灰树花菌丝体不同培养时期代谢组学分析

为了解不同生长时期灰树花(Grifola frondosa)菌丝体的代谢产物差异及其通路,该研究采用HPLC-MS/MS分析方法对培养10、20、30 d的灰树花菌丝体进行分析。结果表明:(1)共42类584种代谢物被鉴定出,其中159种、47种和165种代谢物在对照组(10 d vs 20 d、20 d vs 30 d、10 d vs 30 d)中表现出不同的积累模式,不同培养时间的代谢物成分差异显著。(2)培养10 d产生较多与促进菌丝体生长和氧化供能有关的物质,培养20 d产生或积累了多种对人体有益的次生代谢物,如橄榄苦甙、甘胆酸、N-甲基酪胺、Alprazolam,培养30 d菌丝体中含有多种与产生香气有关的物质。(3)KEGG代谢通路富集分析,10 d vs 20 d比较组、20 d vs 30 d比较组和10 d vs 30 d比较组分别富集到163条、81条、137条代谢通路,氨基酸代谢在菌丝体不同培养时间中的影响最大。该研究初步探索了灰树花菌丝体的差异代谢物及代谢通路,并发现不同培养时间灰树花菌丝体代谢产物具有明显的差异,以及菌丝体中部分成分含量与培养时间有关,对灰树花菌丝体的质量控制和机理研究具有一定的参考价值。     

离体培养生产次级代谢产物

根茎共培养生产高水平茛菪胺颠茄（Ａｔｒａｐａｂｅｌｌａｄｏｎｎａ）和Ｄｕｂｏｉｓｉａｓｐｐ．产生的茛菪烷生物碱阿托品、天仙子胺和茛菪胺具有广谱的药理学效应。全世界茛菪烷销售量估计比阿托品和天仙子胶总和的１０倍还高。在植物中,天仙子胺主要由根产生,然后被转运到植物的气生部分,在那里由６－羟化酶催化转变为茛菪胺。发根土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｒｈｉｚｏｇｅｎｅｓ）－转化的毛根培养物可产生能与完整植株产量相比的天仙子胺,但仅有非常少量的Ａ茛菪胺。在澳大利亚新南威尔士大学和Ｍｏｎａｓｈ大学,Ａ…     

提高植物细胞培养法生产次级代谢物产量的方法

介绍了提高植物细胞培养法生产次级代谢物中需要考虑的几个问题,如外植体的选择,高产细胞系的选育,培养条件的优化,培养技术的选择,前体物的添加,诱导剂和抑制剂的使用。     

提高植物培养细胞中次级代谢产物含量的途径

利用植物细胞培养的方法可以取代人工栽培、天然采集或人工合成的方法大量生产很多具有价值的产物(如药品、农药、香料、色素及食品添加剂等)。目前,利用紫草(Lithospermum erythrochizon)培养细胞生产紫草素,利用人参(Panax ginseng)根培养物生产食品添加剂等已进入商业市场。另外,利用黄连(Coptisjaponica)培养细胞生产小蘗碱、利用长春花(Catharathus roseus)培养细胞生产蛇根碱及阿吗碱和利用毛地黄(Digitalis lanata)培养细胞生产地高辛等亦都进行了工业化生产。尽管这样,由于植物培养细胞亦存在着一些缺点如很多培养物中代谢物含量很低或不存在,生长速度缓慢加上目前发酵成本过高、很多关键性问题未解决等等,使之未能广泛地应用于工业化生产。近年来,为了解决上述诸问题特别是解决培养物中代谢物含量低的问题,各国科研人员进行了不懈的努力,取得了长足的进展。本文着重对这方面近年来采用的新方法及新技术的研究作一概述。     

分批补料培养对L-异亮氨酸发酵的影响

以钝齿棒杆菌(Corynebacterium crenatum Met-,Bio-,AECr,AHVr)的分批补料技术进行L-异亮氨酸(L-ILE)发酵调控的研究.采用间歇恒速补料方式,当初糖浓度由7%下降至1%～2%,补糖为23g/L·h,整个发酵过程糖浓度一直维持在2%,μ、x值都提高,qp为最大;L-亮氨酸产率达17.4g/L,产酸率提高21%,结果明显优于分批培养.     

