基于途径分析的L-异亮氨酸发酵溶氧控制研究

利用途径分析方法对黄色短杆菌（Brevibacterium flavum）TC-21 生产L-异亮氨酸的途径进行了分析,确定了黄色短杆菌TC-21生产L-异亮氨酸的最佳途径的通量分布,根据途径分析的结果,TCA循环的代谢流量对L-异亮氨酸产量有明显影响,而TCA循环与发酵过程中的溶氧密切相关,因此可以通过控制溶氧来提高L-异亮氨酸产量。在发酵过程的不同阶段,根据菌体生长和产酸的需求,改变TCA代谢流量,可以有效提高产酸率。实验证明,通过溶氧分阶段控制发酵生产L-异亮氨酸,比溶氧恒定控制方式发酵产率提高了15.77％。实验结果说明,用途径分析的结果指导发酵过程中的溶氧可以大幅度提高L-异亮氨酸的产量。     

鸟苷产生菌的代谢途径分析

代谢工程要解决的主要问题就是改变某些途径中的碳架物质流量或改变碳架物质流在不同途径中的流量分布,其目标就是修饰初级代谢,将碳架物质流导入目的产物的载流途径以获得产物的最大转化率。利用途径分析方法对枯草芽孢杆菌生产鸟苷的途径进行了分析,建立了3种基础模型,鸟苷理论摩尔产率分别是0．625、0．75和0．667,确定了枯草芽孢杆菌生产鸟苷的最佳途径的通量分布。     

L-色氨酸生物合成的代谢流量分析

建立了谷氨酸棒杆菌合成L-色氨酸(L-Try)的代谢流量平衡模型,应用该模型计算出发酵中后期的代谢流分布并通过MATLAB软件线性规划得到Try理想代谢流分布。结果表明75．15％的碳架进入糖酵解,24．85％的碳架进入HMP途径;但与理想代谢流相比,应从遗传改造和发酵控制方面降低TCA循环的代谢流,减少副产氨基酸的生成,摸索最适的溶氧控制对提高Try产率至关重要。     

不同溶氧条件下L-苏氨酸生物合成菌株的代谢流量分析

[目的]探索L-苏氨酸生物合成机理及影响因素.[方法]建立了大肠杆菌L-苏氨酸的代谢流平衡模型,应用MATLAB软件计算出不同溶氧条件下发酵中后期代谢网络的代谢流分布及理想代谢流分布.[结果]5%溶氧条件下,25.5%碳架进入HMP途径,74.5%碳架进入糖酵解途径,获得33.9%质量转化率;20%溶氧条件下,58.08%碳架进入HMP途径,41.92%碳架进入糖酵解途径,获得46.5%质量转化率;[结论]与理想代谢流(88.23%质量转化率)相比,应从菌种改造和发酵控制方面通过改变6-磷酸葡萄糖异构酶借以增加HMP途径代谢流量,通过增加磷酸烯醇式丙酮酸羧化反应代谢流提高天冬氨酸族合成代谢流,减少TCA循环代谢流量,从而达到减少副产物生成,增加L-苏氨酸生物合成的目的.     

L-色氨酸生物合成的代谢流量分析

建立了谷氨酸棒杆菌合成L色氨酸(LTry)的代谢流量平衡模型,应用该模型计算出发酵中后期的代谢流分布并通过MATLAB软件线性规划得到Try理想代谢流分布。结果表明75.15%的碳架进入糖酵解,24.85%的碳架进入HMP途径;但与理想代谢流相比,应从遗传改造和发酵控制方面降低 TCA循环的代谢流,减少副产氨基酸的生成,摸索最适的溶氧控制对提高Try产率至关重要。     

黄色短杆菌TK0303的L-亮氨酸生物合成途径分析

应用途径分析方法分析了在拟稳态时黄色短杆菌(Brevibacterium flavum)TK0303由葡萄糖发酵生产L-亮氨酸的代谢途径,确定了L-亮氨酸合成的最佳途径和最大理论产率。通过比较途径分析所获得的反应模型,确定了丙酮酸和乙酰辅酶A是L-亮氨酸合成途径的关键节点。在此基础上改变外界环境因子,强化L-亮氨酸生物合成途径中丙酮酸和乙酰辅酶A两个关键节点的代谢流,以期进一步提高L-亮氨酸产率。结果表明,经过谷氨酸以及醋酸铵的调节,代谢途径流量发生显著变化,L-亮氨酸产量有明显提高。     

