Construction of shuttle vectors useful for transforming Clostridium acetobutylicum

The symbiotic plasmid (pSym) DNA present in bacteroids of strain RCR1001 of Rhizobium leguminosarum biovar viceae has been compared qualitatively and quantitatively with that present in free living bacteria by hybridization experiments with appropriate probes. A decrease in the relative amount of pSym DNA was observed in bacteroids as compared to bacteria. No rearrangements of the symbiotically expressed pSym borne genes were detected in bacteroids.     

Plasmid transformation of Clostridium perfringens by electroporation methods

Two electroporation methods were compared and modified to improve the frequencies of transfer of plasmid DNA into Clostridium perfringens. A plasmid shuttle vector, pSB92A2, containing chloramphenicol and ampicillin resistance genes and a clostridial origin of replication isolated from a cryptic C. perfringens plasmid, was constructed and successfully introduced into C. perfringens by both electrotransformation methods. Modifications which improved frequencies by 15-28 fold are described and may improve frequencies sufficiently for some vector/host combinations to consider the future use of more direct cloning strategies for the clostridia.     

丙酮丁醇梭菌磷酸化蛋白质组分析

近年的研究揭示,细菌细胞中蛋白质的磷酸化状态可能调节信号或代谢通路的生物活性。丙酮丁醇梭菌是一个重要的工业菌株,在酸性条件下能够生成大量的有机溶剂。然而,调节丙酮丁醇梭菌有机溶剂生成的分子机制尚未完全阐明。采用双向电泳和质谱联用的技术,比较了该菌在产酸期与产有机溶剂期间的差异蛋白质谱图。特别关注了那些分子量接近但具有不同等电点的蛋白质。在高有机溶剂生成速率的丙酮丁醇梭菌中,发现了8个电泳斑点簇呈现明显的酸移而且伴随光密度强度的变化。质谱分析数据表明,这些蛋白质均含有磷酸化修饰的肽段。生物信息学分析预示,这些蛋白质参与了有机溶剂的生成过程。但究竟它们的磷酸化状态如何调控有机溶剂生成仍需更为深入地研究。     

丙酮丁醇梭菌丁醇耐受性

丁醇在发酵培养基中的积累所产生的毒性问题是限制丁醇产量的重要因素,然而对于Clostridium acetobutylicum是如何适应丁醇胁迫,进而调节菌体生长和代谢的,目前尚缺乏系统研究,不能全面揭示C.acetobutylicum的丁醇耐受性机制.对丙酮丁醇梭菌丁醇耐受性有关的研究成果进行了综述,旨在深入理解菌株丁醇耐受性发生改变的相关分子基础.希望为进行微生物丁醇耐受性分子机制的改造、提高菌株的丁醇耐受性提供新的研究思路.     

丙酮丁醇梭菌的遗传操作系统

丙酮丁醇梭菌是极具潜力的替代燃料——生物丁醇的合成菌,受到各国研究者的普遍关注。丙酮丁醇梭菌菌株改造是生物丁醇产业化进程中的一项重要工作,其中遗传操作是核心内容之一。以下对丙酮丁醇梭菌的遗传操作系统的发展历史、种类和原理进行了综述,分析了目前几种遗传操作系统的局限性,并对其发展进行了展望。     

Recognition sequence of a new methyl-specific restriction system from Clostridium acetobutylicum strain ABKn8

A new type II restriction endonuclease, named Cac8I was detected in Clostridium acetobutylicum strain ABKn8. Cac8I cleaved the hexanucleotide sequence [5'-GCN decreases NGC-3'] and generated blunt ends. Up to now no isoschizomer of Cac8I has been described [corrected].     

丙酮丁醇梭菌XY16丁醇发酵特性

以诱变选育的1株突变菌株丙酮丁醇梭菌XY16为对象,对影响该菌发酵特性的相关因素(N源、生长因子、热激)进行研究。结果显示:无机N源乙酸铵比其他N源更有利于丙酮丁醇的发酵,玉米浆或玉米蛋白可以直接替代生长因子进行丙酮丁醇发酵,热激可以提高总溶剂产量,最高可以达到21.28 g/L。该菌还可以同时利用葡萄糖和木糖,当葡萄糖利用完后,木糖才能被有效利用。     

