基于代谢流量分析的L-谷氨酸发酵过程优化

目的通过构建L-谷氨酸生产菌的代谢网络,依据代谢流分析理论,得到L-谷氨酸生产菌不同发酵时期的代谢流分配;通过对节点及代谢流的分析,为发酵过程控制提供理论指导.方法测定并计算发酵中、后期L-谷氨酸等代谢物的胞外浓度和积累(或消耗)速率;应用代谢流分析方法,通过MATLAB软件线性规划得到发酵中、后期胞内代谢流分布.结果表明在L-谷氨酸发酵过程中,99.84%的葡萄糖进入糖酵解途径.41.66%的碳架进入乙醛酸循环途径,76.35%用于合成L-谷氨酸,CO2固定反应的代谢流量为32.86%与途径分析获得的理想代谢流分布相比,试验测定的CO2固定反应代谢流量偏低,合成L-谷氨酸的代谢流远低于理想代谢流(100%);采用脉冲流加补料方式,控制溶氧量5%左右,发酵液中L-谷氨酸最高产酸达141 g/L.结论根据代谢流分析结果,通过优化发酵过程控制(如流加方式、溶氧水平等)来减少副产物的生成,增强CO4固定反应.降低乙醛酸循环途径的代谢流量,从而显著提高L-谷氨酸的产率.     

谷氨酸棒杆菌ATCC 14067中L-精氨酸合成途径关键酶的过表达对L-精氨酸合成的影响

谷氨酸棒杆菌中与精氨酸合成相关的酶主要是由2个操纵子基因簇argCJBDFR和argGH编码合成。此外,在L-精氨酸合成途径的前体物谷氨酸和氨甲酰磷酸的合成中,由gdh、icd、gltA、carAB 4个基因分别编码的谷氨酸脱氢酶、异柠檬酸脱氢酶、柠檬酸合酶、氨甲酰磷酸合成酶是谷氨酸和氨甲酰磷酸合成的关键酶。研究以解除了L-精氨酸的别构抑制作用的谷氨酸棒杆菌为出发菌株,利用3种不同抗性的表达质粒分别对这些关键酶的编码基因进行串联共表达。在最终得到的13个菌株中,L-精氨酸产量最高的菌株是出发菌株L-精氨酸产量的6.23倍,这表明串联过表达菌株L-精氨酸合成途径中,不同节点关键酶的编码基因可以更有效地提高菌株中L-精氨酸的产量,为进一步改造L-精氨酸的合成途径、优化L-精氨酸的产量奠定了基础。     

L-色氨酸产生菌TQ2223的途径分析

应用途径分析方法分析了在稳态时谷氨酸棒杆菌TQ2223菌株由葡萄糖发酵生产L 色氨酸的途径,确定了L 色氨酸合成的最佳途径及其代谢流分布和最大理论产率.通过刺激所选途径的酶活来提高L 色氨酸产率,结果表明,经NH4+调节后,代谢途径流量发生显著变化,可以使色氨酸的代谢流从6提高到8.8.     

发酵过程流加L-谷氨酸提高ε-聚赖氨酸的产量

为了提高Streptomyces sp.M-Z18合成ε-聚赖氨酸(ε-PL)能力,在考察L-谷氨酸添加浓度和添加时机基础上,提出发酵过程中流加L-谷氨酸的策略。结合甘油补料-分批发酵方式,该策略实现174 h内ε-PL发酵产量和产率分别达到31.65 g/L和4.36 g/(L·d),较原发酵工艺分别提高49.2%和43.9%。实验结果表明,发酵过程流加L-谷氨酸是提高ε-PL发酵水平的有效策略之一。     

基于代谢流量分析的黄色短杆菌TK0303合成L-亮氨酸发酵过程的优化

测定并计算了在发酵中后期L-亮氨酸等代谢物的胞外浓度和积累(或消耗)的速率。应用代谢流分析方法,通过MATLAB软件线性规划得到发酵中后期胞内代谢流分布及L-亮氨酸合成的理想代谢流分布。结果表明,在L-亮氨酸分批发酵过程中,有98.73%的葡萄糖进入糖酵解途径,仅1.27%进入HMP途径,55.10%的碳架进入TCA循环,25.21%用于合成L-亮氨酸。实验测定的合成L-亮氨酸的代谢流远低于理想代谢流(66.67)。根据代谢流分析结论,文中通过优化发酵过程控制如流加方式、溶氧水平等方面来减少副产物的生成,控制TCA循环代谢流,从而提高L-亮氨酸的产率。实验采用脉冲补料方式,控制溶氧浓度在20%左右,L-亮氨酸最高产酸达到38g/L。     