培养时间对蛹拟青霉(Paecilomyces militaris)菌丝体中代谢产物的影响

[目的]通过测定不同培养时间蛹拟青霉(Paecilomyces militaris)菌丝体中次级代谢产物的变化,分析蛹拟青霉次级代谢产物与培养时间之间的关系.[方法]液体种子接入SDAY固体培养基 ;培养温度为25℃,培养周期为9d,从第2天开始每天取样 ;用甲醇与乙酸乙酯分别提取菌丝体中次级代谢产物,离心、过滤后合并提取液,用液质联用仪进行分析,用MetaboAnalyst software软件进行数据采集分析.[结果]主成分分析结果表明供试菌株在不同培养时间内其菌丝中次级代谢产物差异显著.聚类分析结果显示,供试拟青霉对生物碱、肽类和核苷等易形成阳离子类物质的代谢物可分为前中后三个阶段.供试拟青霉对糖类和有机酸等易形成阴离子类物质的代谢主要分为前后两个阶段.差异代谢物及热图分析结果表明,在培养的第2和第3天含量显著增加的代谢产物种类较多,主要有酯类及其水解产物、细胞破坏素B和拟青霉素,以及多种尚未鉴定的含氮化合物等 ;在培养第4和第5天含量显著增加的差异代谢物质种类较少,主要有细胞破坏素A和团囊虫草素等肽类抗菌杀虫物质 ;在培养第6天至第9天含量显著增加的代谢物种类较多,除多种白僵菌交酯和细胞破坏素等肽类抗生素外,显著增加的还有多种脂肪酸、氨基酸、鼠李糖、海藻糖、脑苷脂类化合物和核黄素等物质.[结论]培养时间对蛹拟青霉菌丝中次级代谢产物的产生有显著影响.在培养初期,菌体中酯类及细胞破坏素B和拟青霉素等含氮化合物的合成旺盛.在培养中期,次生代谢物明显减少,但细胞破坏素A2和团囊虫草素等肽类抗生素的合成却仍显著增多.在培养后期,供试蛹拟青霉除代谢出多种白僵菌交酯等肽类抗生素外,还大量产生有机酸、氨基酸和海藻糖等物质.虽然在整个培养过程中都有肽类抗生素产生,但这些抗生素并不相同,同时后一阶段新的抗生素的产生常常伴随着前一阶段的抗生素减少,因此前期的抗生素可能是后期产生的抗生素的前体.     

放线菌中腺苷三磷酸结合盒转运蛋白的研究进展

放线菌由于能产生多种结构新颖、活性独特的次级代谢产物,在医药工业、农业和环境保护上具有重要作用。全基因组测序的数据显示,放线菌中含有数目众多的腺苷三磷酸结合盒(ATP-binding cassette,ABC)转运蛋白基因,在营养摄入、次级代谢产物转运、外源毒素解毒等一系列过程中发挥着重要的作用。本文概述了ABC转运蛋白的结构和作用机制,并结合本实验室的研究工作,对近年来放线菌中ABC转运蛋白的研究进展进行了比较全面的综述,着重介绍了负责次级代谢产物跨膜转运的ABC外排蛋白,并展望了放线菌ABC转运蛋白的研究热点和应用前景。     

假蜜环菌液体种子培养条件的优化及其应用研究

目前国内多采用固体菌种进行假蜜环菌的发酵生产。在假蜜环菌液体种子的培养及培养条件优化等方面进行了探讨,优化条件为:玉米淀粉为较适宜碳源;酵母浸出粉为较适宜氮源;添加KH2PO4、吐温-80对菌丝生长有一定的促进作用。正交试验结果显示,适合假蜜环菌菌丝体生长的培养基配方为玉米淀粉30 g/L,酵母浸出粉3g/L,KH2PO41.5 g/L。此外,还探讨了液体种子与传统的固体种子对固体发酵产物的影响,结果表明,接种液体种子能提高固体发酵的主要代谢产物的产量。该实验为改善假蜜环菌现行的生产工艺提供参考。     

