产琥珀酸放线杆菌发酵生产琥珀酸的研究进展

近年来,因瘤胃微生物产琥珀酸放线杆菌Actinobacillus succinogenes具有高的琥珀酸产量,并能够利用多种碳源进行发酵等优点,在利用发酵法生产琥珀酸领域具有广泛的应用前景和商业化价值,因而其代谢途径和发酵工艺等基础研究成为国内外研发的热点。近年来,人们在产琥珀酸放线杆菌的代谢途径、琥珀酸发酵动力学模型、新型经济培养基以及高产菌株选育等方面的研究取得了很大进展,对研发琥珀酸发酵工艺、降低生产成本和节能减耗等具有重要的理论意义。     

产琥珀酸放线杆菌的原生质体制备与再生

基因组重组技术是一项重要的菌种改造技术,原生质体制备和再生是进行基因组重组的前提和基础。目前少有关于产琥珀酸放线杆菌(Actinobacillus succinogenes)CGMCC2650原生质体研究的报道。为了优化该菌的原生质体制备和再生条件,及利用基因组重组技术构建优良菌种提供参考,研究了甘氨酸预处理,菌龄,酶浓度,作用时间,温度对产琥珀酸放线杆菌原生质体制备和再生的影响,并考察了不同渗透压稳定剂对其再生的影响。结果表明,菌体在添加了0.6mg/ml甘氨酸的TSB培养基中培养5h后收集,用SMM稀释到OD660=1.0,用0.025mg/ml溶菌酶在37℃下酶解45min制备原生质体,将原生质体涂布于含0.3mol/L蔗糖的再生培养基中,再生率最大,达到40.9%。确定了产琥珀酸放线杆菌原生质体制备和再生的最佳条件,所用的原生质体制备的方法对琥珀酸的产生没有影响,这为进一步开展该菌的原生质体诱变及基因组重组等研究奠定了基础。     

超高静压在琥珀酸生产菌株选育中的应用

采用超高静压对一株产琥珀酸放线杆菌A3进行诱变育种, 进一步提高其生产性能。考察了压力、变压速度、生长期对菌株致死率的影响。在压力为200 MPa, 50 MPa/min变压速度的诱变条件下对稳定期菌株进行诱变, 筛选到一株突变菌株产琥珀酸放线杆菌B19, 琥珀酸产量达到 32.2 g/L, 比出发菌株A3提高了17.9%, 代谢副产物乙酸的产量降低了11.5%, 经过6次传代表明突变菌株具有稳定的遗传特性。     

最新专利文摘

CN1814747:一种微生物发酵生产丁二酸的菌种和方法本专利具体涉及一种发酵糖质原料产生丁二酸的微生物琥珀酸放线杆菌(Actinobacillus succinogenes)SW 0580,保藏号CGMCC No·1593,以及利用该微生物发酵生产丁二酸的方法。该微生物是从瘤胃中分离并鉴定的琥珀酸放线杆菌(Actino     

一株丁二酸高产菌株的筛选和鉴定

从瘤胃中筛选到一株高产丁二酸生产菌株,为革兰氏阴性菌,短杆状,无芽孢,不运动,兼性厌氧.经形态学、生理生化鉴定和基于16S rRNA序列的系统发育分析,该菌株为巴斯德菌科的产琥珀酸放线杆菌,是产琥珀酸放线杆菌CCUG 43843的变种,二者的序列相似性为99.93%,命名为产琥珀酸放线杆菌A3.5L发酵罐分批发酵实验表明,当发酵培养基中葡萄糖浓度为50g/L时,产琥珀酸放线杆菌A3可以产25.8g/L丁二酸,具有较好的丁二酸生产潜力.     