α-淀粉酶抑制剂生物合成途径和代谢流分析

为提高糖类的利用效率,加强糖类代谢向生成α-淀粉酶抑制剂的方向流动,提高α-淀粉酶抑制剂产量,对天蓝黄链霉菌合成α-淀粉酶抑制剂的代谢网络进行分析,找出影响α-淀粉酶抑制剂合成的代谢流量分配规律和关键节点,并且应用代谢流分析的方法研究了谷氨酸钠对α-淀粉酶抑制剂发酵中后期胞内代谢流分布的影响。结果表明：在α-淀粉酶抑制剂分批发酵过程中,未流加谷氨酸钠时合成α-淀粉酶抑制剂的代谢流量为1.84;在发酵培养基中流加谷氨酸钠使其质量浓度维持在4.0 g/L后,α-淀粉酶抑制剂生物合成的代谢流增长至3.18。因此发酵过程中流加谷氨酸钠能够改变α-淀粉酶抑制剂生物合成途径的关键节点6-磷酸葡萄糖、7-磷酸景天庚酮糖及谷氨酸的代谢流分布,提高α-淀粉酶抑制剂生物合成途径的代谢流量。     

基于代谢流量分析的黄色短杆菌TK0303合成L-厂亮氨酸发酵过程的优化

测定并计算了在发酵中后期L-亮氨酸等代谢物的胞外浓度和积累(或消耗)的速率.应用代谢流分析方法,通过MATLAB软件线性规划得到发酵中后期胞内代谢流分布及L-亮氨酸合成的理想代谢流分布.结果表明,在L-亮氨酸分批发酵过程中,有98.73%的葡萄糖进入糖酵解途径,仅1.27%进入HMP途径,55.10%的碳架进入TCA循环,25.21%用于合成L-亮氨酸.实验测定的合成L-,亮氨酸的代谢流远低于理想代谢流(66.67).根据代谢流分析结论,文中通过优化发酵过程控制如流加方式、溶氧水平等方面来减少副产物的生成,控制TCA循环代谢流,从而提高L-亮氨酸的产率.实验采用脉冲补料方式,控制溶氧浓度在20%左右,L-亮氨酸最高产酸达到38g/L.     

α-淀粉酶抑制剂生物合成途径和代谢流分析（英文）

为提高糖类的利用效率,加强糖类代谢向生成α-淀粉酶抑制剂的方向流动,提高α-淀粉酶抑制剂产量,对天蓝黄链霉菌合成α-淀粉酶抑制剂的代谢网络进行分析,找出影响α-淀粉酶抑制剂合成的代谢流量分配规律和关键节点,并且应用代谢流分析的方法研究了谷氨酸钠对α-淀粉酶抑制剂发酵中后期胞内代谢流分布的影响。结果表明:在α-淀粉酶抑制剂分批发酵过程中,未流加谷氨酸钠时合成α-淀粉酶抑制剂的代谢流量为1.84;在发酵培养基中流加谷氨酸钠使其质量浓度维持在4.0 g/L后,α-淀粉酶抑制剂生物合成的代谢流增长至3.18。因此发酵过程中流加谷氨酸钠能够改变α-淀粉酶抑制剂生物合成途径的关键节点6-磷酸葡萄糖、7-磷酸景天庚酮糖及谷氨酸的代谢流分布,提高α-淀粉酶抑制剂生物合成途径的代谢流量。     

