摘要
L-苯丙氨酸(L-phenylalanine, L-Phe)是一种重要的必需氨基酸,广泛应用于食品、饲料添加剂以及医药等领域中。微生物发酵法由于具有原料廉价易得、环境污染较小、产物纯度高等优点成为目前国内外工业化生产L-Phe的主要方法。但微生物中L-Phe的合成途径和调控方式较复杂,L-Phe的合成效率受到限制。
本研究利用代谢工程手段构建了一株新的L-Phe高效生产菌株Escherichia coli WSH-Z06 (pAP-B03),在此基础上筛选到抗噬菌体BP-1的突变株BR-42 (pAP-B03),同时研究了葡萄糖流加方式对BR-42 (pAP-B03)生产L-Phe的影响。为了提高L-Phe对葡萄糖的转化率,分别敲除了BR-42 (pAP-B03)的丙酮酸激酶(Pyruvate kinase, PYK)基因(pykA和/或pykF),并优化了pykA缺失菌株BR-42ΔpykA (pAP-B03)生产L-Phe的诱导温度,进一步提高产物对底物的转化率。其主要研究内容及结果如下:
1.通过NTG诱变获得了一个新的抗反馈抑制的分支酸变位酶-预苯酸脱水酶(CM-PDT)编码基因pheAfbr,将该基因和编码3-脱氧-D-阿拉伯庚酮糖酸7-磷酸合成酶(DS)的aroF基因插入一个低拷贝、温度诱导表达的质粒pAP-B中,构建了一个重组表达质粒pAP-B03,转化到L-酪氨酸(L-Tyr)营养缺陷型的E. coli WSH-Z06中进行表达,构建了L-Phe生产菌株E. coli WSH-Z06 (pAP-B03)。在3-L发酵罐上采用pH-stat分批补料培养方式比较了WSH-Z06 (pAP-B03)和WSH-Z06生长及生产L-Phe的发酵过程。结果表明,质粒pAP-B03的引入使得E. coli WSH-Z06生产L-Phe的能力得到显著提高,发酵58 h时,L-Phe产量达到35.38 g/L,对底物的转化率为0.26 mol/mol,生产强度为0.61 g/L/h,分别比对照菌提高了181%、100%和178%,且在整个发酵过程中,质粒的稳定性保持在95%以上,从而保证了合成L-Phe的关键酶的过量表达。
2.利用双层平板法从WSH-Z06 (pAP-B03)发酵生产L-Phe的异常发酵液中分离、纯化到一株噬菌体BP-1,以该噬菌体为筛子,逐一检测经不同浓度的NTG (0.2 mg/mL、0.4 mg/mL、0.6 mg/mL和0.8 mg/mL)诱变处理后的WSH-Z06突变株对该噬菌体的敏感性,获得了两株对BP-1不敏感的菌株BR-42和BR-130。将生产L-Phe的重组质粒pAP-B03转化到BR-42和BR-130中,并在摇瓶水平上进一步考察了两突变株生产L-Phe的能力。通过与原始菌株WSH-Z06 (pAP-B03)的发酵特性进行比较发现,BR-130的生长和生产L-Phe的水平较原始菌明显降低,而BR-42 (pAP-B03)无论在菌体生长还是生产L-Phe能力方面都无显著变化。因此将BR-42 (pAP-B03)作为目的菌株用于L-Phe的发酵生产中。为了进一步考察BR-42 (pAP-B03)在噬菌体存在条件下的发酵特性,在3-L发酵罐中分别对WSH-Z06 (pAP-B03)和BR-42 (pAP-B03)进行了pH-stat分批补料培养,并在发酵初始时向发酵罐中添加1% (v/v)效价为1×1010 pfu/mL的BP-1裂解液。结果表明,WSH-Z06 (pAP-B03)在发酵进行到14 h时,发酵液变得澄清,菌体大量裂解,葡萄糖消耗缓慢;而BR-42 (pAP-B03)则没有受到BP-1的影响,最大菌体浓度达到15.41 g/L,在52 h时,L-Phe产量达到34.87 g/L,该水平与WSH-Z06 (pAP-B03)正常发酵时无显著差别,说明BR-42 (pAP-B03)是一株可以抗噬菌体BP-1并能生产L-Phe的优良菌株。
