【作者】 胡云；

【导师】 堵国成；

【作者基本信息】 江南大学 ， 发酵工程， 2011， 硕士

【摘要】 大肠杆菌在有氧发酵过程中能产生胞外副产物乙酸,乙酸在中性条件下能以乙酸盐的形式存在于发酵液中。即使低浓度的乙酸(0.5 g/L)也能抑制大肠杆菌的生长和蛋白质的合成。并且,乙酸的积累将消耗本可用于菌体生长和蛋白产物合成的碳源。本文在利用大肠杆菌发酵生产L-苯丙氨酸(L-Phe)过程中发现发酵液中有较高水平的乙酸积累。为了降低副产物乙酸的积累和提高L-Phe的产量,分别优化了L-Phe发酵的初始葡萄糖浓度和葡萄糖的指数流加策略。结果表明,细胞的比生长速率直接受葡萄糖浓度的影响,同时比生长速率亦和乙酸的积累有密切的联系。过量的乙酸不仅抑制细胞的生长,并且对L-Phe的合成会产生负面影响。当葡萄糖浓度过高时,细胞的实际比生长速率μ会超过临界值(0.3 h-1),造成乙酸的大量积累,最终抑制L-Phe的合成。20 g/L的初始葡萄糖浓度可以控制细胞的比生长速率低于临界值0.3 h-1,显著地降低了乙酸的积累,提高了L-Phe的产量。采用预设比生长速率μ3*=0.4 h-1进行葡萄糖的指数流加取得了实际最大比生长速率0.29 h-1,使细胞的比生长速率低于临界值,促进了L-Phe的合成,最终获得L-Phe的产量达48.45 g/L,比本实验室原有水平提高了37%。由于葡萄糖指数流加操作中比较繁琐且难于控制,因此我们考虑从培养基优化角度来实现对乙酸水平的控制或降低乙酸产生的负面影响。我们考察了20种氨基酸添加对大肠杆菌E. coli WSH-Z06(pAP-B03)生长的影响,发现只有甘氨酸,脯氨酸,甲硫氨酸和精氨酸能够促进大肠杆菌的生长。进一步的实验发现,甲硫氨酸能够缓解乙酸对大肠杆菌生长的抑制作用:向培养基中添加0.5 g/L的甲硫氨酸使得菌体在2.5 g/L的乙酸存在条件下能够获得0.37 h-1的最大比生长速率,这个值与细胞在未添加乙酸和甲硫氨酸的对照组中取得的比生长速率的最大值相当。在3 L发酵罐中,在不同时间点添加0.5 g/L的甲硫氨酸对菌体的生长、乙酸的积累和L-Phe的合成均有重要的影响。在8 h添加甲硫氨酸使得即使在乙酸含量积累达2.58 g/L环境中L-Phe的生产强度达1.06 g/L/h ,最大菌体量达17.52 g/L和最终L-Phe浓度达42.37 g/L,分别比对照提高了89%,12%和29%。而在0 h添加甲硫氨酸虽然对菌体的生长有很强的促进作用,使其最大比生长速率达0.55 h-1,最大DCW达18.36 g/L,但其最大的L-Phe积累量为36.34 g/L,特别是此条件下L-Phe的积累主要集中在对数生长期,平衡期的L-Phe积累量较少,可能是由于发酵时间过长,甲硫氨酸对乙酸抑制缓解作用减弱。从总体来看,由于甲硫氨酸能够缓解乙酸过量积累所带来的抑制作用,甲硫氨酸的添加确能促进菌体的生长和L-Phe合成。在发酵生产L-Phe过程中,我们发现当pH偶然高于7.0时,氨水的消耗显著上升,我们假设大肠杆菌在此条件下产酸增加。因此,我们在接下来的实验中考察了高pH胁迫对大肠杆菌生产L-Phe的影响。实验研究了E. coli WSH-Z06(pAP-B03)对数生长末期改变发酵液的pH对菌体存活率和L-Phe合成的影响。摇瓶实验结果表明大肠杆菌生长的最适pH值为7.0,当对数生长末期改变发酵液的pH值至8.0和9.0后的4个小时之内L-Phe的合成分别达到3.72 g/L和4.25 g/L,比pH值为7.0时分别提高了13%和29%,且同时葡萄糖的消耗量比pH值为7.0时分别增加了25%和43%。然而,在对数生长末期提高发酵液的pH值,对细胞的生存产生负面影响,特别是当pH值调至9.0,DCW出现明显的下降。在3 L发酵罐中在pH值为8.0和7.0之间进行pH脉冲变化实验时,当以2 h的时间间隔进行实验对L-Phe的合成有显著地促进作用,至发酵结束时L-Phe的产量达48.35 g/L,比对照提高了14.2%。更多还原