谷氨酸棒杆菌S9114在不同溶氧条件下发酵生产谷氨酸的代谢流分析

建立了谷氨酸棒杆菌S9114合成L-谷氨酸(L-Glu)的代谢流平衡模型,应用该模型并结合S9114在10%和30%溶氧条件下发酵至中后期时胞外相关产物的分泌情况计算出其代谢流分布;通过MATLAB线性规划得到了谷氨酸合成的理想代谢流分布。代谢流分析结果表明,减少HMP和TCA循环的代谢流量,增加CO2固定的代谢流将有利于谷氨酸的合成;在谷氨酸发酵工艺控制中,溶氧是关键因素,发酵中后期提高溶氧有利于谷氨酸的生成,同时可抑制副产氨基酸的产生;主要副产物乳酸在理想代谢流分布中仍占有一定比例,说明乳酸在整个代谢过程中起到一定的作用。     

以葡萄糖为底物合成2-吡咯烷酮重组谷氨酸棒杆菌的构建及发酵研究

2-吡咯烷酮(2-pyrrolidone)因在纺织和制药行业的广泛应用而受到越来越多的关注。通过对合成2-吡咯烷酮途径的关键酶Co A转移酶的挖掘及功能的研究,首次探索了从谷氨酸棒杆菌(Corynebacterium glutamicum)中建立2-吡咯烷酮合成途径,为未来可持续合成2-吡咯烷酮工业指明了方向。首先通过敲除N-乙酰谷氨酸激酶基因(arg B)阻断L-精氨酸合成途径,促使更多葡萄糖流向L-谷氨酸。其次,通过表达将L-谷氨酸转化为γ-氨基丁酸(γ-aminobutyric acid,GABA)的谷氨酸脱羧酶(Gad)突变体,获得合成GABA的重组菌。同时,异源表达经N-端RBS优化的丙酸厌氧菌(Anaerotignum propionicum)来源的Co A转移酶,实现了GABA向2-吡咯烷酮的转化。最终,构建的C. glutamicum EAGAN2重组菌株在5 L发酵罐补料分批发酵72 h时,积累了(8±0. 3)g/L的2-吡咯烷酮。该研究首次在谷氨酸棒杆菌中建立了2-吡咯烷酮合成途径,实现了以廉价原料葡萄糖为底物一步法合成2-吡咯烷酮。     

提高L- 谷氨酸产量的后期发酵工艺参数研究

以谷氨酸生产菌S9114 为供试菌株,利用50m3 发酵罐研究了L- 谷氨酸的发酵过程,确定发酵后期产酸速率过低是影响L- 谷氨酸产量的主要原因。优化发酵工艺的参数以提高L- 谷氨酸后期发酵的比产酸速率,结果表明：采用溶氧控制的葡萄糖流加方式,控制发酵后期的pH 值,在发酵的适当时期流加一定量的生物素和KCl 等措施可有效提高L- 谷氨酸的后期产酸水平。在最优条件下,单罐最高产量可达148g/L,糖酸转化率为60.5%。     

Metabolic flux analysis for efficient pyruvate fermentation using vitamin-auxotrophic yeast of Torulopsis glabrata

The metabolism of a vitamin-auxotrophic pyruvate-producing microorganism, Torulopsis glabrata IFO 0005, was investigated by metabolic flux analysis. Particular attention was focused on the effect of culture conditions, such as dissolved oxygen (DO) concentration and thiamine concentration, on specific pathway activities. The results of metabolic flux analysis indicate that the thiamine concentration significantly affected pyruvate dehydrogenase and pyruvate decarboxylase activities, and plays an important role in cell growth and pyruvate production. Metabolic flux analysis was also utilized to clarify the metabolism of this strain during pyruvate fermentation under different oxygen supply conditions, and the reason for the enhanced pyruvate production under conditions of 30-40% DO concentration was clarified from the viewpoint of intracellular flux distributions. Based on the analysis of the effect of thiamine concentration on the metabolic fluxes, we conducted a fed-batch experiment where the initial thiamine concentration was reduced to 30 microg/l and thiamine was added at 10 microg/l during fermentation when the cell growth rate decreased to 0.2 h(-1). With separate addition of thiamine, the overall pyruvate yield could be improved by 15% due to the decrease of ethanol production.     