丙酮丁醇梭菌(Clostridium acetobutylicum)原生质体融合育种研究

为了提高丙酮丁醇梭菌(Clostridium acetobutylicum)的丁醇耐受能力和培养基总糖产丁醇的转化率,通过原生质体融合的方法,研究了溶菌酶浓度及其作用时间、再生培养基种类、55℃条件下菌体致死时间、不同PEG分子量以及作用时间、Ca^2+和Mg^2+不同的添加量对丙酮丁醇梭菌原生质体制备、融合、再生的影响,得到了一套比较系统的丙酮丁醇梭菌的原生质体融合条件,同时通过气相色谱检测了融合菌的产溶剂能力并计算总糖转化率。结果显示,最终得到的215I菌株的总糖转化率比原始菌株提高了34.7%,产丁醇能力比原始菌株提高了32.2%,并且发现1株融合菌能产生新物质。原生质体融合方法在丙酸丁醇梭菌育种方面有广泛的应用潜力,通过融合得到的菌株为丁醇生产奠定了基础。     

The improvement of glucose/xylose fermentation by Clostridium acetobutylicum using calcium carbonate

Batch fermentation of 60g/l glucose/xylose mixture by Clostridium acetobutylicum ATCC 824 was investigated on complex culture medium. Different proportions of mixtures, ranged between 10 and 50g of each sugar/l, were fermented during pH control at 4.8 (optimum pH for solventogenesis) or during CaCO₃ addition. Using xylose-pregrown cells and pH control, an important amount of xylose was left over at the end of the fermentation when the glucose concentration was higher than that of xylose. The addition of 10g of CaCO₃/l (to prevent the pH dropping below 4.8) increased xylose uptake: a substantial decrease of residual xylose was observed when xylose-pregrown cells as well as glucose-pregrown cells were used as inoculum for all the mixture proportions studied. MgCO₃ (Mg²⁺-containing compound) and CaCl₂ (Ca²⁺-containing compound) reduced residual xylose only during pH control at 4.8 by NaOH addition. As butanol is the major limiting factor of xylose uptake in C. acetobutylicum, fermentations were carried out with or without CaCO₃ in butanol-containing media or in iron deficient media (under iron limitation, butanol synthesis occurred early and could inhibit xylose uptake). Results showed that an excess of CaCOCaCO₃ could increase butanol tolerance which resulted in an increase in xylose utilization. This positive effect seem to be specific to Ca²⁺- or Mg²⁺-containing compounds, going beyond the buffering effect of carbonate.     

Pervaporative butanol fermentation by Clostridium acetobutylicum B18

Extractive acetone-butanol-ethanol (ABE) fermentation was carried out successfully using pervaporation and a low-acid-producing Clostridium acetobutylicum B18. A pervaporation module with 0.17 m(2) of surface area was made of silicone membrane of 240 mum thickness. Pervaporation experiments using make-up solutions showed that butanol and acetone fluxes increased linearly with their concentrations in the aqueous phase. Fickian diffusion coefficients were constants for fixed air flow rates, and increased at higher sweep air flow rates. During batch and fed-batch fermentations, pervaporation at an air flow rate of 8 L/min removed butanol and acetone efficiently. Butanol concentration was maintained below 4.5 g/L even though Clostridium acetobutylicum B18 produced butanol steadily. Pervaporation could not remove organic acids efficiently, but organic acids did not accumulate because strain B18 produced little organic acid and recycled added organic acids efficiently. With pervaporation, glucose consumption rate increased compared to without pervaporation, and up to 160 g/L of glucose was consumed during 80 h. Cell growth was not inhibited by possible salt accumulation or oxygen diffusion through the silicone tubing. The culture volume was maintained relatively constant during fed-batch operation because of an offsetting effect of water and product removal by pervaporation and addition of nutrient supplements. (c) 1994 John Wiley & Sons, Inc.     

丙酮丁醇发酵菌的分子遗传改造

丙酮丁醇梭菌及拜氏梭菌是重要的ABE（丙酮、丁醇和乙醇）工业生产菌株,其发酵产物中的丙酮和丁醇均为重要的化工原料,汽车发动机试验证明丁醇还是一种性能优于乙醇的极具潜力的生物燃料和燃料添加剂。随着新生物技术的不断发展及工业生产的需求,遗传工程改造不断应用于丙酮丁醇生产菌株。在前人研究及工业实践的基础上,对丙酮丁醇生产菌株的遗传特性及其分子遗传改造取得的进展进行了详细概述。     

In situ butanol recovery from Clostridium acetobutylicum fermentations by expanded bed adsorption

