Muconic acid (MA) is a valuable compound for adipic acid production, which is a precursor for the synthesis of various polymers such as plastics, coatings, and nylons. Although MA biosynthesis has been previously reported in several bacteria, the engineered strains were not satisfactory owing to low MA titers. Here, we generated an engineered Corynebacterium cell factory to produce a high titer of MA through 3-dehydroshikimate (DHS) conversion to MA, with heterologous expression of foreign protocatechuate (PCA) decarboxylase genes. To accumulate key intermediates in the MA biosynthetic pathway, aroE (shikimate dehydrogenase gene), pcaG/H (PCA dioxygenase alpha/beta subunit genes) and catB (chloromuconate cycloisomerase gene) were disrupted. To accomplish the conversion of PCA to catechol (CA), a step that is absent in Corynebacterium, a codon-optimized heterologous PCA decarboxylase gene was expressed as a single operon under the strong promoter in a aroE-pcaG/H-catB triple knock-out Corynebacterium strain. This redesigned Corynebacterium, grown in an optimized medium, produced about 38 g/L MA and 54 g/L MA in 7-L and 50-L fed-batch fermentations, respectively. These results show highest levels of MA production demonstrated in Corynebacterium, suggesting that the rational cell factory design of MA biosynthesis could be an alternative way to complement petrochemical-based chemical processes.

3-Dehydroshikimate (DHS) is a useful starting metabolite for the biosynthesis of muconic acid (MA) and shikimic acid (SA), which are precursors of various valuable polymers and drugs. Although DHS biosynthesis has been previously reported in several bacteria, the engineered strains were far from satisfactory, due to their low DHS titers. Here, we created an engineered Escherichia coli cell factory to produce a high titer of DHS as well as an efficient system for the conversion DHS into MA. First, the genes showing negative effects on DHS accumulation in E. coli, such as tyrR (tyrosine dependent transcriptional regulator), ptsG (glucose specific sugar: phosphoenolpyruvate phosphotransferase), and pykA (pyruvate kinase 2), were disrupted. In addition, the genes involved in DHS biosynthesis, such as aroB (DHQ synthase), aroD (DHQ dehydratase), ppsA (phosphoenolpyruvate synthase), galP (D-galactose transporter), aroG (DAHP synthase), and aroF (DAHP synthase), were overexpressed to increase the glucose uptake and flux of intermediates. The redesigned DHS-overproducing E. coli strain grown in an optimized medium produced ~117 g/L DHS in 7-L fed-batch fermentation, which is the highest level of DHS production demonstrated in E. coli. To accomplish the DHS-to-MA conversion, which is originally absent in E. coli, a codon-optimized heterologous gene cassette containing asbF, aroY, and catA was expressed as a single operon under a strong promoter in a DHS-overproducing E. coli strain. This redesigned E. coli grown in an optimized medium produced about 64.5 g/L MA in 7-L fed-batch fermentation, suggesting that the rational cell factory design of DHS and MA biosynthesis could be a feasible way to complement petrochemical-based chemical processes.

Shikimate is a key intermediate in high-demand for synthesizing valuable antiviral drugs, such as the anti-influenza drug, oseltamivir (Tamiflu®). Microbial-based shikimate production strategies have been developed to overcome the unstable and expensive supply of shikimate derived from traditional plant extraction processes. Although shikimate biosynthesis has been reported in several engineered bacterial species, the shikimate production yield is still unsatisfactory. This study designed an Escherichia coli cell factory and optimized the fed-batch culture process to achieve a high titer of shikimate production. Using the previously constructed dehydroshikimate (DHS)-overproducing E. coli strain, two genes (aroK and aroL) responsible for converting shikimate to the next step were disrupted to facilitate shikimate accumulation. The genes with negative effects on shikimate biosynthesis, including tyrR, ptsG, and pykA, were disrupted. In contrast, several shikimate biosynthetic pathway genes, including aroB, aroD, aroF, aroG, and aroE, were overexpressed to maximize the glucose uptake and intermediate flux. The shiA involved in shikimate transport was disrupted, and the tktA involved in the accumulation of both PEP and E4P was overexpressed. The rationally designed shikimate-overproducing E. coli strain grown in an optimized medium produced approximately 101 g/L of shikimate in 7-L fed-batch fermentation, which is the highest level of shikimate production reported thus far. Overall, rational cell factory design and culture process optimization for microbial-based shikimate production will play a key role in complementing traditional plant-derived shikimate production processes.