代谢副产物对L-苏氨酸发酵的影响及应对措施

研究了L-苏氨酸的发酵过程,通过高效液相色谱和氨基酸分析仪测定了L-苏氨酸发酵液中的主要副产物为乙酸、乳酸、缬氨酸、天冬氨酸、丙氨酸等。利用外源添加实验研究了各种代谢副产物对L-苏氨酸发酵的影响。结果表明乙酸浓度高于2 g/L、乳酸浓度高于10 g/L时对L-苏氨酸生产菌的生长和产酸有抑制作用,采用提高溶解氧和葡萄糖限制性脉冲流加措施将乙酸、乳酸的浓度控制在0.5 g/L以下,L-苏氨酸产量明显提高。     

Effects of pH and dissolved oxygen on cellulose production by Acetobacter xylinum BRC5 in agitated culture

Acetobacter xylinum BRC5 was cultivated in a jar fermentor using glucose as the sole carbon source. Strain BRC5 oxidized almost all of the glucose to gluconic acid; thereafter, it biosynthesized cellulose by utilizing gluconic acid accumulated in the broth. The optimal pH for metabolizing glucose to gluconic acid was 4.0, while a pH of 5.5 was preferred for cell growth and cellulose production from the accumulated gluconic acid in the medium. Shifting the pH from 4.0 to 5.5 during the cellulose production phase in batch cultures improved cellulose production and reduced the total fermentation time, compared to batch cultures at constant pH. In constant fed-batch culture, 10 g/l of cellulose was obtained from 40 g/l of glucose, a yield which was approximately 2-fold higher than in batch culture with the same initial glucose concentration, even without control of the level of dissolved oxygen. The highest cellulose yield was obtained in fed-batch cultures in which the dissolved oxygen concentration was controlled at 10% saturation. Control of pH and dissolved oxygen to optimal levels was effective for improving the production rate and yield of cellulose, to achieve a high cellulose productivity of 0.3 g cellulose/l x h. Approximately 15 g/l of cellulose was considered to be the highest yield obtainable using conventional fermentors because the culture broth then became too viscous to allow satisfactory aeration.     

Production of extracellular bifidogenic growth stimulator (BGS) from Propionibacterium shermanii using a bioreactor system with a microfiltration module and an on-line controller for lactic acid concentration

Production of a bifidogenic growth stimulator (BGS) by Propionibacterium freudenreichii subsp. shermanii (Propionibacterium shermanii) using lactic acid as a carbon source was investigated using different cultivation methods. When a continuous bioreactor system with a filtration device was used at a dilution rate of 0.075 h(-1), the average BGS concentration was 2.4 mg/l, which corresponds to a BGS productivity per cultivation time of 1.8 x 10(-1) mg x l(-1) x h(-1). The BGS productivity per cultivation time in continuous cultivation with filtration was 1.9-fold that (9.4 x 10(-2) mg x l(-1).h(-1)) in a conventional batch cultivation. In fed-batch cultivation with feed-back control using an on-line lactic acid controller with a lactic acid biosensor, it was possible to prevent substrate inhibition by maintaining the lactic acid concentration in culture broth low at 3.3 g/l, and an enhanced BGS production (31 mg/l) was successfully attained. The BGS productivity per cultivation time (2.1x10(-1) mg x l(-1) x h(-1)) in the fed-batch cultivation with feed-back control was 2.2-fold that in the conventional batch cultivation. A new bioreactor system was developed by coupling a continuous bioreactor system with a filtration device to an on-line lactic acid controller. Using the new bioreactor system, we produced BGS continuously at a high level of 47 mg/l. The BGS productivities per cultivation time (3.5 mg.l(-1) x h(-1)) and the total volume of medium used (1.7 x 10(-1) mg x l(-1) x h(-1)) obtained in the new bioreactor system were 37-fold and 2.1-fold those in the conventional batch cultivation, respectively. These results described above clearly demonstrate the positive effects of both the continuous filtration for removal of metabolites (propionic and acetic acids) inhibitory to cell growth and feed-back control of lactic acid concentration in the culture broth on BGS production by P. shermanii. This paper is the first report on BGS production by the propionic acid bacterium using lactic acid as a carbon source.     