Although butanol is a promising biofuel, its fermentative production suffers from inhibition caused by end product toxicity. The in situ removal of butanol from cultures via expanded bed adsorption offers an effective strategy for mitigating the effects of product toxicity while eliminating the need to clarify cultures via microfiltration. The hydrophobic polymer resin Dowex Optipore L‐493 was found to be both an effective butanol adsorbent and suitable for use in expanded bed adsorption. Recirculation rates through the adsorption column were strongly correlated with and ultimately controlled rates of butanol uptake from the media which, reaching as high as 41.1 g/L h, easily exceed those of its production in a typical fermentation. Vacuum application with vapor collection was found to be an effective means of adsorbent regeneration, with an average of 81% butanol recovery possible, with butanol concentrations in the cold trap reaching as high as 85.8 g/L. Integration of expanded bed adsorption with a fed‐batch Clostridium acetobutylicum ATCC 824 fermentation and its continuous operation for 38.5 h enabled the net production (i.e., in solution and adsorbed) of butanol and total solvent products at up to 27.2 and 40.7 g/L of culture, respectively, representing 2.2‐ and 2.3‐fold improvements over conventional batch culture. While adsorbent biofouling was found to be minimal, further investigation of biofouling in longer‐term studies will provide useful and further insight regarding the robustness of the process strategy. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:68–78, 2014     

丙酮丁醇梭菌发酵菊芋汁生产丁醇

对丙酮丁醇梭菌Clostridium acetobutylicum L7发酵菊芋汁酸水解液生产丁醇进行了初步研究。实验结果表明,以该水解液为底物生产丁醇,不需要添加氮源和生长因子。当水解液初始糖浓度为48.36 g/L时,其发酵性能与以果糖为碳源的对照组基本相同,发酵终点丁醇浓度为8.67 g/L,丁醇、丙酮和乙醇的比例为0.58∶0.36∶0.06,但与以葡萄糖为碳源的对照组相比,发酵时间明显延长,表明该菌株葡萄糖转运能力强于果糖。当水解液初始糖浓度提高到62.87 g/L时,发酵终点残糖浓度从3.09 g/L增加到3.26 g/L,但丁醇浓度却提高到11.21 g/L,丁醇、丙酮和乙醇的比例相应为0.64∶0.29∶0.05,表明适量糖过剩有助于C.acetobutylicum L7胞内代谢从丙酮合成向丁醇合成途径调节;继续提高水解液初始糖浓度,发酵终点残糖浓度迅速升高,丁醇生产的技术经济指标受到明显影响。     

The regulation of thiomethylgalactoside transport in Clostridium acetobutylicum P262 by inducer exclusion and inducer expulsion mechanisms

Abstract Clostridium acetobutylicum P262 had phosphotransferase systems for glucose and lactose, and the lactose system was inducible. When C. acetobutylicum P262 was provided with glucose and lactose, the cultures grew in a diauxic fashion, and glucose was used preferentially. Cells grown on lactose took up thiomethylgalactoside, and retained this non-metabolizable lactose analog for long periods of time. Because glucose inhibited thiomethylgalactoside uptake and caused the efflux of thiomethylgalactoside that had already been taken up, it appeared that C. acetobutylicum P262 had inducer exclusion and inducer expulsion mechanisms similar to those found in lactic acid bacteria.     

Induction of acetoacetate decarboxylase in Clostridium acetobutylicum

Abstract Factors that may initiate the biosynthesis of acetoacetate decarboxylase were investigated in resting cells of Clostridium acetobutylicum . Linear acids from C₁ to C₄ were inducers, whereas branched acids and linear acids from C₅ to C₇ were not inducers of acetoacetate decarboxylase biosynthesis. Induction of acetoacetate decarboxylase was maximal at pH 4.8 in the presence of acid concentrations comparable with those found during fermentation. In growth conditions repression of acetoacetate decarboxylase biosynthesis was found. This fact explains that acetone production by Clostridium acetobutylicum occurs when growth slows down.     

玉米黄浆水在丙酮-丁醇厌氧梭菌发酵中的应用

为降低丙酮-丁醇厌氧梭菌发酵生产丁醇的成本,研究了不同添加量玉米黄浆水对发酵的影响。与葡萄糖培养基相比,在发酵培养基中添加少量玉米黄浆水对发酵产量无显著影响。当添加体积分数为25％的玉米黄浆水时,丙酮、丁醇和乙醇的最终质量浓度分别是0．31、2．70和8．00g/L,总溶剂量为11．01g／L。通过成本核算,每生产1kg溶剂,添加体积分数25％的玉米黄浆水可比葡萄糖培养基节约成本2．11元。