以葡萄糖为生长相基质的重组毕赤酵母表达植酸酶发酵

甲醇营养型毕赤酵母表达外源蛋白是在醇氧化酶（alcohol oxidase,AOX）启动子（PAOXI）严格调控下进行的,然而这种启动子在转录水平受到葡萄糖的阻遏。本文研究了毕赤酵母在葡萄糖替代甘油为生长相碳源时表达重组植酸酶蛋白的发酵特征。结果表明：初始葡萄糖浓度为20dL的细胞得率高,为0．39g[DCW]／g。通过基于实时参数（溶氧和呼吸商）调控的葡萄糖补料策略,生长相40h后细胞密度达到100g[DCW]／L,甲醇诱导100h后植酸酶产量达到2200FTUphytase／mL,甲醇得率系数为0．25FTU phytase／gmethnol。因此,在毕赤酵母高表达重组蛋白培养中葡萄糖能够用作生长相基质,并能实现重组蛋白的高效表达。     

Use of respiratory quotient as a control parameter for optimum oxygen supply and scale-up of 2,3-butanediol production under microaerobic conditions

The respiratory quotient (RQ) was found to be a suitable control parameter for optimum oxygen supply for the production of 2,3-butanediol + acetoin under microaerobic conditions. In laboratory scale continuous cultures optimum production of 2,3-butanediol + acetoin was obtained at an RQ value between 4.0 to 4.5. This agreed well with optimum RQ value (4.0) stoichiometrically derived from the bioreactions involved. In fed-batch cultures product concentrations as high as 102.9 g/L (96.0 g/L butanediol + 6.9 g/L acetoin) can be achieved within 32 h cultivation with an RQ control algorithm for oxygen supply. Under similar conditions only 85.7 g/L product (77.6 g/L butanediol + 8.1 g/L acetoin) was obtained with control of constant oxygen supply rate throughout the cultivation.In pilot scale batch cultures under identical oxygen supply rate the achievable RQ value was found to be strongly influenced by the reactor type and scale. The initial oxygen supply rate influenced the achievable RQ as well. However, in all the reactors studied the specific product formation rate of cells in the exponential growth phase was only a function of RQ. The same optimum RQ value as found in continuous cultures was obtained. It was thus concluded that RQ can be used as a control parameter for optimum production of 2,3-butanediol + acetoin in both laboratory and pilot plant scale reactors. (c) 1994 John Wiley & Sons, Inc.     

Enhancing xanthine oxidase fermentation with pH-shift strategy based on kinetic analysis by Arthrobacter M3

The effect of initial culture pH and inducer concentration on xanthine oxidase (XOD) fermentation in shake flasks was first carried out. The results showed that the optimum initial culture pH and inducer concentration were 8.6 and 3.6 g/l, respectively. Batch fermentation of XOD by Arthrobacter M3 in a 7.5-l fermentor was then tested under various pH conditions ranging from 7.6 to 8.6. Based on the analysis of the obtained kinetic parameters, a pH-shift strategy in batch fermentation was implemented to enhance the XOD fermentation. In this strategy, the initial culture pH was set at 8.6 without control and was maintained at 7.6 after the biomass reached 2.0 g/l DCW. XOD production (P) and final average yield coefficient for production on biomass (FAY_p/x) in this strategy reached 7,415.3 U/l and 1,229.7 U/g, respectively, which were significantly higher than the results from the other four protocols. In pH-shift batch fermentation, the Luedeking–Piret equation for product accumulation and the Luedeking–Piret-like equation for substrate consumption fit well with the experimental values. The correlation coefficients (R ²) of these two fitting curves were 0.977 and 0.992, respectively.     

An alkaline pH control strategy for methionine adenosyltransferase production in Pichia pastoris fermentation

Pichia pastoris is a successful system for expressing heterologous proteins and its fermentation pH is always maintained below 7.0. However, particular proteins are unstable under acidic conditions, such as methionine adenosyltransferase (MAT), and thus fermentation under acidic pH conditions is unsuitable because protein activity is lost owing to denaturation. Here, a strategy employing alkaline pH in the late fermentation period was developed to improve MAT production. Initially, P. pastoris KM71 was transformed with the mat gene to overexpress MAT. After 72 h of in vitro incubation at different pH values, the expressed MAT displayed highest stability at pH 8.0; however, pH 8.0 inhibited cell growth and induced cell rupture, thus affecting protein production. To balance MAT stability and Pichia cell viability, different pH control strategies were compared. In strategy A (reference), the induction pH was maintained at 6.0, whereas in strategy B, it was gradually elevated to 8.0 through a 25 h transition period (80 ～ 105 h). MAT activity was 0.86 U/mg (twofold higher than the control). However, MAT content was reduced by 50% when compared with strategy A, because of proteases released upon cell lysis. To improve cell viability under alkaline conditions, glycerol was added in addition to methanol (strategy C). When compared with strategy B, the MAT-specific activity remained nearly constant, whereas the expression level increased to 1.27 g/L. The alkaline pH control strategy presented herein for MAT production represents an excellent alternative for expressing proteins that are stable only under alkaline conditions.     

