Abstract
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.
Highlights
Aromatic amino acid biosynthetic pathways have long been a potential source for commercially relevant chemicals with diverse industrial applications (Tzin and Galili, 2010)
To further improve DHS accumulation, we first disrupted the key genes involved in DHS accumulation in E. coli AB2834
Strategies to redesign metabolic pathways constitute the primary goal of metabolic engineering, which is a prerequisite to produce certain valuable compounds
Summary
Aromatic amino acid biosynthetic pathways have long been a potential source for commercially relevant chemicals with diverse industrial applications (Tzin and Galili, 2010). One of the core metabolites that facilitate the production of useful intermediates in aromatic amino acid biosynthetic pathways is 3dehydroshikimate (DHS), a key intermediate in the biosynthesis of shikimic acid (SA), muconic acid (MA), vanillin, and protocatechuate (PCA) (Li et al, 2019). Bio-based production of these compounds is a promising alternative to the current petrochemical-based production routes that are a cause of environment and energy concerns (Jan and Cavallaro, 2015). SA is an initial precursor for the production of a well-known anti-virial compound named oseltamivir phosphate, known as Tamiflu R
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