溶氧对L-缬氨酸发酵过程的影响

目的:以黄色短杆菌XV0505为供试菌,探索溶氧对L-缬氨酸发酵过程的影响及其控制策略。方法:利用5L发酵罐,考察了不同溶氧浓度对L-缬氨酸发酵的影响,并采用代谢流分析对其结果进行阐述,提出分阶段溶氧控制策略。结果:采用分阶段溶氧控制策略,即在0～24h溶氧浓度为20%,24～60h溶氧浓度为5%,发酵60h,L-缬氨酸可达到58.36g/L,比5%和20%溶氧浓度下分别提高了18.95%和13.56%。结论:溶氧浓度对L-缬氨酸发酵有重要影响。     

L-缬氨酸的发酵工艺研究

目的:以黄色短杆菌XV0505为生产菌株,探讨发酵培养基和发酵控制条件对L-缬氨酸的产量和糖酸转化率的影响。方法:应用单因素实验确定发酵的工艺条件;利用纸层析-色斑洗脱比色法测定发酵液中L-缬氨酸的含量。结果:在最优发酵条件下,通过10L罐流加发酵72h,产酸量可达53.4g/L,糖酸转化率为26.7%,分别比补料分批发酵提高11.9%和3.5%。结论:环境因子和发酵控制工艺对发酵生产L-缬氨酸具有重要影响。     

运用重组细菌生产L-异亮氨酸

用遗传工程细菌进行好气发酵,生产出了L-异亮氨酸,比传统用的人工诱变法获得的突变株的产率高。本专利提供的方法,是把含有短杆菌(Breoibacterium)或棒状杆菌(Corynebacterium)L-异亮氨酸产生菌染色体DNA的重组质粒,插     

不同碳源生物转化合成L-亮氨酸的代谢计量分析

目的:建立黄色短杆菌利用不同碳源生物合成L-亮氨酸的代谢网络模型,并进行代谢网络计量分析.方法:通过对所构建的L-亮氨酸代谢网络模型进行途径分析,确定以果糖、葡萄糖、蔗糖或木糖为碳源时L-亮氨酸生物合成的基元模型、最大理论产率和不同模型的呼吸熵.结果:通过途径分析得到了L-亮氨酸生物合成的基元模型.以果糖、葡萄糖、蔗糖和木糖为碳源时L-亮氨酸的最大理论产率均为66.7%,其对应的最大呼吸熵分别为18、16、19、18.结论:L-亮氨酸理论得率与碳源种类无关;呼吸熵增加,能够有效提高L-亮氨酸合成代谢流,限制菌体量的过量生成.与其他碳源相比,蔗糖能够避免碳架溢流出现,合成L-亮氨酸能量代谢需求低;而葡萄糖能够较好地满足菌体生长和产酸的需求.     

Application of metabolic flux analysis for the identification of metabolic bottlenecks in the biosynthesis of penicillin-G

A detailed stoichiometric model was developed for growth and penicillin-G production in Penicillium chrysogenum. From an a priori metabolic flux analysis using this model it appeared that penicillin production requires significant changes in fluxes through the primary metabolic pathways. This is brought about by the biosynthesis of carbon precursors for the beta-lactan nucleus and an increased demand for NADPH, mainly for sulfate reduction. As a result, significant changes in flux partitioning occur around four principal nodes in primary metabolism. These are located at: (1) glucose-6-phosphate; (2) 3-phosphoglycerate; (3) mitochondrial pyruvate; and (4) mitochondrial isocitrate. These nodes should be regarded as potential bottlenecks for increased productivity. The flexibility of these principal nodes was investigated by experimental manipulation of the fluxes through the central metabolic pathways using a high-producing strain of P. chrysogenum. Metabolic fluxes were manipulated through growth of the cells on different substrates in carbon-limited chemostat culture. Metabolic flux analysis, based on measured input and output fluxes, was used to calculate the fluxes around the principal nodes. It was found that, for growth on glucose, ethanol, and acetate, the flux partitioning around these nodes differed significantly. However, this had hardly any effect on penicillin productivity, showing that primary carbon metabolism is not likely to contain potential bottlenecks. Further experiments were performed to manipulate the total metabolic demand for the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). NADPH demand was increased stepwise by cultivating the cells on glucose or xylose as the carbon source combined with either ammonia or nitrate as the nitrogen source, which resulted in a stepwise decrease of penicillin production. This clearly shows that, in penicillin fermentation, possible limitations in primary metabolism reside in the supply/regeneration of cofactors (NADPH) rather than in the supply of carbon precursors.     

