Enhanced Synthesis Of P-coumaric Acid In Escherichia Coli By Directed Evolution Of Tyrosine Ammonia Lyase

p-Coumaric acid(p CA) has many important biological activities such as heart protection, cholagogic effects, antioxidant and anti-cancer properties. It has been widely used in industries of food, cosmetic and pharmaceutical. p CA also serves as a precursor for production of many value-added chemicals, such as stilbenes and flavonoids. Currently, the production of p CA relies heavily on plant extraction and chemical synthesis, which has the disadvantages of low efficiency, high energy consumption and intense pollution. This limits the widely application of p CA. This dissertation focused on microbial production of p CA from a cheaper and renewable substrate, L-tyrosine. A tyrosine ammonia lyase gene(tal) from Rhodotorula glutinis was heterogeneously expressed in Escherichia coli BL21(DE3). To further improve the production of p CA, random mutagenesis, high throughput screening and rational modification of enzyme was conducted. The main results were described as follows:(1) The tal gene was codon-optimized and heterogeneously expressed in E. coli BL21(DE3). After treatment with ammonium sulfate precipitation, the recombinant TAL was purified with anion exchange chromatography and gel filtration chromatography. The purified enzyme exhibited a specific activity of 1.78 U×mg-1, an optimum p H of 8.5 and an optimum temperature of 40oC. The kinetic parameters Km and kcat/Km were 0.382 mmol×L-1 and 298 mmol-1×L×s-1, respectively. To further improve the production of p CA, the expression level of TAL was optimized based on different plasmids including p ET-32a(+), p ETDuet-1, p ET-28a(+), p ACYCDuet-1, p RSFDuet-1 and p CDFDuet-1. Because the solubility expression of TAL was enhanced by thioredoxin from the plasmid p ET-32a(+), the recombinant strain harboring p ET-32a(+)/tal gained the highest yield of p CA(196.3 mg×L-1) from L-tyrosine after a total fermentation time of 24 h.(2) Directed evolution of TAL was conducted by random mutagenesis to improve the activity of TAL and enhance the production of p CA. Protocols of random mutagenesis and high throughput screening were optimized. To control the mutant frequency and percentage of active mutants, 100 ng DNA template was added in a 50-mL error-prone PCR reaction. To increase transformation efficiency, p CDFDuet-1/tal was chosen for whole-plasmid PCR. For being more sensitive and efficient, the high throughput screening was conducted based on product detection. Through random mutagenesis and high throughput screening, three mutants were selected for higher p CA production, named MT-1H11(S9N/A125V), MT-5C4(E518V/S626C) and MT-1G9(A11T/S106C/N170D/M227T/D542G), respectively. The p CA production titer from MT-1H11, MT-5C4 and MT-1G9 was 231.5 mg×L-1, 232.9 mg×L-1 and 238.4 mg×L-1, respectively. These titers were improved by 37.1%, 38.0% and 41.2% compared to the wild type strain.(3) To investigate which mutation made significant effects in enhancing p CA production, site-directed mutagenesis was conducted based on 9 sites from 3 random mutants. The mutants S9 N, A11 T and E518 V yielded p CA production of 234.6 mg×L-1, 212.5 mg×L-1 and 239.7 mg×L-1, respectively. These three mutants exhibited an over 20.0% increase in p CA production than wile type strain, while other 6 mutants didn’t show significant increase. Further analysis showed that the apparent enzyme activity and protein expression level of S9 N was increased, because the N-terminal second structure of TAL was reduced. Meanwhile, the specific activity and catalytic efficiency of A11 T and E518 V was enhanced. Furthermore, combination of mutations between S9 N, A11 T and E518 V was conducted in the same way. It was found that the mutant S9N/A11T/E518 V yielded the highest p CA production of 280.1 mg×L-1 after 24 h fermentation, which was increased by 65.9% than wild type strain.