3.在pH-stat分批补料培养模式下,考察了诱导时间对BR-42 (pAP-B03)发酵生产L-Phe的影响。结果表明,最优的诱导时间为对数中期,即DCW为9.60 g/L左右时,L-Phe产量最高,达到52.75 g/L。在此基础上考察了葡萄糖流加方式即pH-stat、F=16 mL/h和10 mL/h的恒速流加、F= (-0.55×t+23) mL/h的速率线性下降的流加以及μset=0.12 h-1和0.18 h-1的指数流加对BR-42 (pAP-B03)发酵生产L-Phe的影响。通过分析各种流加方式下的发酵参数,提出并采用了在20 h前采用μset=0.18 h-1的指数流加,20 h后采用F= (-0.55×t+18.6) mL/h的速率线性下降的流加方式,显著促进了L-Phe的生产,发酵时间缩短到50 h,L-Phe最高产量达到57.63 g/L,生产强度为1.153 g/L/h,平均L-Phe比合成速率为0.100 h-1,分别比采用pH-stat流加时高了34.81%、48.20%和42.86%。
4.通过Red同源重组的方法,敲除了L-Phe生产菌株E. coli BR-42 (pAP-B03)的PYK基因(pykA和/或pykF),构建了3株PYK突变株,分别为BR-42ΔpykA (pAP-B03)、BR-42ΔpykF (pAP-B03)和BR-42ΔpykA/pykF (pAP-B03)。通过检测3株PYK突变株的PYK相对酶活,发现pykA单基因敲除后保留有73.2%的PYK酶活;pykF单基因敲除后PYK的活性降低至21.8%;pykA和pykF双基因敲除后,PYK的酶活仅为12.8%。在3-L发酵罐上进行发酵生产L-Phe的研究和分析,结果表明,BR-42ΔpykF (pAP-B03)和BR-42ΔpykA/pykF (pAP-B03)菌体生长缓慢,不利于L-Phe的合成;而pykA单缺失对菌体生长没有明显的影响,同时,L-Phe的产量也和出发菌株无显著差异,L-Phe的生产强度和L-Phe对葡萄糖的得率分别提高了6.1%和3.5%,达到了0.714 g/L/h和0.265 mol/mol。pykA的单缺失虽然没有明显地提高L-Phe的产量,但在L-Phe的生产强度和对碳源的得率方面具有一定的积极作用。
5.考察了不同诱导温度对E. coli BR-42ΔpykA (pAP-B03)菌体生长和L-Phe生产的影响,通过对发酵参数进行动力学分析,发现几乎整个诱导阶段,菌体比生长速率(μ)在36oC时最高;而在不同的诱导阶段,L-Phe比合成速率(qp)对于不同的诱导温度呈现不同变化趋势,根据平均qp的大小,将发酵过程分为3个阶段,分别为13.5-20 h (第1阶段)、20-37 h (第2阶段)和37-46 h (第3阶段)。在第1阶段诱导温度采用40oC;第2阶段采用38oC,第3阶段采用36oC。结果表明,分阶段控制诱导温度可以显著改善菌株生产L-Phe的能力,发酵48 h时,L-Phe的产量达到52.70 g/L,分别比在40oC、38oC和36oC时提高了22.39%、13.77%和12.01%。qp和L-Phe生产强度都有不同程度的提高。采用分阶段温度控制策略解决了较高的诱导温度下菌体浓度下降和质粒不稳定等问题,显著提高了菌体合成L-Phe的能力。
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.
引文
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