Enhancement and stabilization of desulfurization activity of Rhodococcus erythropolis KA2-5-1 by feeding ethanol and sulfur components

We developed a fed-batch culture system fed with ethanol and restricted amounts of sulfur compounds to enhance and stabilize the desulfurizing activity in bacterial cells. In this system using dibenzothiophene (DBT) as the sole sulfur source, a desulfurizing bacterium Rhodococcus erythropolis KA2-5-1 cultivated with small amounts of sulfur showed stable desulfurizing activity and a low rate of growth. However, the cells cultured with excess amounts of sulfur showed unstable activity and a high growth rate. DBT had disadvantages as a sulfur source for cultivation because it is immiscible with water and toxic to cells. We then investigated water-soluble sulfur compounds for use as the sole sulfur source for the cultivation of R. erythropolis KA2-5-1 with desulfurizing activity, and found 2-aminoethanesulfonic acid to be the most effective. When 2-aminoethanesulfonic acid was used instead of DBT as the sole sulfur source in the fed-batch fermentation system, R. erythropolis KA2-5-1 showed the highest desulfurizing activity, 111 mmol of 2-HBP/kg-cells/h, a high growth rate (mu = 0.37/h) and a final cell concentration of 20.0 g-dry cells/l during 89 h of cultivation. The production levels of the desulfurizing enzymes in the bacterial cells cultivated with DBT or 2-aminoethanesulfonic acid were evaluated by immunoblot analysis with specific antisera, indicating that the same quantity of desulfurizing enzymes was expressed in both cases.     

Production of highly concentrated xylitol by Candida magnoliae under a microaerobic condition maintained by simple fuzzy control

Microbial production of xylitol from xylose was investigated using Candida magnoliae. In particular, the effect of the oxygenation condition on the xylitol production yield was examined and the significance of maintaining a microaerobic condition was demonstrated. A simple system of fuzzy logic control (FLC) was devised to maintain the microaerobic condition in the xylitol production phase by regulating the proportion of air (air flow rate) supplied to the fermentor. The input variables to the fuzzy control system were the dissolved oxygen (DO) concentration in the culture broth and the CO2 concentration in the exit gas. A batch cultivation test using the FLC system confirmed the importance of maintaining a constant microaerobic condition throughout the xylitol production phase, and indicated it would be advantageous for this phase to be prolonged. An intermittent fed-batch culture was therefore carried out. The FLC system allowed a constant microaerobic condition to be maintained, resulting in minimal cell mass production and constant xylitol accumulation in the culture medium. As a consequence, a very high xylitol concentration of 356 g/dm3 could be attained. The xylitol yield in the fed-batch culture was 0.75, which corresponded to 82% of the theoretical yield.     

谷氨酸连续等电结晶中主要污染微生物野生酵母的鉴定与防治

对从谷氨酸连续等电结晶罐中分离得到的野生酵母菌株进行了初步的鉴定,并探寻了防治其污染的可能途径。结果表明,该酵母属于假丝酵母属,增殖过程中能够同化利用谷氨酸,是引起发酵液中谷氨酸含量下降的主要原因。通过对该野生酵母生长特性的分析可知,当温度高于50℃时其生长几乎停滞,pH在下降至等电点3.2时也能一定程度抑制其快速增殖。结合谷氨酸连续等电结晶过程特点,尝试缩短结晶停留时间来防治其污染,当停留时间低于9.12 h时,野生酵母被稀释洗脱,从而减少提取过程主产品谷氨酸的损失。     

谷氨酸全营养流加发酵新工艺

全营养流加主要是选择适当的全营养培养,在合适的时间进行营养的补加,通过补加的营养来弥补菌体因生长代谢而消耗的营养物质,同时也可以降低发酵培养基的浓度,避免富营养对于菌体活力的抑制。因此,采用全营养流加策略能够解决L-谷氨酸发酵后期菌体活力不足和产酸能力下降等问题。实验结果表明,最佳流加条件为从发酵2 h开始流加,持续24 h流加体积分数为60%的流加培养基。在此条件进行L-谷氨酸发酵,生物量(OD 600)达到了66,提升了29.4%,菌体转型时间提前了2 h,L-谷氨酸产量为168 g/L,提高了22.6%,乳酸含量为3.1 g/L,降低了13.8%,丙氨酸含量为2.06 g/L,降低了17.6%,糖酸转化率为63%,提高了1.5%。全营养流加发酵对于加快菌体转型,提高菌体活力、谷氨酸产量及糖酸转化率均有积极作用。