Biocatalytic production of adipic acid from glucose using engineered Saccharomyces cerevisiae

Abstract

Adipic acid is an important industrial chemical used in the synthesis of nylon-6,6. The commercial synthesis of adipic acid uses petroleum-derived benzene and releases significant quantities of greenhouse gases. Biocatalytic production of adipic acid from renewable feedstocks could potentially reduce the environmental damage and eliminate the need for fossil fuel precursors. Recently, we have demonstrated the first enzymatic hydrogenation of muconic acid to adipic acid using microbial enoate reductases (ERs) - complex iron-sulfur and flavin containing enzymes. In this work, we successfully expressed the Bacillus coagulans ER in a Saccharomyces cerevisiae strain producing muconic acid and developed a three-stage fermentation process enabling the synthesis of adipic acid from glucose. The ability to express active ERs and significant acid tolerance of S. cerevisiae highlight the applicability of the developed yeast strain for the biocatalytic production of adipic acid from renewable feedstocks.

Keywords: Adipic acid; Biosynthesis; Renewable resources; Synthetic biology; Yeast.

Fig. 1

The proposed pathway for adipic acid biosynthesis from glucose in S. cerevisiae. Metabolites and enzymes not native to yeast are shown in green. Abbreviations: DHS-D, 3-Dehydroshikimate Dehydratase; PCA-D, Protocatechuate Decarboxylase; Cat-DO, Catechol 1,2 – Dioxygenase; ER, Enoate Reductase.

Fig. 2

Induction of ER and adipic acid production by the S. cerevisiae strain ADP1 during incubation with glutathione and ccMA. A: Cell densities (Optical Density at 600 nm) after induction with galactose at time 0. B: Adipic acid titers obtained during incubation. In both plots, “Anaerobic” refers to the cultures that were maintained anaerobic throughout, while “Anaerobic/Aerobic” refers to cultures that were maintained anaerobic for the first 12 h, followed by incubation under aerobic conditions.

Fig. 3

The integrated adipic acid producing ADP2 strain and a fermentation strategy for adipic acid production. A: The plasmids present in the adipic acid producing ADP2 strain have been depicted. The names of the plasmids have been shown within them, followed by their auxotrophic markers (given in paranthesis). The genes encoding for various enzymes required for the adipic acid pathway have also been shown on each of the plasmids in colored boxes. Abbreviations - TKL1: Transketolase, DHS-D: 3-Dehydroshikimate Dehydratase, Cat-DO: Catechol Dioxygenase, PCA-D: Protocatechuate Decarboxylase, ER: Enoate Reductase, URA: Uracil, HIS: Histidine, LEU: Leucine. B: The proposed three-stage process for adipic acid production involves alternating between sparging the fermentation media with air and nitrogen to maintain aerobic and anaerobic conditions respectively.