抗性突变他克莫司高产菌株的选育

研究利用核糖体工程育种方法结合紫外诱变处理产他克莫司链霉菌SIIA-9818,以期筛选得到发酵水平有较大提高的新菌株。首轮采用链霉素抗性筛选,第二轮组合链霉素和利福平抗性结合紫外诱变育种,进一步巩固育种成效。采用链霉素抗性诱变得到突变株T-122,其气生菌丝丰满程度明显好于原对照株,发酵效价提高22.8%;采用组合链霉素和利福平抗性结合紫外诱变T-122,得到多株高产菌株,其中H-493发酵水平较原出发菌株提高82.6%;在50 L发酵罐通过增大搅拌转速,使菌种H-493发酵单位进一步提高31.0%。本方法简单经济,得到的突变株发酵单位显著提高,组分无明显变化,传代稳定。     

基于自联想神经网络的谷氨酸发酵故障诊断

研究了用自联想神经网络对谷氨酸发酵进行故障诊断。自联想神经网络采用一种带有瓶颈层的特殊结构,且具有单位总增益。在经过大量样本的训练之后,各变量之间能够建立起内在联系。输入信息通过瓶颈层前的压缩及瓶颈层后的解压缩过程,信息中的精华将被提取。应用自联想神经网络对发酵过程变量进行预处理,可以准确及时的进行谷氨酸发酵故障诊断。     

链霉素抗性突变理性筛选新霉素高产菌株

采用化学诱变剂NTG结合链霉素抗性筛选法获得新霉素高产菌株。出发菌株费氏链霉菌(Streptomyces fradiae)FS1109的孢子悬液经不同剂量的化学诱变剂NTG处理后,涂布在含链霉素最小抑制浓度(3μg/m L)的培养基平板上培养,获得大量的链霉素抗性突变株。经影印法初筛和摇瓶发酵复筛,正突变率高于负突变率,获得一株遗传性状稳定的Streptomyces fradiae Str 63菌株,其新霉素生物活性单位比出发菌株提高了50%以上,且C组分较出发菌株的低。     

电场诱导棘孢小单胞菌原生质体融合

以小诺霉素（Micromomicin,MCR)产生菌棘孢小单胞菌（Micromonosporaechinospora）为出发菌株,诱变筛选得到一株链霉素抗性菌株,抗性菌株的原生质体分别用紫外线照射和热处理致死,通过单亲致死原生质体融合,以链霉素为选择条件选出融合株,经摇瓶发酵并结合薄层层析扫描（ThinLayerCharamotography,TLC）分析,筛得4株MCR百分含量高于亲株的融合子,MCR百分含量达到90％,效价1076u／ml,分别比亲株提高15％和11％。     

链霉素发酵过程中的溶氧问题

链霉素产生菌—灰色链霉菌(Streptomyces griseus)是一种高度需氧菌,它在整个代谢过程中以葡萄糖做为主要碳源,只有以氧做为最终电子受体时方能获得大量能量,来满足菌体生长,繁殖和合成链霉素的需要。物质代谢与能量代谢是相辅相成的。据文献记载,空气中,氧在培养液中的饱和浓度(1 atm,25℃)大约只有0.2毫克分子(O_2)/升,而链霉素发酵液中菌体的摄氧率在10～50毫克分子(O_2)/升小时,因此向发酵液中迅速地补充溶解氧,是链霉素发酵中的重要问题。氧分子首先要溶解在水中,然后透过细胞壁和细胞膜进入细胞,它进入细胞内和排出细胞外,是简单的渗透作用,其推动力是培养液和细胞中溶解氧的浓度差。当发酵液中溶氧浓度较低时,进入细胞内的氧就少,当溶氧浓度降到临界值以下时,就不能满足微生物对氧的需求,成为生产中的限制因素。所以研究发酵过程中的溶氧问题是十     

链霉素发酵补料前后某些生物化学方面的变化

在链霉素发酵中,中间补料是极为重要的,补料成分以葡萄糖和无机氮较为适宜。补料后观察出菌丝量稍有增加,更主要是提高了单位菌体的生产能力。菌丝体内含物增生,相应地延缓了菌丝自溶期,蛋白酶活力有所下降。发酵液中氨基葡萄糖含量增加,链霉素发酵单位也显著提高。     