补料方式对酵母菌生产谷胱甘肽的影响

比较了酵母菌发酵生产谷胱甘肽（GSH）的几种补料分批培养方式。实验发现补料可以明显地促进酵母菌的生长和谷胱甘肽的合成,同时还发现不同的补料方式对发酵液中的菌体浓度和GSH浓度有不同的影响。采用指数流加方式可获得极高的菌体浓度,但菌体中的GSH浓度较低;而采用恒－pH补料分批培养既可以达到较高菌体浓度,菌体中又含有较高的GSH含量,因此,其总的GSH产量最高,可达到977.8mg/L。     

赖氨酸流加发酵最优控制的研究

在赖氨酸发酵动力学模型和庞特里金明小值原理的基础上,得出流加发酵的最优化底物流加方式。并进一步对流加发酵的全过程进行了分析,得出了在实际控制中较为可行的流加发酵全过程的总控制策略,实际控制表明在小型反应器中赖氨酸产生菌FB42的发酵水平为81．6g／l。、转化率为0．418％、生产强度为1．16g／h·L,和分批发酵相比分别提高了45．4％、9．7％和28．4％。     

Fed-batch fermentation of Lactobacillus lactis for hyper-production of l-lactic acid

A fed-batch fermentation of Lactobacillus lactis to produce l-lactic acid was developed in which the residual glucose concentration in the culture was used to control a continuous feeding strategy. Up to 210 g l-lactic acid l^–1 (97% yield) was obtained. The maximal dry cell was 2.7 g l^–1 and the average l-lactic acid productivity was 2.2 g l^–1 h^–1.     

Fed-batch fermentation for production of nitrile hydratase byRhodococcus rhodochrous M33

To enhance the productivity and activity of nitrile hydratase inRhodococcus rhodochrous M33, a glucose-limited fed-batch culture was performed. In a fed-batch culture where the glucose was controlled at a limited level and cobalt was supplemented during the fermentation period, the cell mass and total activity of nitrile hydratase both increased 3.3-fold compared to that in the batch fermentation. The productivity of nitrile hydratase also increased 1.9-fold compared to that in the batch fermentation. The specific activity of nitrile hydratase in the whole cell preparation when using a fed-batch culture was 120 units/mg-DCW, which was similar to that in the batch culture.     

Self-cycling fermentation in a stirred tank reactor

Self-cycling fermentations (SCFs) were conducted in a stirred tank apparatus using Bacillus subtilis and Acinetobacter calcoaceticus. The systems were very stable and the experiments lasted through many cycles. The variation of parameters such as biomass and doubling time from cycle to cycle was small. The stirred tank reactor (STR) allowed a much better control of the working volume in the fermentor from cycle to cycle, compared to the cyclone column, and it was not necessary to make periodic corrections.The production of surfactin from B. subtilis was achieved without extending the cycle time. The harvested broth at the end of each cycle was allowed to remain in a secondary vessel, at ambient temperature, before being collected. It is exhaustion of the limiting nutrient which causes an increase in dissolved oxygen (DO). At this point, the computer, which constantly monitors the DO, triggered the harvesting sequence to end the cycle. Thus, the mature culture in the secondary vessel experienced appropriate conditions for the production of the secondary metabolite. Meanwhile, the next batch of cells was being grown in the primary reactor.The response of a gas analyzer on the effluent paralleled that of the DO measurements in the fermentor. These data for oxygen and carbon dioxide exhibited less noise than the DO readings. Either would be a more reliable parameter for feedback control of the SCF because the problem of fouling of the DO probe after extended runs of many cycles would be eliminated. (c) 1993 John Wiley & Sons, Inc.     