【Abstract】 E. coli cells produce acetic acid as an extracellular co-product of aerobic fermentation, and this exists as the ion acetate at the neutral pH used in E. coli fermentations. Acetate is undesirable because it retards growth even at concentrations as low as 0.5 g/L, and it inhibits protein formation. Moreover, acetate production represents a diversion of carbon that might otherwise have generated biomass or the protein product. In this study, high level of acetic acid was detected during fermentative production of L-Phe. In order to reduce acetic acid accumulation and improve L- Phenylalanine production, we optimized the initial glucose concentration and its exponential feeding strategy during L-Phe fermentation in 3 L fermentor. The results showed that glucose concentration had direct impact on specific growth, which was closely related with acetic acid accumulation. Not only would excessive acetic inhibit cell growth, but also exert negative effect on L-Phe production. Under high concentration of glucose, cell would exceed critical specific growth rate (0.3 h-1), causing remarkable acetic acid accumulation, and ultimately inhibiting L-Phe synthesis. Firstly, we optimized initial glucose concentration, the results indicated that initial glucose of 20 g/L could control specific growth rate under critical value of 0.3 h-1, leading to great reduction in acetic acid accumulation and significant improvement in L-Phe production. Real maximal specific growth rate of 0.29 h-1 was achieved when preset specific growth rate of 0.4 h-1 was employed. The combination of initial glucose concentration of 20 g/L and preset specific growth rate of 0.4 h-1 greatly promoted L-Phe synthesis, gaining ultimate L-Phe concentration of 48.45 g/L, which was 37% higher than previously reported value in our lab.Acetate formation is strongly affected by the composition of the culture medium. Previous reports showed that supplementing the medium with yeast extract, methionine or glycine can similarly reduce acetate formation and enhance protein production. The effects of 20 amino acid supplements on growth of E.coli WSH-Z06(pAP-B03) were tested. Only Gly, Pro, Met and Arg brought about enhancement of cell growth. In shaken flasks, adding 0.5 g/L of Gly, Pro, Met and Arg could respectively improved maximal specific growth rate to 0.50, 0.52, 0.53 and 0.56 h-1. However, further study found that only Met could relieve cell growth from inhibitory effects of acetic acid: addition of 0.5 g/L Met into medium led to 0.37 h-1 maximal specific growth rate in presence of 2.5 g/L acetic acid, which was equivalent to the value achieved in absence of external acetic acid. Addition of Gly, Pro and Arg could not mitigate the significant reduction in specific growth in presence of acetic acid from 0.5-2.5 g/L. Previous studies suggested that the methionine biosynthetic pathway in E. coli can be perturbed by excessive acetic acid, leading to a reduced growth rate, which is caused by partial methionine auxotrophy. In 3 L fermentor experiment, addition of 0.5 g/L Met at different points during E.coli fermentation for production of L-Phe exhibited remarkable effects on cell growth, acetic acid accumulation as well as L-Phe synthesis. The addition of 0.5 g/L Met at 8 h achieved L-Phe productivity of 1.06 g/L/h, maximal DCW of 17.52 g/L, maximal L-Phe concentration of 42.47 g/L in presence of maximal acetic acid accumulation of 2.58 g/L, which were respectively 89%, 12% and 29% higher than the control without Met addition. However, despite the significant enhancement of cell growth (maximal specific growth rate was 0.55 h-1 and maximal DCW was 18.36 g/L) when 0.5 g/L Met was added at 0 h, the ultimate L-Phe concentration was 36.34 g/L, in particular, L-Phe formation mainly focused in exponential phase. In general, Met addition could indeed enhance cell growth and L-Phe synthesis, possibly due to its reliving effects on acetic acid inhibition.It is well known that E.coli cells need proper pH for normal growth. The effects of changes in pH value on DCW and L-Phe production were investigated. The results of our study showed that the optimal pH for E.coli WSH-Z06(pAP-B03) growth was 7.0, under which 7.4 g/L maximal DCW could be achieved. However, the maximal DCW under pH of 6.0, 8.0 were respectively 3.20 and 4.55 g/L. Interestingly, in shaken flasks, 3.72 g/L and 4.25 g/L L-Phe were synthesized within 4 h after changing pH to 8.0 and 9.0, respectively, which were increased by 13% and 29% compared to those achieved at pH 7.0. At the same time, glucose consumption rates were respectively increased by 25% and 43%.pH impulse between 8.0 and 7.0 with interval time of 2 h could remarkably enhance L-Phe production in 3 L fermentor: the maximal L-Phe concentration of 48.35 g/L, 14.2% higher than the control, was achieved at 64 h.更多还原