Construction of a novel phenol synthetic pathway in Escherichia coli through 4-hydroxybenzoate decarboxylation

Abstract

Phenol is a bulk chemical with lots of applications in the chemical industry. Fermentative production of phenol had been realized in both Pseudomonas putida and Escherichia coli by recruiting tyrosine phenol-lyase (TPL). The TPL pathway needs tyrosine as the direct precursor for phenol production. In this work, a novel phenol synthetic pathway was created in E. coli by recruiting 4-hydroxybenzoate decarboxylase, which can convert 4-hydroxybenzoate to phenol and carbon dioxide. Activating 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase and chorismate pyruvate lyase (UbiC) through plasmid overexpression led to 7- and 69-fold increase of phenol production, respectively, demonstrating that these two enzymes were the rate-limiting steps for phenol production. Genetically stable strains were then obtained by gene integration and gene modulation directly in chromosome. Phenol titer increased 147-fold (from 1.7 to 250 mg/L) after modulating the DAHP synthase, UbiC, and 4-hydroxybenzoate decarboxylase genes in chromosome. Five solvents were tested for two-phase extractive fermentation to eliminate phenol toxicity to E. coli cells. Tributyrin and dibutyl phthalate were the best two solvents for improving phenol production, leading to 23 and 30 % increase of total phenol production, respectively. Two-phase fed-batch fermentation of the best strain Phe009 was performed in a 7 L fermentor, which produced 9.51 g/L phenol with a yield of 0.061 g/g glucose.

Expanding the repertoire of aromatic chemicals by microbial production

“…Wei and coworkers 53 first constructed an L-DOPA-producing strain, E. coli DOPA-1, by a singleplex genome engineering approach. 20,58 L-Tyr and L-Phe have the same chemical structure except for the -OH group of C4 position on the benzene ring. Our research group also constructed a L-DOPA-overproducing strain by a series of singleplex genome editing based on our established 3-dehydroshikimate-overproducing strain and the titer of L-DOPA reached 57 g L −1 within 72 h by fed-batch fermentation (Unpublished data).…”

“…13,20,23,49,76 Therefore, several approaches have been implemented to reduce aromatic toxicity to microbial cells. 13,20,23,49,76 Therefore, several approaches have been implemented to reduce aromatic toxicity to microbial cells.…”

Creating pathways towards aromatic building blocks and fine chemicals

“…However, TPL is both reversible (actually favoring Tyr formation) and subject to feedback inhibition by phenol, and has accordingly been suggested as the principal limiting factor in phenol bioproduction [46]. Figure 3) [47 ].…”

Emerging tools, enabling technologies, and future opportunities for the bioproduction of aromatic chemicals

“…Microbial production of aromatic chemicals has largely been enabled via pathway engineering, generally consisting of either: (i) the functional reconstruction of naturally‐occurring but non‐native (often plant) pathways; or (ii) the bottom‐up construction of novel pathways comprised of individual enzymes derived from a diversity of heterologous sources. Recent examples include the successful engineering of microbes capable of the de novo production of, in the first case, flavonoids (usually consisting of two phenyl groups and a heterocyclic ring), stilbenes (ethylene moiety with two phenyl groups), and coumarins (containing a 1,2‐benzopyrone backbone), and, in the second case, numerous aromatic aldehydes, alcohols, and acids, styrenics, and phenolics . In most cases, these heterologous pathways stem from natively produced aromatic chemicals such as the aromatic amino acids (i.e.…”

“…Recent examples include the successful engineering of microbes capable of the de novo production of, in the first case, flavonoids (usually consisting of two phenyl groups and a heterocyclic ring), 10,11 stilbenes (ethylene moiety with two phenyl groups), 12,13 and coumarins (containing a 1,2-benzopyrone backbone), 14,15 and, in the second case, numerous aromatic aldehydes, alcohols, and acids, [16][17][18][19][20][21] styrenics, [22][23][24][25] and phenolics. [26][27][28][29][30][31][32][33] In most cases, these heterologous pathways stem from natively produced aromatic chemicals such as the aromatic amino acids (i.e. L-phenylalanine, L-tyrosine, and L-tryptophan) or their precursors (e.g.…”