Fed-batch high-cell-density fermentation strategies for Pichia pastoris growth and production

Pichia pastoris is extensively used to produce various heterologous proteins. Amounts of biopharmaceutical drugs and industrial enzymes have been successfully produced by fed-batch high-cell-density fermentation (HCDF) of this cell factory. High levels of cell mass in defined media can be easily achieved and therefore large quantities of recombinant proteins with enhanced activities and lower costs can be obtained through HCDF technology. A robust HCDF process makes a successful transition to commercial production. Recently, efforts have been made to increase the heterologous protein production and activity by the HCDF of P. pastoris. However, challenges around selecting a suitable HCDF strategy exist. The high-level expression of a specific protein in P. pastoris is still, at least in part, limited by optimizing the methanol feeding strategy. Here, we review the progress in developments and applications of P. pastoris HCDF strategies for enhanced expression of recombinant proteins. We focus on the methanol induction strategies for efficient fed-batch HCDF in bioreactors, mainly focusing on various stat-induction strategies, co-feeding, and the limited induction strategy. These processes control strategies have opened the door for expressing foreign proteins in P. pastoris and are expected to enhance the production of recombinant proteins.     

Optimization of fed-batch fermentation for xylitol production by Candida tropicalis

Xylitol, a functional sweetener, was produced from xylose by biological conversion using Candida tropicalis ATCC 13803. Based on a two-substrate fermentation using glucose for cell growth and xylose for xylitol production, fed-batch fermentations were undertaken to increase the final xylitol concentration. The effects of xylose and xylitol on xylitol production rate were studied to determine the optimum concentrations for fed-batch fermentation. Xylose concentration in the medium (100 g l⁻¹) and less than 200 g l⁻¹ total xylose plus xylitol concentration were determined as optimum for maximum xylitol production rate and xylitol yield. Increasing the concentrations of xylose and xylitol decreased the rate and yield of xylitol production and the specific cell growth rate, probably because of an increase in osmotic stress that would interfere with xylose transport, xylitol flux to secretion to cell metabolism. The feeding rate of xylose solution during the fed-batch mode of operation was determined by using the mass balance equations and kinetic parameters involved in the equations in order to increase final xylitol concentration without affecting xylitol and productivity. The optimized fed-batch fermentation resulted in 187 g l⁻¹ xylitol concentration, 0.75 g xylitol g xylose⁻¹ xylitol yield and 3.9 g xylitol l⁻¹ h⁻¹ volumetric productivity. Journal of Industrial Microbiology & Biotechnology (2002) 29, 16–19 doi:10.1038/sj.jim.7000257 Received 15 October 2001/ Accepted in revised form 30 March 2002     

High cell density fed-batch fermentation for the production of a microbial lipase

Extracellular lipase of the yeast Candida rugosa was produced via high cell density fed-batch fermentations using palm oil as the sole source of carbon and energy. Feeding strategies consisted of a pH-stat operation, foaming-dependent control and specific growth rate control in different experiments. Compared to foaming-dependent feeding and the pH-stat operation, the specific growth rate control of feeding proved to be the most successful. At the specific growth rate control set at 0.05 h⁻¹, the final lipase activity in the culture broth was the highest at ∼700 U L⁻¹. This was 2.6-fold higher than the final enzyme activity obtained at a specific growth rate control set at 0.15 h⁻¹. The peak enzyme concentration achieved using the best foaming-dependent control of feeding was around 28% of the peak activity attained using the specific growth rate control of feeding at 0.05 h⁻¹. Similarly, the peak enzyme concentration attained using the pH-stat feeding operation was a mere 9% of the peak activity attained by specific growth rate control of feeding at a set-point of 0.05 h⁻¹. Fed-batch fermentations were performed in a 2 L stirred-tank bioreactor (30 °C, pH 7) with the dissolved oxygen level controlled at 30% of air saturation.     

