroshikimate-derived" route in parallel with the "4-hydroxybenzoic acid-derived" route to create a synthetic "metabolic funnel". Finally, on the basis of the glycerol feeding strategies, the muconic acid yield reached 0.122 mol/mol glycerol. The results suggest that the construction of synthetic pathway with a plasmid-free strategy in P. chlororaphis displays a high biotechnological perspective.

Topics: Adipates; Biosynthetic Pathways; Gene Expression Regulation, Bacterial; Glycerol; Metabolic Engineering; Microorganisms, Genetically-Modified; Parabens; Plasmids; Pseudomonas chlororaphis; Shikimic Acid; Sorbic Acid; Ubiquinone

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