Construction Of Escherichia Coli Cell Factories For Phenol Production

Phenol is one of the most important raw materials with applications as chemical engineering and pharmaceuticals. So far,97% of the phenol has been produced via the chemical oxidation process of cumene, which is synthesis from benzene. However, the ever-rising oil price and increasing of safety problems from these chemical industries urges us to develop an alternative bio-based phenol production method to replace the energy intensive and even dangerous phenol synthesis process.In this work, a novel phenol biosynthesis pathway was constructed by cloning the enzyme 4-hydroxybenzoate decarboxylase. And the key enzymes in this pathway were identified by detecting phenol production after overexpressing these enzymes. E. coli cell factories for phenol production were obtained via integrating the ycl operon into E. coli chromosome, followed by introducing a point mutation to aroG gene and modulating aroG, ubiC genes as well as ycl operon directly in E. coli chromosome.Firstly, the phenol biosynthesis pathway was constructed by cloning the yclBCD genes cluster into the vector pTrc99A-M. Then the phenol production was tested after cloning aroGfbr (a mutant of aroG gene) and ubiC genes into the expresss vector pACYC184-M, which encodes 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase and chorismate lyase (UbiC), respectively. Results revealed that both of these two enzymes were of great importance, and they could introduce an increase of 5.8-fold and 68.2-fold, respectively.For the construction of a genetically stable phenol-production E. coli strain, the ycl operon was integrated into E. coli ATCC 8739 chromosome at ldhA (encoding lactate dehydrogenase) site, resulting in the original phenol-production strain Phe002. The phenol production of this strain was 1.7 mg/L. The feedback inhibition of DAHP synthase was relieved by mutation and modulation of aroG gene, leading to a 4.3-fold increase of phenol titer. Another 19.2-fold increase of phenol titer was achieved by the same integration into pflB (encoding pyruvate formate lyase) site and modulation of ubiC gene. In addition, the promoter of the ycl operon was replaced to the Ml-93 artifitial regulatory part, bringing about a 36% increase of phenol titer, again. Finally, a two-phase extractive fed-batch fermentation of the strain Phe009 was performed, and the phenol titer was increased to 9.5 g/L, with a yield of 0.061 g phenol/g glucose.A genetically stable phenol-production E. coli cell factory was achieved after the integration of the 4-hydroxybenzoate decarboxylase, reliving the feedback inhibition of DAHP synthase, the improvement of the supply of precursor 4-hydroxybenzoate, and the overexpression of the 4-hydroxybenzoate decarboxylase. There was a 147-fold increase of the phenol titer compared to the original phenol-production strain. The final phenol titer was increased to 9.5 g/L after the two-phase extractive fed-batch fermentation. To the best of our knowledge, this was the highest phenol titer and yield obtained for engineered E. coli.