腈水解酶psn基因工程菌的发酵及产酶条件优化

来自恶臭假单胞菌的腈水解酶具有高效催化3-氰基吡啶产烟酸的能力,对表达该酶的基因psn进行发酵和产酶条件优化,通过对C源、N源、磷酸盐、金属离子、温度、诱导剂浓度和诱导时间进行单因素考察,获得最适培养基条件(g/L):葡萄糖5、蛋白胨15、酵母粉5、(NH4)2SO45、K2HPO424.5、KH2PO45.76、MgSO40.48;最佳诱导条件:培养2.5 h后添加IPTG诱导,浓度0.2 mmol/L,诱导温度30℃。在该条件下培养,重组大肠杆菌的腈水解酶比酶活可达到45.67 U/mL,比优化前提高了2.26倍。在此基础上,于5 L发酵罐上进行C、N源的补料研究,获得最适分批补料策略,发现其腈水解酶活力可达到75.40 U/mL,是优化前的3.74倍。     

Fed-batch sorbose fermentation using pulse and multiple feeding strategies for productivity improvement

Microbial oxidation of D-sorbitol tol-sorbose byAcetobacter suboxydans is of commercial importance since it is the only biochemical process in vitamin C synthesis. The main bottleneck in the batch oxidation of sorbitol to sorbose is that the process is severely inhibited by sorbitol. Suitable fed-batch fermentation designs can eliminate the inherent substrate inhibition and improve sorbose productivity. Fed-batch sorbose fermentations were conducted by using two nutrient feeding strategies. For fed-batch fermentation with pulse feeding highly concentrated sorbitol (600 g/L) along with other nutrients were fed intermittently in four pulses of 0.5 liter in response to the increased DO signal. The fed-batch fermentation was over in 24 h with a sorbose productivity of 13.40 g/L/h and a final sorbose concentration of 320.48 g/L. On the other hand, in fed-batch fermentation with multiple feeds, two pulse feeds of 0.5 liter nutrient medium containing 600 g/L sorbitol was followed by the addition of 1.5 liter nutrient medium containing 600 g/L sorbitol at a constant feed rate of 0.36 L/h till the full working capacity of the reactor. The fermentation was completed in 24 h with an enhanced sorbose productivity of 15.09 g/L/h and a sorbose concentration of 332.60 g/L. The sorbose concentration and productivity obtained by multiple feeding of nutrients was found to be higher than that obtained by pulse feeding and was therefore a better strategy for fed-batch sorbose fermentation.     

冬虫夏草固态培养菌丝中纤溶酶的纯化和酶学性质

【背景】血栓性疾病发病率逐年递增,发病人群呈现低龄化趋势,严重地影响着人们的身心健康。因此研发高效、安全、特异性强的溶栓药物具有重要的意义,是血栓性疾病预防与治疗研究领域的热点。【目的】对冬虫夏草菌丝固态培养过程中产生的纤溶酶进行分离纯化,并对纯化的纤溶酶进行酶学性质分析。【方法】通过硫酸铵盐析、阳离子交换色谱和Superdex 75凝胶过滤色谱分离纯化虫草纤溶酶。采用Bradford法测定样品中总蛋白质浓度,纤维蛋白平板法测定纤溶酶活性,Native-PAGE检测纯度,SDS-PAGE测定相对分子量。【结果】在固态培养中冬虫夏草菌丝可以产生至少两种纤溶酶,分别命名为OSP-1和OSP-2。纯化后OSP-1比活力达到4186.25U/mg,纯化倍数为41.69倍。OSP-1由两个亚基构成,相对分子量分别为27.60k D和23.83k D,是一种丝氨蛋白酶。酶学性质分析表明,该酶最适作用温度为40°C,最适pH为4.0。Cu2+可促进OSP-1酶活,而Zn2+会抑制酶活。除了具有较高的纤溶酶活性,OSP-1还可发挥激活纤维蛋白酶原的作用。在水解纤维蛋白原的过程中,该酶可依次降解γ、Aα、Bβ链。【结论】研究发现的OSP-1具有开发成新型溶栓药物的潜力。