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.     

灵芝悬浮培养中添加微颗粒Talc对灵芝酸产生的影响

灵芝酸是灵芝中重要的药理活性物质,其低产量限制了它的广泛应用和深入研究,高效提高液体发酵中灵芝酸的含量十分必要。以CGMCC 5.65为材料,在悬浮培养条件下,研究添加微颗粒Talc对灵芝酸产生的影响。结果表明,微颗粒Talc添加显著减小了灵芝细胞粒径,对照组为(3.33±0.16)mm,15g/L微颗粒Talc添加组为(2.04±0.12)mm。灵芝细胞中单体灵芝酸和总灵芝酸的含量在微颗粒Talc添加条件下也显著提高。15g/L微颗粒Talc添加组的总灵芝酸含量达到(1.51±0.02)mg/100mg细胞干重,GA-Mk、GA-T和GA-Me的含量最高为(6.02±0.29)、(5.08±0.14)和(1.71±0.09)μg/100mg细胞干重,分别是对照的1.6、4、1.9和1.4倍。另外,15g/L微颗粒Talc添加条件下鲨烯和羊毛甾醇的最大积累量分别为(3.69±0.23)和(34.86±6.41)μg/100mg细胞干重,是对照的2.6和4.2倍;灵芝酸生物合成途径关键基因fps和cyp-5150l8的表达量最高为对照的2.35和1.53倍。     

Biocatalytic resolution of benzyl glycidyl ether and its derivates by Talaromyces flavus: effect of phenyl ring substituents on enantioselectivity

Talaromyces flavus containing a constitutive epoxide hydrolase (EH) resolved racemic benzyl glycidyl ether and nine derivatives into their (R)-enantiomers. After optimization of the fermentation conditions, the specific EH activity and biomass concentration were improved from 13.5 U/g DCW and 14.8 g DCW/l to 26.2 U/g DCW and 31.3 g DCW/l, respectively, with final values for e.e. ( s ) of 96 % and E of 13 with (R)-benzyl glycidyl ether. Substituents on the phenyl ring, however, gave low enantioselectivities.     

重组大肠杆菌生产谷胱甘肽发酵条件的研究

研究了重组Ｅ．Ｃｏｌｉ产ＧＳＨ的发酵条件,重点考察了添加酵母膏、前体氨基酸和ＡＴＰ的影响。结果发现,前体氨基酸和ＡＴＰ均能促进胞内ＧＳＨ的积累,若在发酵０ｈ和１２ｈ分别加入２０ｇ／ＬＡＴＰ和９ｍｍｏｌ／Ｌ前体氨基酸,则细胞干重和胞内ＧＳＨ含量可分别比对照提高２４％和１４倍。应用正交试验得出的针对细胞干重和ＧＳＨ总量的最佳组合,最大细胞干重和ＧＳＨ总量比原试验中的最好结果分别提高了１０％和２６％。在分析了该菌对葡萄糖利用情况的基础上,对该菌进行了指数流加培养,２５ｈ细胞干重与发酵液内ＧＳＨ总量分别达到８０ｇ／Ｌ和８８０ｍｇ／Ｌ,比摇瓶最好结果分别提高了８３和４６倍。     

Production of a platelet aggregation inhibitor, salmosin, by high cell density fermentation of recombinant Escherichia coli

Optimal conditions for a high cell density fermentation were investigated in a recombinant Escherichia coli producing salmosin, a platelet aggregation inhibitor. The optimized carbon and nitrogen sources were glycerol 10 g/l, yeast extract 30 g/l, and bacto-tryptone 10 g/l, yielding the dry cell weight (DCW) of 10.61 g/l in a 500 ml flask culture. The late-stage induction with 1% L-arabinose in a 5 l jar fermentor showed the highest DCW of 65.70 g/l after 27 h of the fed-batch fermentation. Around 2,200 mg/l of the protein was expressed as an inclusion body that was then refolded to obtain the active salmosin of 96 mg/l. We also confirmed the inhibitory activity against platelet aggregation of the active salmosin from the high cell density fermentation.     

