Optimization of L-tryptophan fermentation conditions from recombinant Escherichia coil

为了提高重组大肠杆菌FB-04/pSV-04发酵生产L-色氨酸的产量,减少代谢副产物乙酸的生成,考察了比生长速率和无机盐对重组大肠杆菌发酵生产L-色氨酸的影响.在确定了合适的比生长速率和无机盐浓度之后,乙酸积累很少,L-色氨酸的产量为53.4 g/L,比优化前提高了141.6％.经30 L发酵罐初步放大,L-色氨酸的产量达53.6 g/L,发酵结果稳定,具有工业化应用前景.     

重组大肠杆菌生产L-色氨酸发酵条件优化

为了提高重组大肠杆菌FB讲／pSV－04发酵生产L－色氨酸的产量,减少代谢副产物乙酸的生成,考察了比生长速率和无机盐对重组大肠杆菌发酵生产L．色氨酸的影响。在确定了合适的比生长速率和无机盐浓度之后,乙酸积累很少,L-色氨酸的产量为53．4g／L,比优化前提高了141．6％。经30L发酵罐初步放大,L－色氨酸的产量达53．6g／L,发酵结果稳定,具有工业化应用前景。     

NH4+对L-色氨酸发酵的影响

目的:探究NH₄⁺浓度对大肠杆菌E.coli TRTH发酵生产L-色氨酸的影响。方法:通过外源添加试验,利用30 L发酵罐进行分批补料发酵试验,考察E. coli TRTH发酵生产L-色氨酸过程中生物量、L-色氨酸产量、有机酸含量、耗糖速率、发酵液中NH₄⁺浓度及质粒稳定性变化。建立了大肠杆菌合成L-色氨酸的代谢流平衡模型,应用 MATLAB 软件计算出E. coli TRTH发酵中后期代谢网络的代谢流分布。结果:发酵结果显示,利用NaOH和氨水混合补料,控制NH₄⁺浓度在120 mmol/L以下,菌体能够以较长时间和较高比生长速率保持对数生长,最终菌体生物量和L-色氨酸产量分别提高了12.16%和19.80%。随着NH₄⁺浓度的增加,发酵液中丙酮酸、乳酸及乙酸浓度均略有增加,细胞质粒稳定性下降。控制NH₄⁺浓度在120 mmol/L以下,E. coli TRTH发酵生产L-色氨酸的代谢流量分析结果表明,EMP途径的代谢流量降低7.31%,PP途径的代谢流量增加7.14%,TCA循环的代谢流量降低22.04%。结论:高浓度的NH₄⁺导致菌体生长提前结束,耗糖速率降低,产酸受阻,控制NH₄⁺浓度在120 mmol/L以下,解除了NH₄⁺对菌体生长和产物生成的抑制,使得菌体生物量和L-色氨酸产量大幅提高,实现了高密度发酵培养的目的。     

pta基因敲除对L-色氨酸发酵的影响

[目的]探究pta基因缺失对大肠杆菌发酵生产L-色氨酸的影响.[方法]运用Red重组技术敲除pta基因,构建pta缺失株E.coli TRTH△pta.利用30 L发酵罐进行分批补料发酵试验,考察重组菌E.coliTRTHApta发酵0生产L-色氨酸过程中生物量、L-色氨酸产量、有机酸含量、发酵液中NH4+浓度及变化....     

大肠杆菌色氨酸转运系统多基因敲除对色氨酸生产的影响

大肠杆菌中色氨酸向胞内的转运主要是由mtr、tnaB和aroP 3个基因编码的通透酶进行调控.利用Red重组技术,在mtr单基因敲除菌的基础上,成功构建了mtr.tnaB和mtr.aroP双基因敲除菌以及mtr.tnaB.aroP三基因敲除菌,并通过发酵实验首次考察了色氨酸转运系统多基因缺失对大肠杆菌合成色氨酸的影响.发酵结果表明,mtr.tnaB和mtr.aroP双基因缺失后,色氨酸产量分别达到1.38 g/L和1.27 g/L,与出发菌株相比分别提高了17％和9％,而mtr.tnaB.aroP三基因缺失后,菌体生长受到了明显抑制,发酵后色氨酸产量仅为0.63 g/L.在补料分批发酵实验中,mtr.tnaB双基因敲除菌的色氨酸产量进一步提高至12.2 g/L,与出发菌株相比色氨酸产量提高了27％.     