微波诱变结合抗性突变筛选放线菌素D高产菌株

采用微波结合链霉素抗性筛选法选育放线菌素D的高产菌株。通过考察链霉素对Streptomyces rubiginosohelvolus FIM-N31菌株孢子生长情况的影响确定链霉素致死浓度,出发菌株FIM-N31的孢子经微波辐照处理后,涂布在含链霉素致死浓度（50 μg/mL）的培养基平板上培养,获得了大量的链霉素抗性基因突变株。摇瓶发酵筛选突变株,结果获得一株遗传性状稳定的放线菌素D高产菌Str186,其产放线菌素D的能力比出发菌株提高了8倍以上。     

激光诱变玫瑰孢链霉菌结合链霉素抗性筛选法选育达托霉素高产菌株

将经过20 mW激光辐照20 min的达托霉素(Daptomycin)生产菌株-玫瑰孢链霉菌(Streptomyces roseosporus)D-38的孢子悬液倾注在含有1．9λg／mL链霉素的高氏一号培养平板上。通过链霉素抗性法筛选获得了10％正变率的突变株,其中突变株LC-54摇瓶发酵单位为81．2 mg／L,比出发菌株提高了39％。     

南昌霉素高产菌株的链霉素抗性基因突变诱变筛选研究

通过对链霉素对南昌霉素（Nanchangmycin)产生菌NS－41－80菌株孢子的致死浓度测定基础上,采用诱变剂甲基磺酸乙酯（EMS）的不同诱发剂量对菌株孢子进行诱变处理,诱变处理的孢子涂布在含链霉素（10ug/mL)致死浓度的高氏平板上,获得了大量的链霉素抗性基因（str)突变株。然后从3,000株链霉素抗性基因（str)突变株中通过初筛获得比诱变出发菌株产素能力提高20％以上的菌株202株,再进一步通过摇瓶复筛,获得比出发菌株产素能力分别提高100％,200％,300％高产菌株为48株,7株和1株,分别为复筛菌和初筛菌株的23．76％和1．60％,3．46％和0．23％,0．5％和0．03％,将产素能力提高240％以上5个菌株连同出发菌株连续3批次进行摇瓶发酵结果,5个突变株的产素能力均比出发菌株的产素能力提高57％－96．4％,其中突变株80－5．3－165菌株摇瓶发酵单位达6,000ug/mL以上,3批次摇瓶平均发酵单位达5,855ug/mL,建立了南昌霉素高产菌株的链霉素抗性基因突变诱变快速高效的筛选方法。     

抗药性突变肌苷高产菌的选育

以枯草芽孢杆菌(Bacillur subtilis)22为出发菌株,经紫外线诱变,分别获得对链霉素(Sm)、盐酸羟胺(Hc)、叠氮化钠(Sa)有抗性的突变株,产肌苷均明显高于出发株。其中链霉素抗性突变株Sm-37-18产肌苷最高,在最佳发酵条件下,产肌苷达19.49g/L,较出发株提高38.4%,发酵周期由60小时缩短至48小时。     

Comparative metabolomic analysis on industrial continuous and batch ethanol fermentation processes by GC-TOF-MS

The intracellular metabolic profile characterization of Saccharomyces cerevisiae throughout industrial ethanol fermentation was investigated using gas chromatography coupled to time-of-flight mass spectrometry. A total of 143 and 128 intracellular metabolites in S. cerevisiae were detected and quantified in continuous and batch fermentations, respectively. The two fermentation processes were both clearly distinguished into three main phases by principal components analysis. Furthermore, the levels of some metabolites involved in central carbon metabolism varied significantly throughout both processes. Glycerol and phosphoric acid were principally responsible for discriminating seed, main and final phases of continuous fermentation, while lactic acid and glycerol contributed mostly to telling different phases of batch fermentation. In addition, the levels of some amino acids such as glycine varied significantly during both processes. These findings provide new insights into the metabolomic characteristics during industrial ethanol fermentation processes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