门多萨假单胞菌NK-01合成中长链聚羟基脂肪酸酯的发酵条件优化

为了提高PHA_MCL在门多萨假单胞菌NK-01中的积累,采用单因素实验和正交实验确立了发酵生产PHA_MCL的最佳条件,即以PHA产量为指标的最佳发酵条件为15 g/L葡萄糖浓度、C/N=50、发酵时间48 h,该条件下获得产量0.8 g/L以上的PHA;以PHA占菌体干重百分含量为指标的最佳发酵条件为10 g/L葡萄糖浓度、C/N=60、发酵时间48 h,该条件下获得占菌体干重50%以上的PHA。该研究将为门多萨假单胞菌NK-01用于PHA_MCL的规模化生产提供理论依据。     

表达血管生长抑制素的重组毕赤酵母在诱导阶段混合碳源的流加

为进行高密度发酵并实现外源基因的高表达,在表型为Mut^S的重组毕赤酵母（Pichia pastoris）表达人血管生长抑制素的诱导阶段,采用了甘油甲醇混合补料的培养方式。以溶氧水平作为甘油代谢指针来控制甘油限制性流加既可维持一定菌体生长,又不会发生发酵液中残余甘油及有害代谢产物（乙醇）阻遏蛋白表达。当表达阶段的菌体平均比生长速率控制于0.012h^-1,菌体浓度达150 g/L,血管生长抑制素浓度最高达到108 mg/L,血管生长抑制素的平均比生产速率为0.02 mg/(g·h),菌体关于甘油的表观得率为0.69 g/g,菌体关于甲醇的表观得率为0.93g/g,较没有采用甘油限制性流加时都有所提高。     

Nutrition and bioprocess development for efficient biosynthesis of an antitumor compound from marine-derived fungus

An integrated nutrition and bioprocess strategy was developed for improving the biosynthesis of an antitumor compound, 1403C, by a marine-derived fungus, Halorosellinia sp. (no. 1403). First, statistical design strategies were synthetically applied to optimize the nutritional composition. The resulting 1403C production reached 2.07 g/l, which was 143.5 % higher than the original production. However, it only produced 0.44 g/l of 1403C in 5-l bioreactor fermentation. Thus, the operating parameters including culture pH, dissolved oxygen, agitation speed, impeller type and inoculum level were considered to improve the fermentation process, and an effective control strategy for 1403C production by Halorosellinia sp. submerged in a 5-l bioreactor was established. When inoculating 0.22 g/l dry biomass, controlling dissolved oxygen not lower than 30 % during the growth phase but ranging between 30 and 40 % during the stationary phase, using a double-layer six-flat-blade Rushton disc turbine agitated at 400 rpm, keeping short-term low pH and rapid-rising pH with glucose starvation, the highest 1403C production was finally obtained at 1.32 g/l, which was promoted by 200 % compared to before optimization. Fermentation scale-up was finally performed in a 500-l bioreactor, and 1403C production of 1.09 g/l was obtained.     

Arachidonic acid-rich oil production by Mortierella alpina with different gas distributors

Arachidonic acid (ARA)-rich oil production by Mortierella alpina is a high oxygen demand and shear-sensitive process. In the aerobic fermentation process, oxygen supply is usually a limiting factor owing to the low solubility of oxygen in the fermentation broth. Two kinds of perforated ring gas distributors and a novel microporous ceramic membrane gas distributor were designed and applied to improve oxygen supply. With the decrease of the orifice diameter of perforated ring gas distributors, dry cell weight (DCW), lipids concentration, and ARA content in total fatty acid increased from 17.86 g/L, 7.08 g/L, and 28.08 % to 25.67 g/L, 11.94 g/L, and 36.99 %, respectively. Furthermore, the effect of different dissolved oxygen (DO) on ARA-rich oil production with membrane gas distributor was also studied. The maximum DCW, lipid concentration, and ARA content using membrane gas distributor with DO controlled at 40 % reached 29.67 g/L, 16.74 g/L, and 49.53 %, respectively. The ARA titer increased from 1.99 to 8.29 g/L using the membrane gas distributor to substitute the perforated ring gas distributor. In the further experiment, a novel tubular titanium metal membrane gas distributor was successfully applied in a 7,000 L bioreactor and the results demonstrated that membrane gas distributor was industrially practical.     

Evaluation of feeding strategies in carbon-regulated secondary metabolite production through mathematical modeling

There is now growing evidence that the production of many secondary metabolic by microorganisms is subjected to carbon-catabolite regulation. Even though the exact mode of this regulation is not yet clear, an engineering analysis of the production process is still possible based upon a suitable hypothesis. By way of simulation of penicillin fermentation data obtained from the literature, a mechanistic model involving a substrate inhibition kinetics of product formation has been verified in this paper. Such a model has been found successful not only in predicting simple sugar-feeding strategy, but also a complicated computer guided strategy based upon controlling biomass growth rates in the tropo and idiophases. Using this model, for strategies for sugar feeding into penicillin fermentation have been investigated. These results show that similar penicillin productivities can be obtained using any of these strategies provided fermentations are carried out under optimal conditions corresponding to the strategy chosen. Effect of maximum oxygen transfer capacity of the fermentor under the conditions of fungal growth has been incorporated using an upper limit of biomass concentration on achievement of which the fermentations must be stopped due to serious oxygen limitations. Results of model simulations with such limits throw light upon the way in which different fermentors may behave with respect to product formation.     

