Escherichia coli strain CAR001 that produces β-carotene was genetically engineered to produce lycopene by deleting genes encoding zeaxanthin glucosyltransferase (crtX) and lycopene β-cyclase (crtY) from the crtEXYIB operon. The resulting strain, LYC001, produced 10.5 mg lycopene/l (6.5 mg/g dry cell weight, DCW). Modulating expression of genes encoding α-ketoglutarate dehydrogenase, succinate dehydrogenase and transaldolase B within central metabolic modules increased NADPH and ATP supplies, leading to a 76 % increase of lycopene yield. Ribosome binding site libraries were further used to modulate expression of genes encoding 1-deoxy-D-xylulose-5-phosphate synthase (dxs) and isopentenyl diphosphate isomerase (idi) and the crt gene operon, which improved the lycopene yield by 32 %. The optimal strain LYC010 produced 3.52 g lycopene/l (50.6 mg/g DCW) in fed-batch fermentation.
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.
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