Two-stage oxygen supply strategy for enhanced lipase production by Bacillus subtilis based on metabolic flux analysis

Lipase production by Bacillus subtilis CICC20034 was assessed by metabolic flux distribution analysis. Lipase production was tested under various oxygen supply conditions in a synthetic medium to obtain the optimal oxygen supply profile. Based on the metabolic flux analysis, a two-stage oxygen supply strategy (TOS) that maintained high oxygen supply conditions during early fermentation phase, and then step-wisely reduced aeration to keep a stable, smooth, and adequate changing dissolved oxygen (DO) level profile throughout the production phases was carried out. With the proposed control strategy, the final lipase activity in batch fermentation significantly increased and reached a high level at 0.56 U/ml, corresponding to a 51% increase. The relevant metabolic flux analysis verified the effectiveness of the proposed control strategy. By applying TOS in composite medium, the final lipase activity reached 5.0 U/ml.     

Effects of product inhibition in metabolic pathways: stability and control

The effect of product inhibition in metabolic pathways is examined using (a) an unbranched pathway in the absence of endproduct inhibition and (b) an unbranched pathway with endproduct inhibition. It is shown that product inhibition may be considered an alternative mechanism to endproduct inhibition for reducing the overall logarithmic gain of an unregulated pathway. When product inhibition and endproduct inhibition are both present, they act in concert with each other to lower the overall logarithmic gain and alleviate parameter sensitivities. Product inhibition is also found to exert a stabilizing influence that competes with the destabilizing effect of endproduct inhibition in controlling the dynamic behavior.     

Metabolic pathway analysis of Scheffersomyces (Pichia) stipitis: effect of oxygen availability on ethanol synthesis and flux distributions

Elementary mode analysis (EMA) identifies all possible metabolic states of the cell metabolic network. Investigation of these states can provide a detailed insight into the underlying metabolism in the cell. In this study, the flux states of Scheffersomyces (Pichia) stipitis metabolism were examined. It was shown that increasing oxygen levels led to a decrease of ethanol synthesis. This trend was confirmed by experimental evaluation of S. stipitis in glucose-xylose fermentation. The oxygen transfer rate for an optimal ethanol production was 1.8 mmol/l/h, which gave the ethanol yield of 0.40 g/g and the ethanol productivity of 0.25 g/l/h. For a better understanding of the cell's regulatory mechanism in response to oxygenation levels, EMA was used to examine metabolic flux patterns under different oxygen levels. Up- and downregulation of enzymes in the network during the change of culturing condition from oxygen limitation to oxygen sufficiency were identified. The results indicated the flexibility of S. stipitis metabolism to cope with oxygen availability. In addition, relevant genetic targets towards improved ethanol-producing strains under all oxygenation levels were identified. These targeted genes limited the metabolic functionality of the cell to function according to the most efficient ethanol synthesis pathways. The presented approach is promising and can contribute to the development of culture optimization and strain engineers for improved lignocellulosic ethanol production by S. stipitis.     