Redox potential control and applications in microaerobic and anaerobic fermentations

Many fermentation products are produced under microaerobic or anaerobic conditions, in which oxygen is undetectable by dissolved oxygen probe, presenting a challenge for process monitoring and control. Extracellular redox potentials that can be detected conveniently affect intracellular redox homeostasis and metabolism, and consequently control profiles of fermentation products, which provide an alternative for monitoring and control of these fermentation processes. This article reviews updated progress in the impact of redox potentials on gene expression, protein biosynthesis and metabolism as well as redox potential control strategies for more efficient production of fermentation products, taking ethanol fermentation by the yeast Saccharomyces under microaerobic conditions and butanol production by the bacterium Clostridium under anaerobic conditions as examples.     

L-谷氨酸补料分批发酵的研究

对谷氨酸棒杆菌(Corynebacteriuin glutamicum)HCJ46产L-谷氨酸的补料分批发酵条件进行研究.结果表明:最适初糖质量浓度和最佳残糖维持质量浓度分别为100和(10～20)g/L;对发酵控温方式进行研究,确定了最佳温度控制策略为0～8h维持32℃,8～16h维持34℃、16～32h维持36℃,同时发现相对溶氧控制在30%左右时产酸最高.在以上的优化条件下,L-谷氨酸产量从72g/L提高到95g/L,提高了31.9%.     

假丝酵母发酵玉米芯半纤维素水解液生产木糖醇

采用一株驯化过的假丝酵母(Candida sp.)直接发酵经过简单脱毒处理的玉米芯半纤维素水解液生产木糖醇。确定了水解液的最适浓缩倍数在3.0～3.72的范围内。利用正交实验,确定了摇瓶分批发酵工艺条件的最适组合为：摇床转速180r/min,起始C/N为50,起始pH 5.5,接种量5% (体积比)。在此基础上,重点研究了在发酵罐中通气量对酵母发酵玉米芯水解液生产木糖醇的影响。结果表明采用先高后低的分段通气发酵在木糖醇得率方面明显优于恒定通气发酵;其中,在0～24h,3.75 L/min;24～108h,1.25 L/min的分段通气条件下(装液量为2.5L),木糖醇得率(木糖醇/木糖,g/g) 达到0.75 g/g。该结果将有助于建立一种高效的、大规模的利用玉米芯半纤维素水解液发酵生产木糖醇的工艺。     

过量表达苹果酸脱氢酶对大肠杆菌NZN111产丁二酸的影响

大肠杆菌NZN111是敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸-甲酸裂解酶的编码基因 (pflB) 的工程菌,厌氧条件下由于辅酶NAD(H) 的不平衡导致其丧失了代谢葡萄糖的能力。构建了苹果酸脱氢酶的重组菌大肠杆菌NZN111/pTrc99a-mdh,在厌氧摇瓶发酵过程中通过0.3 mmol/L的IPTG诱导后重组菌的苹果酸脱氢酶 (Malate dehydrogenase,MDH) 酶活较出发菌株提高了14.8倍,NADH/NAD+的比例从0.64下降到0.26,同时NAD+和NADH浓度分别     

Effects of dissolved oxygen concentration and DO-stat feeding strategy on CoQ10 production with Rhizobium radiobacter

The cell growth and CoQ₁₀ (coenzyme Q₁₀) formation of Rhizobium radiobacter WSH2601 were investigated in a 7-1 bioreactor under different dissolved oxygen (DO) concentrations. A maximal CoQ₁₀ content (C/B) of 1.91 mg/g dry cell weight (DCW) and CoQ₁₀ concentration of 32.1 mg/l were obtained at the appropriate DO concentration of 40% (of air saturation). High DO concentration was favourable to the cell growth of Rhizobium radiobacter WSH2601. In order to achieve the maximal yield of CoQ₁₀ production, a new DO-stat feeding strategy was proposed, which significantly improved cell growth and CoQ₁₀ formation. With this strategy, the maximal CoQ₁₀ concentration and DCW reached 51.1 mg/l and 23.9 g/l, respectively, which were 67 and 44.8% higher than those obtained in the batch culture with DO concentration controlled.