谷氨酸棒杆菌生产琥珀酸的代谢工程研究进展

琥珀酸是一种具有重要应用价值的四碳平台化合物。微生物法发酵生产琥珀酸以其社会、环境和经济优势展现出良好的发展前景。谷氨酸棒杆菌被广泛应用于氨基酸、核苷酸等高附加值化学品的工业化生产,在厌氧条件下细胞处于生长停滞状态,但仍能高效利用碳源合成有机酸,通过代谢工程改造的谷氨酸棒杆菌有望成为理想的琥珀酸生产菌株。结合近年来谷氨酸棒杆菌生产琥珀酸取得的最新成果,本文综述了构建高产琥珀酸工程菌株的代谢工程策略、底物的扩展利用,并展望了将来的研究方向。     

琥珀酸：对能量稳态调节具有多效功能的代谢物

琥珀酸是连接三羧酸循环和线粒体呼吸链的重要中间产物，同时也是某些肠道共生菌的重要代谢产物。线粒体和肠道菌来源的琥珀酸均可进入机体循环系统，导致该有机酸水平在生理和病理状态下产生波动。尽管长期以来琥珀酸被认为是一种与炎症相关的危险信号分子，但其还在能量稳态调节中显示多效作用。琥珀酸的多效性除与其来源有关外，还与其作用途径密切相关。本文从琥珀酸的来源、参与能量稳态调节的途径及其与代谢紊乱性疾病间的关系三个方面进行重点论述，以期为全面了解琥珀酸的功能及靶向琥珀酸途径进行代谢性疾病的防治提供重要信息。     

大肠杆菌产琥珀酸基因工程研究进展

生物合成琥珀酸摆脱了对不可再生战略资源石油的依赖,以其社会、经济和环境效益展现出良好的发展前景。野生型大肠杆菌的琥珀酸生产强度难以满足生物合成琥珀酸工业化的要求,但遗传背景清楚,容易改造。近年来,人们深入研究了大肠杆菌的琥珀酸代谢途径,通过强化大肠杆菌琥珀酸合成途径、抑制琥珀酸旁路代谢途径、构建产琥珀酸乙醛酸循环和有氧生产体系等多种基因工程策略,对大肠杆菌进行菌株改造和代谢进化筛选,提高了琥珀酸产量。综述了大肠杆菌产琥珀酸的基因工程研究进展。     

基于代谢通量分析的琥珀酸放线杆菌高产选育研究

在对产琥珀酸放线杆菌代谢分析的基础上选育出高产突变株对琥珀酸的工业生物转化有重要意义.在矩阵分析代谢通量基础上,围绕柔性节点下的副产物乙酸及乙醇的降低分别实施软X诱变及定点突变选育,并对比分析了突变株与出发株相关酶活及基因序列变化.针对出发株的流量分析显示产物琥珀酸的代谢通量为1.78(mmol/g/h),主要副产物乙酸与乙醇的代谢通量分别为(0.60mmol/g/h)和(1.04 mmol/g/h),并发现乙醇代谢加剧了琥珀酸合成中的H电子供体的不足;筛选出的氟乙酸抗性突变株S.JST1的乙酸代谢通量降低了96%,为0.024(mmol/g/h),酶活检测表明磷酸乙酰转移酶(Pta)的酶比活力从602降低到74,进一步的序列对比分析发现pta突变基因中产生了一个突变位点:adh定点复合突变株S.JST2的乙醇代谢通量降低了98%,为0.020(mmol/g/h),酶活检测表明Adh的酶比活力从585降低到62.最终突变株S.JST2琥珀酸累积产量达65.7 g/L.围绕产琥珀酸放线杆菌Pta及Adh酶活的降低实施定向选育,在降低副产物流量的同时,有助于改善细胞H供体代谢平衡进而提高琥珀酸的流量.所获突变株具有工业应用潜力.     

