利用酵母菌生产有机酸的研究进展

有机酸是含有一种或多种低分子量酸性基团(如羧基、磺酸基)的可生物合成的有机化合物,广泛应用于食品、农业、医药、生物基材料工业等领域。酵母菌具有生物安全、抗逆性强、底物谱广泛、方便遗传改造,以及大规模培养技术成熟等独特优点,因此利用酵母菌生产有机酸的研究日益受到国内外学者的关注。目前利用酵母生产有机酸还存在浓度低、副产物多,以及发酵效率低等缺陷。随着酵母菌代谢工程和合成生物学技术的发展,利用酵母菌生产有机酸取得了快速进展。本文总结了利用酵母合成11种有机酸的研究,包括内源和异源合成的大宗羧酸和高价值有机酸,并对该领域的未来研究方向进行了展望。     

微生物有机酸转运蛋白的研究进展

转运蛋白是一类膜蛋白,可介导生物膜内外化学物质的跨膜转运及信号交换。有机酸转运蛋白在微生物有机酸代谢的跨膜转运过程中发挥重要作用,根据转运蛋白有机酸转运的方向不同可以分为摄取转运蛋白和外排转运蛋白。在微生物代谢中,有些有机酸可以作为能源直接参与体内代谢,有些是能量转换过程中的重要中间产物;摄取转运蛋白的过表达,可以促进微生物细胞获取能源物质,高效的生产目标产物;有机酸摄取转运蛋白敲除或外排转运蛋白表达,有利于底盘细胞外排更多目标产物,进而促进有机酸的生物合成。研究有机酸转运蛋白的结构和功能,有助于解析微生物细胞有机酸生物合成及利用的机制,对于提高工业微生物对有机酸的利用及生物合成具有重要作用。本文综述了微生物有机酸转运蛋白分类和结构、转运方式和转运功能等方面,重点综述了转运蛋白在有机酸生产中的应用,为工业微生物有机酸的高效生物合成及未来发展提供参考。     

酿酒酵母生产羧酸的代谢工程策略

羧酸广泛地应用于食品、医药和化工等行业,具有广阔的市场前景.作为真核模式微生物,酿酒酵母作为代谢工程平台用来生产有机酸具有明显优势.本文论述了酿酒酵母生产重要羧酸的策略:首先构建一条能够和糖酵解途径相连接的高效的重要羧酸积累途径,进而探讨如何将碳代谢流由乙醇转向目的产物,在此基础上研究有机酸的转运及涉及到的能量问题.最后,对当前研究存在的问题进行了分析,并对未来研究方向进行了展望.     

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。     

结冷胶生物合成机理研究进展

结冷胶是少动鞘氨醇单胞菌(Sphingomonas paucimobilis)产生的一种新型微生物多糖,其独特的流变特性使结冷胶具有广泛的工业用途。虽然在结冷胶的理化特性方面的研究比较详尽,但是对结冷胶的发酵生产及其生物合成机制还缺乏深入了解。主要关注最近在结冷胶生物合成途径分子生物学方面的研究,用于编码结冷胶生物合成所需蛋白质的基因主要有三类:与糖核苷酸合成有关的基因、与四碳重复单元合成有关的基因及与长链聚合和多糖分泌有关的基因。基因工程是结冷胶分子改造和产量增加最具前景的方法。     

丁醇在大肠杆菌中的生物合成研究进展*

生物丁醇作为一种重要的化学品和石油基燃料的替代品引起了人们的广泛关注。大肠杆菌(Escherichia coli)是生物合成化学品的优良底盘菌株,已在其体内构建了丁醇的生物合成途径。但大肠杆菌合成丁醇存在:(1)代谢通量非最优;(2)辅因子和氧化还原不平衡;(3)丁醇产量和产率低等问题,为此,从高效酶选择、碳代谢流调控、辅因子调控、丁醇生产和工艺等方面已经对丁醇合成途径和丁醇发酵进行了优化。从该角度出发阐述近几年来大肠杆菌生物合成丁醇的研究进展,并展望了利用工程大肠杆菌生产丁醇的研究方向,旨在为应用其进行高效的丁醇生产提供参考。     

