Metabolic Engineering Of Phenol Biosynthetic Engineering Strain

Phenol was an important chemical material and mainly used for the production of some resins, bisphenol A, adipic acid and drug productions, so the production of phenol with good prospects for development. Its chemical synthesis had a lot of advantages, however, there were a series of problems of high production cost, environmental pollution, many by-products. Due to its availability, low-price, and a high degree of reduction of C atom in the production of fuels and chemicals with higher yields than glucose and so on, glycerol had become an attractive carbon source. A new method was explored in this study that phenol was produced from glycerol by Escherichia coli. The major experimental results were as follows:(1) Codon optimization and enzyme activity measurement of TPL gene(encoding tyrosine phenol-lyase). Replacing TPL in Enterobacter agglomerans wild-type gene codon for Escherichia coli preferred codon optimization sequence, recombinant strain Escherichia coli BL21(pET28a-TPL) catalyzed 10 mM substrate tyrosine to 5.66 mM phenol by protein expression. Then the enzyme activity of TPL had been verified.(2) The pathway of phenol produced from glycerol was open-up. The approach involved two key enzymes, TPL and P4M(encoding phenylalanine hydroxylase), phenylalanine hydroxylase catalyzed phenylalanine to tyrosine, then under the catalysis of tyrosine phenol-lyase to produce phenol. The recombinant strain Escherichia coli ATCC31884(pTrc99a-TPL-P4M) was constructed, named PH1, and the yield of phenol was 21.30mg/L.(3) Enhancement of gene expression strength about glycerol utilization. The use of glycerol involved two enzymes, fructose-1,6- phosphatase(GlpX) and transketolase(TktA). The recombinant strain Escherichia coli ATCC31884(pTrc99a-TPL-P4M-trcGlpX-TktA) was constructed, named strain PH2. The yield of phenol was measured 27.68mg/L. Considering P4 M was the rate-limiting enzyme at the pathway of phenol produced, therefore constructed the recombinant strain Escherichia coli ATCC31884(pTrc99a-TPL-trcP4M-trcGlpX-TktA), named strain PH3, the yield of phenol was 57.66mg/L.(4) The increasement of precursor phosphoenolpyruvate in shikimic acid pathway. Firstly pyk F gene was knocked out by using Red homologous recombination technology., then ptsI gene(encoding protein phosphotransferase) and ppc gene(encoding phosphoenol pyruvate carboxylase). Plasmid pTrc99a-TPL-trcP4M-trcGlpX-Tkt A was transformed into recombinant strains, obtained strains PH4, PH5 and PH6. The yield of phenol were 88.38mg/L, 123.85mg/L and 127.60mg/L.(5) The optimization of fermentation. In order to improve the yield of phenol, the fermentation medium was optimized and the toxicity of phenol to the strain was reduced by using the two-phase fermentation. Selecting five organic solvents: octanol, tributyrin, dibutyl phthalate, dioctyl phthalate and isopropyl myristate, each was added to the fermentation broth of strain PH6, measured phenol were 4.31mg/L, 143.10mg/L, 95.27mg/L, 159.01mg/L,116.63mg/L. The results revealed that dioctyl phthalate was the best to the yield of phenol.Finally, the maximum yield of phenol was 159.01mg/L in the study.