溶氧对L-苏氨酸发酵的影响

探索溶氧对L-苏氨酸发酵过程的影响及其控制方法。通过摇瓶装液量试验、不同溶氧控制方式考察发酵过程中溶氧对L-苏氨酸合成的影响。采用补料分批发酵工艺发酵L-苏氨酸,利用氨基酸分析仪测定发酵液中L-苏氨酸的产量,通过10L罐补料分批发酵36h,产酸可达118.9g/L,糖酸转化率为47.6%。可以得出溶氧对L-苏氨酸生物合成有重要影响,并建立了最佳溶氧控制条件。     

ppc基因敲除对大肠杆菌L-色氨酸发酵的影响

目的：减少大肠杆菌L-色氧酸前体物质磷酸烯醇式丙酮酸向草酰乙酸的代谢流,提高其L-色氨酸的产量。方法：以大肠杆菌TRTH0709为出发菌株,利用Red重组敲除技术敲除磷酸烯醇式丙酮酸羧化酶（Ppc）编码基因PPc,并经测序和酶活性检测确证;对出发菌株和基因敲除菌株进行L-色氖酸发酵,对比分析发酵结果。结果：测序和酶活性检测结果表明ppc基因被成功敲除。发酵结果表明,与出发菌株相比,基因敲除菌株TRTH0709△ppc生长速度减慢,最终生物量减少32％,L-色氯酸产量降低27％,但糖酸转化率提高6％;向发酵培养基中添加1％琥珀酸后,TRTH0709△ppc的生长速率和产酸量有所提高,但仍与出发菌株有一定差距。结论：虽然ppc基因敲除对菌体生长和产酸量影响较大,但能有效提高其糖酸转化率;选育Ppc弱化的突变株以达到减弱代谢流且不影响菌体生长,以及增加,L-色氨酸积累的目的,将是本研究今后的主要方向。     

脯氨酸-4-羟化酶在大肠杆菌中的密码子优化表达及对反式-4-羟脯氨酸生物合成的作用

为了使脯氨酸-4-羟化酶基因在重组大肠杆菌中得到高表达,通过调整大肠杆菌密码子偏好性以及mRNA二级结构,使得脯氨酸-4-羟化酶基因得到优化。将优化后的脯氨酸-4-羟化酶基因插入含有色氨酸串联启动子的p UC19质粒,构建重组质粒p UC19-ptrp2-Hyp,并导入大肠杆菌BL21(DE3)中。在摇瓶水平,重组菌以L-脯氨酸为底物发酵8 h,可积累(0.492±0.034)g/L的反式-4-羟脯氨酸。在发酵罐水平,通过补料分批发酵来提高反式-4-羟脯氨酸的产量,当补糖速率为18 g/h时,反式-4-羟脯氨酸的产量高达42.5 g/L,反式-4-羟脯氨酸产率为0.966 g/(L·h)。     

氮源对L-苏氨酸发酵的影响

以L-苏氨酸生产菌TRFC为供试菌株,研究了氮源对L-苏氨酸发酵的产量和糖酸转化率的影响。首先通过摇瓶实验确定发酵的最佳无机氮源和有机氮源分别为硫酸铵和酵母粉,进一步利用10L罐补料分批发酵确定硫酸铵和酵母粉的最佳用量,继续优化培养条件,采用发酵中后期流加硫酸铵和糖氨混合补料等措施,L-苏氨酸产量得到进一步的提高。在最优发酵条件下,通过10L罐补料分批发酵36h,产酸可达118.9g/L,糖酸转化率为47.6%。     

葡萄糖流加策略对基因工程FR-008／杀念菌素衍生物CS103补料分批发酵过程的影响

以组合生物合成技术得到的链霉菌FR-008突变株CS103为研究对象,研究了3．7L发酵罐上维持一定葡萄糖浓度对其次级代谢产物脱羧FR-008／candicidin衍生聚酮抗生素CS103生物合成的影响。当初始葡萄糖浓度20g／L,发酵过程还原糖浓度维持在10g/L时,抗生素CS103最高产量较分批发酵最高产量相比提高30％。研究了3．7L罐上补料分批发酵生产CS103的工艺,主要考察了脉冲补料、间歇流加补料和连续流加补料三种补料分批发酵工艺,并与分批发酵进行了比较。连续流加补料维持糖浓度的效果明显,最高产量达到126．9μg/mL,与分批发酵相比提高了44％左右。     

The role of amino acids in the amplification and quality of DNA vectors for industrial applications