帝斯曼公司与罗盖特公司将共同建设生物琥珀酸中试装置

2008年1月,荷兰帝斯曼（DSM）公司和法国罗盖特（Roquette）公司宣布共同开发发酵法可再生琥珀酸生产技术并实现商业化生产。DSM公司是荷兰的一家以生命科学为重点的综合性特殊化学制品公司,年销售额约为90亿欧元。Roquette公司是法国的一家以生产淀粉及其诱导体而著称的公司．是世界知名的糖酒精生产厂家,年销售额约为20亿欧元。     

Use of Cheese Whey for Vitamin B12 Production: III. Growth Studies and Dry-Weight Activity1

The patterns of growth and vitamin formation by Propionibacterium shermanii in whey were similar to the patterns established in other substrates. The vitamin formation was observed during the latter part of the fermentation after the organism approached maximal growth. Lactose utilization by the organism corresponded to the logarithmic-growth phase of the organism. Analyses of the dried culture showed a large increase of vitamin B(12) in the fermentation solids compared with unfermented dried whey. A feed analysis showed a notable increase of protein and a large decrease in nitrogen-free extract of the dried fermentation solid compared with dried whey.     

Stimulatory Effect of Malo-Lactic Fermentation on the Growth Rate of Leuconostoc oenos

Although l-malic acid is not an energy source for the malo-lactic organism Leuconostoc oenos (L. citrovorum) ML 34, the growth rate of the organism was found to be greatly increased by malo-lactic fermentation (the decarboxylation of l-malic acid to l-lactic acid). The stimulation was especially striking at the low pH (below pH 4) of wine, the natural habitat of this bacterium. The stimulation of growth did not result from changes in pH that accompany malo-lactic fermentation. Thus, these results suggest a biological function of malo-lactic fermentation.     

A new method for studying microaerobic fermentations. I. A theoretical analysis of oxygen programmed fermentation

An experimental method for studying microaerobic fermentation, called oxygen programmed fermentation, is introduced. The method if based on a chemostat. The mathematical equations governing the dynamics of the system are derived and simulations are made for two principally different cases: a purely respirative organism, and an organism capable of fermentation during oxygen limitation. It is shown that at a suitably chosen ramp rate, the dissolved oxygen concentration in the broth can be made to decrease almost linearly. It is suggested that the greatest use of oxygen programmed fermentation will be in initial experiments. Compared with chemostat studies, a scan of different oxygenation rates will provide a time-saving method of finding the interacting regions for metabolic transitions. Furthermore it is shown that the methods makes it possible to study cell physiology at condition which would normally lead to washout. (c) 1994 John Wiley & Sons, Inc.     

Isolation of isoflavones from soy-based fermentations of the erythromycin-producing bacterium Saccharopolyspora erythraea

A search for an abundant and economical source of isoflavones, particularly genistein, led to the discovery that the erythromycin-producing organism Saccharopolyspora erythraea also produces this promising new cancer-prevention agent. Erythromycin fermentation is a large-scale, soybean-based process used world-wide for the commercial production of this medically important antibiotic. Results from this study indicate that genistin (the glucoside form of genistein), which is added to the fermentation in the soybean media, was converted to genistein through the action of a β-glucosidase produced by the organism. Genistein was co-extracted with erythromycin from the fermentation broth, then separated from erythromycin during the second step of the purification process for the production of erythromycin. Received 10 September 1996 / Received revision: 22 November 1996 / Accepted: 7 December 1996     

Transformation of 1-Menthene by a Cladosporium: Accumulation of beta-Isopropyl Glutaric Acid in the Growth Medium

An organism isolated from soil samples collected near a terpene plant grew in the presence of 1-menthene, with no other major source of carbon and energy. The organism was tentatively identified as a member of the genus Cladosporium. When this organism was grown in the presence of 1-menthene, with no other major source of carbon, substantial quantities of β-isopropyl glutaric acid accumulated in the fermentation medium. β-Isopropyl glutaric acid is probably an end product of the degradation of 1-menthene by the organism.     

