Strain Construction, Metabolic Regulation And Process Optimization For L-Phenylalanine Production

L-phenylalanine (L-Phe), an essential amino acid for human being, is widely used in food, feed additives and medical industries. With lower cost of raw materials, less environment pollution and higher purity of product, fermentative production of L-Phe is becoming more promising. The complicacy of L-Phe biosynthesis pathway and its regulation mechanism results in low efficiency of L-Phe production.In this thesis, a new strain for L-Phe production was constructed, and a mutant able to resist bacteriophage was subsequently screened. We also investigated the effect of glucose feeding on L-Phe production with mutant as the producing strain. Engineered strains with pyruvate kinase gene (pykA and/or pykF) knocked-out were constructed and the induction temperature was optimized to further improve L-Phe yield against glucose. The main contents and results of this thesis are as following:1. A new L-Phe-producing strain E. coli WSH-Z06 (pAP-B03) was constructed by introducing aroF gene encoding DS and pheAfbr gene encoding feedback inhibition resistant enzymes CM-PDT, into plasmid pAP-B, a low-copy number and temperature inducible expression plasmid. In 3-L fermentor, the pH-stat based fed-batch culture of WSH-Z06 (pAP-B03) and WSH-Z06 was conducted for L-Phe synthesis. The results indicated that the capability of E. coli WSH-Z06 for L-Phe production was greatly enhanced with the introduction of plasmid pAP-B03. At 58 h, the titer of L-Phe reached 35.38 g/L, and L-Phe yield on glucose was 0.26 mol/mol, and L-Phe productivity was 0.61 g/L/h, increased by 181%, 100% and 178% compared to the control, respectively. Moreover, during the whole fermentation process, plasmid stability was maintained above 95%, guaranteeing the over-expression of the key enzymes for L-Phe synthesis.2. With a double-layer agar method, bacteriophage BP-1 was screened and purified from the contaminated fermentation broth using WSH-Z06 (pAP-B03) as a producing strain. With bacteriophage BP-1 as the sieve, WSH-Z06 was treated with different concentrations of NTG (0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, and 0.8 mg/mL), yielding two strains (BR-42 and BR-130) resistant to bacteriophage BP-1. The plasmid pAP-B03 was introduced to BR-42 and BR-130. In shake flasks, the biomass and L-Phe production of BR-130 were greatly lower than those of WSH-Z06 (pAP-B03); on the contrary, BR-42 didn't exhibit great difference in term of biomass and L-Phe titer compared to WSH-Z06 (pAP-B03). Therefore, BR-42 (pAP-B03) was used in subsequent L-Phe production. To further study the fermentation characteristics of BR-42 (pAP-B03) in the presence of bacteriophage BP-1, WSH-Z06 (pAP-B03) and BR-42 (pAP-B03) were cultured, respectively, by using pH-stat fed-batch approach in a 3-L fermentor added with 1% (v/v) BP-1 lysate with a titer of 1×1010 pfu/mL. The results showed that the culture broth of WSH-Z06 (pAP-B03) became clear at 14 h, and cell significantly lysed, and the glucose consumption was remarkablely slowed. However, BR-42 (pAP-B03) was not affected by the bacteriophage, and the maximal biomass reached 15.41 g/L and L-Phe titer was 34.87 g/L at 52h, almost the same as those achieved by WSH-Z06 (pAP-B03) without bacteriophage affection, indicating that BR-42 (pAP-B03) was an appropriate strain able to resist BP-1 as well as produce L-Phe.3. The effect of induction time on L-Phe production by E. coli BR-42 (pAP-B03) in pH-stat fed-batch culture was investigated. The results demonstrated that the optimal induction time was mid-logarithm stage at which DCW of 9.60 g/L and the maximal L-Phe yield of 52.75 g/L achieved. To enhance L-Phe production by E. coli BR-42 (pAP-B03), the effects of different feeding strategies including pH-stat, constant rate feeding, linear decreasing rate feeding, and exponential feeding on L-Phe production were investigated. A two-stage feeding strategy, namely, exponential feeding atμset=0.18 h-1 in the first 20 h and a following linear varying rate feeding with F= (-0.55×t+18.6) mL/h was developed to improve L-Phe production. The results showed that the two-stage feeding strategy could remarkably improve L-Phe production and reduce fermentation period to 50 h. Maximal L-Phe yield of 57.63 g/L, L-Phe productivity of 1.153 g/L/h and average specific L-Phe production rate of 0.100 h-1 were achieved, which were 34.81%, 48.20% and 42.86% higher than those of pH-stat feeding method, respectively.4. The PYK encoding gene (pykF/pykA) of BR-42 (pAP-B03) was knocked out through Red homologous recombination. The capability of the constructed strains for L-Phe production in 3-L fermentor was investigated and analyzed. The results showed that the deletion of pykF and pykA/pykF resulted in slow cell growth and low L-Phe titer, while, the single deletion of pykA didn't have significant impacts on cell growth and L-Phe production. however, the L-Phe productivity and the L-Phe yield against glucose reached 0.714 g/L/h and 0.265 mol/mol which were 6.1% and 3.5% higher than those of the original strain BR-42 (pAP-B03). The single deletion of pykA could slightly enhance L-Phe produciton and more efforts are needed to optimize the fermentative performance of the PYK mutant strains.5. The effect of induction time on E. coli BR-42ΔpykA (pAP-B03) cell growth and L-Phe production was investigated. It was found that during the entire fermentation process, the specific cell growth rateμwas the highest at 36oC, and the specific L-Phe production rate qp varied much in different fermentation period. According to the average qp, the induction process was divided into three stages, the first stage was 13.5-20 h, and the second stage was 20-37 h, and the third stage was 37-46 h. A three-stage induction time controlled strategy was proposed, in these three stages, the temperature was controlled at 40oC, 38oC, and 36oC, respectively. With this strategy, L-Phe production by E. coli BR-42ΔpykA (pAP-B03) was remarkably improved. At 48 h, L-Phe titer reached 52.70 g/L, which was 22.39%, 13.77%, and 12.01% higher than that at 40oC, 38oC and 36oC, respectively. The specific L-Phe production rate and L-Phe productivity were also increased. With the three-stage induction time controlled strategy, the cell growth, plasmid maintainance and the capacity of L-Phe biosynthesis by E. coli BR-42ΔpykA (pAP-B03) were greatly improved.