Understanding the Adaptive Growth Strategy of Lactobacillus plantarum by In Silico Optimisation

In the study of metabolic networks, optimization techniques are often used to predict flux distributions, and hence, metabolic phenotype. Flux balance analysis in particular has been successful in predicting metabolic phenotypes. However, an inherent limitation of a stoichiometric approach such as flux balance analysis is that it can predict only flux distributions that result in maximal yields. Hence, previous attempts to use FBA to predict metabolic fluxes in Lactobacillus plantarum failed, as this lactic acid bacterium produces lactate, even under glucose-limited chemostat conditions, where FBA predicted mixed acid fermentation as an alternative pathway leading to a higher yield. In this study we tested, however, whether long-term adaptation on an unusual and poor carbon source (for this bacterium) would select for mutants with optimal biomass yields. We have therefore adapted Lactobacillus plantarum to grow well on glycerol as its main growth substrate. After prolonged serial dilutions, the growth yield and corresponding fluxes were compared to in silico predictions. Surprisingly, the organism still produced mainly lactate, which was corroborated by FBA to indeed be optimal. To understand these results, constraint-based elementary flux mode analysis was developed that predicted 3 out of 2669 possible flux modes to be optimal under the experimental conditions. These optimal pathways corresponded very closely to the experimentally observed fluxes and explained lactate formation as the result of competition for oxygen by the other flux modes. Hence, these results provide thorough understanding of adaptive evolution, allowing in silico predictions of the resulting flux states, provided that the selective growth conditions favor yield optimization as the winning strategy.     

A two-stage oxygen supply strategy for enhanced l-arginine production by Corynebacterium crenatum based on metabolic fluxes analysis

In this study, the metabolic flux distribution analysis of a new l-arginine (Arg) overproducing strain, Corynebacterium crenatum, was carried out under various oxygen supply conditions in order to explore the optimized oxygen supply profile. The metabolic flux analysis indicated that a relatively higher l-arginine production could be obtained under high oxygen supply (HOS) condition overall. However, during the late fermentation phases, a much more stable l-arginine production could be rather achieved under medium oxygen supply (MOS) condition. As a result, a two-stage oxygen supply strategy, which maintained HOS condition during early fermentation phase, and then step-wisely reduced agitation to keep a stable, smooth and moderate dissolve oxygen levels (DO) changing profile throughout the production phases, was proposed. With the proposed control strategy, the final l-arginine concentration of the batch fermentation was largely increased and reached to a high level of 36.6 g L^?1, which was 16% and 51% higher than those obtained under the HOS and MOS conditions. The two-stage oxygen supply strategy could also accelerate glucose consumption rate and thus shorten fermentation time under the same batch initial fermentation condition. The relevant metabolic flux analysis verified the effectiveness of the proposed control strategy.     

Control of glycolytic flux in directed biosynthesis of uridine-phosphoryl compounds through the manipulation of ATP availability

Adenosine triphosphate (ATP), the most important energy source for metabolic reactions and pathways, plays a vital role in control of metabolic flux. Considering the importance of ATP in regulation of the glycolytic pathway, the use of ATP-oriented manipulation is a rational and efficient route to regulate metabolic flux. In this paper, a series of efficient ATP-oriented regulation methods, such as changing ambient temperature and altering reduced nicotinamide adenine dinucleotide (NADH), was developed. To satisfy the different demand for ATP at different phases in directed biosynthesis of uridine-phosphoryl compounds, a multiphase ATP supply regulation strategy was also used to enhance to yield of target metabolites.     

Analysis of the maximum theoretical yield for the synthesis of erythromycin precursors in Escherichia coli

The heterologous biosynthesis of complex polyketides in Escherichia coli was recently achieved through metabolic engineering. However, it was observed that less than 10% of the propionate carbon source is transformed into the erythromycin precursor, 6-deoxyerythronolide B (6dEB), resulting in a 1.4% molar yield. Therefore, metabolic flux analysis was performed using a model of the Escherichia coli metabolism with the addition of the enzymes required for 6dEB synthesis. The analysis shows that the maximum theoretical yield for 6dEB synthesis in E. coli is 11%. The maintenance energy requirement of E. coli and limitations in the specific oxygen uptake rate can further decrease the yield, suggesting that the observed 6dEB yield of 1.4% can be the result of these two factors. In addition, the results suggest that an increase in the specific carbon and oxygen uptake rates will increase the yield of 6dEB. The use of glucose as an alternative carbon source was also evaluated using metabolic flux analysis and the results suggest that the choice of glucose as the carbon source will allow a small improvement in performance relative to a propionate-based process.