拉曼光谱分析技术在资源微生物发酵性能评价中的应用

微生物发酵过程是细胞新陈代谢进行物质转化的过程,为了提高目标产物的转化率,需要对微生物发酵动态特性进行实时分析,以便实时优化发酵过程。拉曼光谱(Raman spectroscopy)量化测试作为一种有应用前景的在线过程分析技术,可以在避免微生物污染的条件下,实现精准监测,进而用于优化控制微生物发酵过程。【目的】以运动发酵单胞菌(Zymomonas mobilis)为例,建立微生物发酵过程中葡萄糖、木糖、乙醇和乳酸浓度拉曼光谱预测模型,并进行准确性验证。【方法】采用浸入式在线拉曼探头,收集运动发酵单胞菌发酵过程中多个组分的拉曼光谱,采用偏最小二乘法对光谱信号进行预处理和多元数据分析,结合离线色谱分析数据,对拉曼光谱进行建模分析和浓度预测。【结果】针对运动发酵单胞菌,首先实现拉曼分析仪对单一产品乙醇发酵过程的精准检测,其次基于多元变量分析,建立葡萄糖、乙醇和乳酸浓度变化的预测模型,实现对发酵过程中各成分浓度变化的准确有效分析。【结论】成功建立了一种评价资源微生物尤其是工业菌株发酵液多种组分的拉曼光谱分析方法。该方法为运动发酵单胞菌等工业菌株利用多组分底物工业化生产不同产物的实时检测,以及其他微生物尤其工业菌株的选育和过程优化提供了新方法。     

Combined use of proteomic analysis and enzyme activity assays for metabolic pathway analysis of glycerol fermentation by Klebsiella pneumoniae

The fed-batch fermentation of glycerol to 1,3-propanediol by Klebsiella pneumoniae displayed an unusual dynamic behavior that can be clearly divided into four distinct phases according to cell growth and CO(2) evolution rate. Metabolism changed significantly during the different phases as reflected by the varied specific rates of substrate consumption and product formation. An assay of activities of the three initial enzymes of glycerol metabolism, namely glycerol dehydratase (GDHt), glycerol dehydrogenase (GDH), and 1,3-propanediol-oxidoreductase (PDOR), showed apparently different patterns of expression. To understand the culture dynamics and patterns of enzyme formation at a more systemic level we analyzed the expression patterns of intracellular proteins of K. pneumoniae from different phases of the fed-batch fermentation using two-dimensional gel electrophoresis (2DE). Two new enzymes, namely a phosphoenolpyruvate-dependent dihydroxyacetone kinase (DHAK II) and a hypothetical oxidoreductase (HOR), which are directly related to glycerol metabolism and 1,3-propanediol formation, were identified among the highly expressed proteins. The changes in expression of these new enzymes and several other proteins identified from the 2DE analysis helped to understand not only the dynamic behavior of the fed-batch fermentation reported in this work but also some previously insufficiently understood phenomena related to this fermentation process. In particular, we demonstrated the combined use of proteomic analysis and enzyme activity assay data for metabolic pathway analysis and for a better identification of targets for bioprocess improvement.     

Detection of phase shifts in batch fermentation via statistical analysis of the online measurements: a case study with rifamycin B fermentation

Industrial production of antibiotics, biopharmaceuticals and enzymes is typically carried out via a batch or fed-batch fermentation process. These processes go through various phases based on sequential substrate uptake, growth and product formation, which require monitoring due to the potential batch-to-batch variability. The phase shifts can be identified directly by measuring the concentrations of substrates and products or by morphological examinations under microscope. However, such measurements are cumbersome to obtain. We present a method to identify phase transitions in batch fermentation using readily available online measurements. Our approach is based on Dynamic Principal Component Analysis (DPCA), a multivariate statistical approach that can model the dynamics of non-stationary processes. Phase-transitions in fermentation produce distinct patterns in the DPCA scores, which can be identified as singular points. We illustrate the application of the method to detect transitions such as the onset of exponential growth phase, substrate exhaustion and substrate switching for rifamycin B fermentation batches. Further, we analyze the loading vectors of DPCA model to illustrate the mechanism by which the statistical model accounts for process dynamics. The approach can be readily applied to other industrially important processes and may have implications in online monitoring of fermentation batches in a production facility.     