Product inhibition and its removal on josamycin fermentation by Streptomyces narbonensis var. josamyceticus

Josamycin formation was shown to be closely associated with the degree of mycelial growth. Media using glycerol or glucose were slight in josamycin accumulation. However, the mycelial growth and product formation were interrupted in the course of the fermentation. The self-resistance level of the producing organism against josamycin was shown to be not higher than 1 mg per ml. In media using fatty oil, the organism grew smoothly and gave a concentration of josamycin at least 5 times higher than the self-resistance levelMost of the josamycin was distributed in the oily phase of the broth. The josamycin concentration of the water phase of the broth was less than the self-resistance level, except in the later period of the fermentation. Unsaturated fatty acids remaining in the broth were found to be the best extractors of josamycin. It was concluded that the enhanced josamycin accumulation of this fermentation resulted from the removal of the product inhibition.     

Transformation of 1-Menthene by a Cladosporium: Accumulation of β-Isopropyl Glutaric Acid in the Growth Medium

An organism isolated from soil samples collected near a terpene plant grew in the presence of 1-menthene, with no other major source of carbon and energy. The organism was tentatively identified as a member of the genus Cladosporium. When this organism was grown in the presence of 1-menthene, with no other major source of carbon, substantial quantities of β-isopropyl glutaric acid accumulated in the fermentation medium. β-Isopropyl glutaric acid is probably an end product of the degradation of 1-menthene by the organism.     

Optimization of protease production from Bacillus halodurans under solid state fermentation using agrowastes

Marine microbes are potential source for novel metabolites. They are efficient in producing these metabolites utilizing agrowastes. Protease is one of the enzymes which find wide industrial applications. In the present study, protease producing bacteria was isolated from marine sediments and the organism was identified as Bacillus halodurans. The organism was subjected to protease production under solid state fermentation (SSF) using different agrowastes as substrates. Among the substrates used, wheat bran yielded maximum quantity of protease. The fermentation process was carried out under different cultural conditions to optimize the parameters influencing the enzyme production. The results of the stain removal studies by the enzyme revealed the increased efficiency of the microbial enzyme than the commercial detergent.     

Effects of pH and acetic acid on homoacetic fermentation of lactate by Clostridium formicoaceticum

Clostridium formicoaceticum homofermentatively converts lactate to acetate at 37 degrees C and pH 6.6-9.6. However, this fermentation is strongly inhibited by acetic acid at acidic pH. The specific growth rate of this organism decreased from a maximum at pH 7.6 to zero at pH 6.6. This inhibition effect was found to be attributed to both H(+) and undissociated acetic acid. At pH values below 7.6, the H(+) inhibited the fermentation following non-competitive inhibition kinetics. The acetic acid inhibition was found to be stronger at a lower medium pH. At pH 6.45-6.8, cell growth was found to be primarily limited by a maximum undissociated acetic acid concentration of 0.358 g/L (6mM). This indicates that the undissociated acid, not the dissociated acid, is the major acid inhibitor. At pH 7.6 or higher, this organism could tolerate acetate concentrations of higher than 0.8M, but salt (Na(+)) became a strong inhibitor at concentrations of higher than 0.4M. Acetic acid inhibition also can be represented by noncompetitive inhibition kinetics. A mathematical model for this homoacetic fermentation was also developed. This model can be used to simulate batch fermentation at any pH between 6.9 and 7.6.     

Paraffinübergang aus der Kohlenwasserstoffphase zur Hefezelle

The capability of the yeast Lodderomyces elongisporus to utilize solved paraffins in fermentation brothes could be demonstrated. The growth rate of this microorganism in the case of utilization of solved paraffins is higher as the most known dates. The saturated concentrations of solved hydrocarbons in the fermentation brothes are higher as in real solvent systems. The part of the solved hydrocarbon is a function of the power input, the diameter of oil drops, the fermentation conditions and the length of the paraffin chain. The organism growth rate depends on the solved paraffin concentration in the fermentation broth. This fact is one of the reasons for the variability of the consumption coefficients by utilization of paraffins with different chain lenghts. The results confirm the assumption that the transport of the paraffins from the oil drops to the cells takes place over water soluble phase.