In this study, we have demonstrated that the type and feeding regimen of amino acids have a significant impact on the quality as well as the quantity of DNA vectors produced. Nutrient pool and factorial design experiments were carried out in order to identify the amino acids involved in increased biomass and induction of plasmid amplification. Leucine, glycine, and histidine were responsible for increased biomass and leucine starvation in the presence of histidine was implicated in plasmid amplification. Supercoiling of the plasmid was optimized using a dual feeding strategy. As a result of this, a fed-batch fermentation strategy for the production of a 6.9 kb plasmid, pSVß, in Escherichia coli DH5α was developed. In batch fermentation, a maximum plasmid yield of 39.4 mg/L equivalent to 11.3 mg/g dry cell weight (DCW) was achieved with casein hydrolysate limitation. About 90% of plasmid was in the supercoiled (SC) form after 31 hr of fermentation but only remained so for a short period, leading to a very brief window for harvesting cells at scale. Subsequently, a fed-batch fermentation using a dual feeding strategy was employed. A mean maximum plasmid yield of 44 mg/L equivalent to 9.1 mg plasmid/g DCW was achieved. After 25 hr, 90% of plasmid was in the SC form and remained at this level for the remaining 10 hr of the fermentation, allowing adequate time for the harvesting of cells without the loss of supercoiling of product. This study emphasized that optimizing fermentation strategy and identifying the essential nutrients are beneficial for bioprocessing of plasmid DNA for therapeutic applications.     

重组质粒pVAX1-PENK大规模制备研究

目的：建立质粒pVAX1-PENK的大规模制备2--艺。方法：对大肠杆菌工程菌DH5α-pVAX1-PENK进行补料发酵,利用自行发明的连续碱裂解过程对菌体进行裂解,经超滤浓缩后,用Sepharnse 6 Fast Flow层析柱分离DNA与RNA,再经Plasmidselect Xtra层析柱分离超螺旋质粒DNA与开环或线性质粒DNA,最后经Source 15Q层析柱精制质粒DNA。结果：发酵获得质粒pVAX1-PENK的产率为182mg／L,经碱裂解及层析分离后,最终制备的质粒DNA超螺旋比例大于98％,总回收率为60.5％,纯度（D260nm／D280nm）为1.8～2.0。结论：建立的质粒DNA生产工艺可以制备大量高纯度的质粒DNA,并避免了使用动物源性的酶及有毒试剂。     

Improvement of mannitol production by Lactobacillus brevis mutant 3-A5 based on dual-stage pH control and fed-batch fermentations

Lactobacillus brevis 3-A5 was isolated and expected to produce mannitol efficiently by regulating pH in batch and fed-batch fermentations. In 48 h batch fermentations with free and constant pH, the optimal pH for cell growth and mannitol production in the first 24 h of incubation was 5.5, whereas that for mannitol production in the second 24 h of incubation was 4.5. To achieve high cell density and mannitol yield simultaneously, a dual-stage pH control strategy was proposed based on the kinetic analysis of mannitol production. The pH value was controlled at 5.5 for the first 12 h of fermentation and subsequently shifted to 4.5 until the fermentation was completed. Under dual-stage pH control fermentation, a 103 g/L yield of mannitol with a volumetric production rate of 3.7 g/L/h was achieved after 28 h. The dual-stage pH control fed-batch fermentation strategy was further developed to improve mannitol yield, wherein the yield increased by 109 % to 215 g/L after 98 h of fermentation. This value is the highest yield of mannitol ever reported using L. brevis.     

Dissolved-oxygen-stat fed-batch fermentation of 1,3-dihydroxyacetone from glycerol by Gluconobacter oxydans ZJB09112

This study investigated the effects of DO concentration on DHA fermentation and of DO-stat fed-batch fermentation using a pH control strategy, on 1,3-dihydroxyacetone (DHA) production. The results showed that DO-stat fed-batch fermentation with pH-shift control was the optimal bioprocess for DHA production. DO-stat fed-batch fermentation was carried out at 30% air saturation, and the culture pH was automatically maintained at pH 6.0 during the first 20 h and then shifted to pH 5.0 until the end of the fermentation. An optimal DHA concentration of 175.9 ± 6.7 g/L, with a production yield to glycerol of 0.87 ± 0.04 g/g, was obtained at 72 h of DO-stat fed-batch fermentation at 30°C in a 15 L fermenter.     

Mixed-feed exponential feeding for fed-batch culture of recombinant methylotrophic yeast

A simple, accurate model capable of predicting cell growth and methanol utilization during the mixed substrate fed-batch fermentation of Mut^S recombinant Pichia pastoris was developed and was used to design an exponential feeding strategy for mixed substrate fed-batch fermentation at a constant specific growth rate. Mixed substrate feeding has been shown to boost productivity in recombinant fed-batch culture of P. pastoris, while fixed growth rate exponential feeding during fed-batch culture is a useful tool in process optimization and control.     