Dynamic metabolic flux analysis (DMFA): a framework for determining fluxes at metabolic non-steady state

Metabolic flux analysis (MFA) is a key tool for measuring in vivo metabolic fluxes in systems at metabolic steady state. Here, we present a new method for dynamic metabolic flux analysis (DMFA) of systems that are not at metabolic steady state. The advantages of our DMFA method are: (1) time-series of metabolite concentration data can be applied directly for estimating dynamic fluxes, making data smoothing and estimation of average extracellular rates unnecessary; (2) flux estimation is achieved without integration of ODEs, or iterations; (3) characteristic metabolic phases in the fermentation data are identified automatically by the algorithm, rather than selected manually/arbitrarily. We demonstrate the application of the new DMFA framework in three example systems. First, we evaluated the performance of DMFA in a simple three-reaction model in terms of accuracy, precision and flux observability. Next, we analyzed a commercial glucose-limited fed-batch process for 1,3-propanediol production. The DMFA method accurately captured the dynamic behavior of the fed-batch fermentation and identified characteristic metabolic phases. Lastly, we demonstrate that DMFA can be used without any assumed metabolic network model for data reconciliation and detection of gross measurement errors using carbon and electron balances as constraints.     

On the methodology of dissolved oxygen saturation concentration measurements

Summary An experimental method for the determination of the concentration of dissolved oxygen saturation in real fermentation media is described. It is based on a joint analysis of gas and liquid phases, applying a mass spectrometer for gas analyses and an oxygen electrode for the liquid phase measurements. This method enables the experimental measurement of oxygen solubilities in real fermentation broth within the bioreactor during the process and its application seems to be of general validity.     

Fermentation Studies with Streptomyces griseus: II. Synthetic Media for the Production of Streptomycin

Synthetic media for streptomycin fermentation were studied to determine which media gave highest yields of streptomycin. The effect of salts on streptomycin production by Streptomyces griseus was examined, and a suitable combination of salts was established in a glucose-casein medium. This medium yielded 3,000 μg/ml of the antibiotic with an inoculum of 1.6%. Substitution of amino acids for casein was examined. Of 17 amino acids tested, best results were obtaind with sodium aspartate. Substitution of ammonium salts was tried, and an excellent streptomycin yield was obtained with a medium containing ammonium citrate.     

Purification and characterization of aminoglycoside phosphotransferase APH(6)-Id,a streptomycin-inactivating enzyme

As part of an overall project to characterize the streptomycin phosphotransferase enzyme APH(6)-Id, which confers bacterial resistance to streptomycin, we cloned, expressed, purified, and characterized the enzyme. When expressed in Escherichia coli, the recombinant enzyme increased by up to 70-fold the minimum inhibitory concentration needed to inhibit cell growth. Size-exclusion chromatography gave a molecular mass of 31.4 ± 1.3 kDa for the enzyme, showing that it functions as a monomer. Activity was assayed using three methods: (1) an HPLC-based method that measures the consumption of streptomycin over time; (2) a spectrophotometric method that utilizes a coupled assay; and (3) a radioenzymatic method that detects production of ³²P-labeled streptomycin phosphate. Altogether, the three methods demonstrated that streptomycin was consumed in the APH(6)-Id-catalyzed reaction, ATP was hydrolyzed, and streptomycin phosphate was produced in a substrate-dependent manner, demonstrating that APH(6)-Id is a streptomycin phosphotransferase. Steady-state kinetic analysis gave the following results: K _m(streptomycin) of 0.38 ± 0.13 mM, K _m(ATP) of 1.03 ± 0.1 mM, V _max of 3.2 ± 1.1 μmol/min/mg, and k _cat of 1.7 ± 0.6 s^?1. Our study demonstrates that APH(6)-Id is a bona fide streptomycin phosphotransferase, functions as a monomer, and confers resistance to streptomycin.     

Critical evaluation of sampling techniques for residual glucose determination in carbon-limited chemostat culture of Saccharomyces cerevisiae

In this paper, three sampling techniques for rapid quenching of cellular metabolism and subsequent separation of cells from fermentation broth are compared: (i) quick freezing of fermentation broth directly in liquid nitrogen; (ii) quenching metabolism by exposing the fermentation broth to stainless steel beads (4-mm diameter) in a filter syringe precooled to -18 degrees C; and (iii) withdrawal of the filtrate through a 0.45 microm filter attached to a syringe and a needle inserted directly into the fermentor. It was concluded that use of liquid nitrogen as a quenching method to rapidly arrest cellular metabolism, for quantitative analysis of extracellular glucose, is not a very reliable method and that the filter syringe steel beads work very well.