Mineralization of pentachlorophenol in a contaminated soil by Pseudomonas sp UG30 cells encapsulated in κ-carrageenan

A fermentation process in Escherichia coli for production of supercoiled plasmid DNA for use as a DNA vaccine was developed using an automated feed-back control nutrient feeding strategy based on dissolved oxygen (DO) and pH. The process was further automated through a computer-aided data processing system to regulate the cell growth rate by controlling interactively both the nutrient feed rate and agitation speed based on DO. The process increased the total yield of the plasmid DNA by approximately 10-fold as compared to a manual fed-batch culture. The final cell yield from the automated process reached 60 g L⁻¹ of dry cell weight (OD₆₀₀ = 120) within 24 h. A plasmid DNA yield of 100 mg L⁻¹ (1.7 mg g⁻¹ cell weight) was achieved by using an alkaline cell lysis method. Plasmid yield was confirmed using High Performance Liquid Chromatography (HPLC) analysis. Because cells had been grown under carbon-limiting conditions in the automated process, acetic acid production was minimal (below 0.01 g L⁻¹) throughout the fed-batch stage. In contrast, in the manual process, an acid accumulation rate as high as 0.36 g L⁻¹ was observed, presumably due to the high nutrient feed rates used to maintain a maximum growth rate. The manual fed-batch process produced a low cell density averaging 10–12 g L⁻¹ (OD₆₀₀ = 25–30) and plasmid yields of 5–8 mg L⁻¹ (approximately 0.7 mg g⁻¹ cells). The improved plasmid DNA yields in the DO- and pH-based feed-back controlled process were assumed to be a result of a combination of increased cell density, reduced growth rate (μ) from 0.69 h⁻¹ to 0.13 h⁻¹ and the carbon/nitrogen limitation in the fed-batch stage. The DO- and pH-based feed-back control, fed-batch process has proven itself to be advantageous in regulating cell growth rate to achieve both high cell density and plasmid yield without having to use pure oxygen. The process was reproducible in triplicate fermentations at both 7-L and 80-L scales. Received 22 March 1996/ Accepted in revised form 20 September 1996     

Temperature gradient-based high-cell density fed-batch fermentation for the production of pyruvate oxidase by recombinant E. coli

Pyruvate oxidase (PyOD) is a very powerful enzyme for clinical diagnostic applications and environmental monitoring. Influences of temperature on cell growth, plasmid stability, and PyOD expression during the PyOD fermentation process by recombinant Escherichia coli were investigated. Based on the influences of temperature on the physiological metabolism, a novel high-cell density fed-batch cultivation with gradient temperature decrease strategy for effective PyOD production was achieved, under which the biomass (OD₆₀₀) of recombinant E. coli could reach to 71 and the highest PyOD activity in broth could reach to 3,307 U/L in 26?hr fermentation.     

High-cell-density fermentation of recombinant Saccharomyces cerevisiae using glycerol

To obtain a high cell density of recombinant Saccharomyces cerevisiae (INVSc 1 strain bearing a 2 microm plasmid, pYES2 containing a GAL1 promoter for expression of the beta-galactosidase gene), the yeast was grown with glycerol as the substrate by fed-batch fermentation. The feeding strategy was based on an on-line response of the medium pH to the consumption of glycerol. The approach was to feed excess carbon into the medium to create a benign environment for rapid biomass buildup. During cell growth in the presence of glycerol, the release of protons in the medium caused a decrease in pH and the consumption rate of ammonium phosphate served as an on-line indicator for the metabolic rate of the organism. The extent of glycerol feeding in a fed-batch mode with pH control at 5.0 +/- 0.1 was ascertained from the automatic addition of ammonium phosphate to the medium. The glycerol feeding to ammonium phosphate addition ratio was found to be 2.5-3.0. On the basis of the experiments, a maximum dry cell biomass of 140 g per liter and a productivity of 5.5 g DCW/L/h were achieved. The high cell density of S. cerevisiae obtained with good plasmid stability suggested a simple and efficient fermentation protocol for recombinant protein production.     

On-line control of fed-batch fermentation of dilute-acid hydrolyzates

Dilute-acid hydrolyzates from lignocellulose are, to a varying degree, inhibitory to yeast. In the present work, dilute-acid hydrolyzates from spruce, birch, and forest residue, as well as synthetic model media, were fermented by Saccharomyces cerevisiae in fed-batch cultures. A control strategy based on on-line measurement of carbon dioxide evolution (CER) was used to control the substrate feed rate in a lab scale bioreactor. The control strategy was based solely on the ratio between the relative increase in CER and the relative increase in feed rate. Severely inhibiting hydrolyzates could be fermented without detoxification and the time required for fermentation of moderately inhibiting hydrolyzates was also reduced. The feed rate approached a limiting value for inhibiting media, with a corresponding pseudo steady-state value for CER. However, a slow decrease of CER with time was found for media containing high amounts of 5-hydroxymethyl furfural (HMF). The success of the control strategy is explained by the conversion of furfural and HMF by the yeast during fed-batch operation. The hydrolyzates contained between 1.4 and 5 g/l of furfural and between 2.4 and 6.5 g/l of HMF. A high conversion of furfural was obtained (between 65-95%) at the end of the feeding phase, but the conversion of HMF was considerably lower (between 12-40%).