丁醇在大肠杆菌中的生物合成研究进展*

生物丁醇作为一种重要的化学品和石油基燃料的替代品引起了人们的广泛关注。大肠杆菌(Escherichia coli)是生物合成化学品的优良底盘菌株,已在其体内构建了丁醇的生物合成途径。但大肠杆菌合成丁醇存在:(1)代谢通量非最优;(2)辅因子和氧化还原不平衡;(3)丁醇产量和产率低等问题,为此,从高效酶选择、碳代谢流调控、辅因子调控、丁醇生产和工艺等方面已经对丁醇合成途径和丁醇发酵进行了优化。从该角度出发阐述近几年来大肠杆菌生物合成丁醇的研究进展,并展望了利用工程大肠杆菌生产丁醇的研究方向,旨在为应用其进行高效的丁醇生产提供参考。     

化学品生物合成专刊序言

以酶及微生物细胞催化剂结合工程学方法将廉价、废弃原料进行高效生物转化可实现化学品的可持续生产。近年来,合成生物学、系统生物学及酶工程等技术的快速发展大大推动了化学品的可持续生物制造,既实现了多种新型化学品的生物合成,又显著提高化学品的生物合成效率。为展示化学品生物合成的最新进展并促进绿色生物制造的发展,《生物工程学报》特组织出版化学品生物合成专刊,从酶催化与生物合成机制、微生物细胞合成、一碳生物炼制以及关键核心技术等方面,介绍化学品生物合成的最新前沿、挑战以及潜在解决方案。     

代谢工程改造大肠杆菌一碳模块高效合成L-甲硫氨酸

L-甲硫氨酸又名L-蛋氨酸,是人体必需8种氨基酸之一,在饲料、医药、食品领域具有重要应用。以实验室前期构建的M2(Escherichia coli W3110?IJAHFEBC/PAM)为出发菌株,以模块化代谢工程策略构建了一株L-甲硫氨酸高产菌株。首先通过过表达亚甲基四氢叶酸还原酶(methylenetetrahydrofolate reductase,MetF)和筛选不同来源的丝氨酸羟甲基转移酶(hydroxymethyltransferase,GlyA),增强了一碳模块甲基供体的生成,优化了一碳模块。随后针对一碳模块的前体供应,过表达了胱醚裂解酶(cysteamine lyase,MalY)和半胱氨酸内运基因(fliY),有效地提高了L-高半胱氨酸和L-半胱氨酸的供应。最终摇瓶发酵L-甲硫氨酸的产量由2.8 g/L提高至4.05 g/L,5 L发酵罐中达到18.26 g/L。研究结果表明,一碳模块对L-甲硫氨酸的生物合成具有十分重要的影响,在细胞内通过优化一碳模块,可以实现L-甲硫氨酸的高效生物合成。本研究为进一步提高微生物发酵生产L-甲硫氨酸的水平奠定了基础。     

解脂耶氏酵母合成萜烯的进展:见微知著的改造策略

萜烯是一类基于五碳单元类异戊二烯的天然化合物,种类繁多且具有多种生物活性,被广泛应用于食品、医药和化工领域.传统萜类物质生产依赖于化学合成或植物组织提取,存在产率低、资源浪费的缺点.近年来,代谢工程和合成生物学的发展促进了微生物细胞工厂的高效构建,为化学品的微生物合成提供了新的选择.解脂耶氏酵母因前体甲羟戊酸途径的内源...     

Effect of clay pretreatment on photofermentative hydrogen production from olive mill wastewater

The aim of this paper was to gain further insight into the effect of the clay pretreatment process on photofermentative hydrogen production. This two-stage process involved a clay pretreatment step followed by photofermentation which was performed under anaerobic conditions with the illumination by Tungsten lamps. Rhodobacter sphaeroides O.U.001 was used for photofermentation. Higher amounts of color (65%), total phenol (81%) and chemical oxygen demand (31%) removal efficiencies were achieved after clay pretreatment process. During photofermentative hydrogen production with the effluent of clay pretreatment process, the main organic compounds resulting higher hydrogen production rates were found to be acetic, lactic, propionic, and butyric acids. Compared to photofermentation using raw olive mill wastewater ( 16LH2/LOMW), the amount of photofermentative hydrogen production was doubled by using the effluent of the clay pretreatment process (31.5LH2/LOMW). The reasons for the improvement of hydrogen production by clay treatment can be attributed to the high removal of the hardly biodegradable compounds such as phenols; minor removal of organic acids, sugars and amino acids that are known to enhance photofermentative hydrogen production; and the color depletion of raw OMW which might cause a shadowing effect on the photosynthetic bacteria.     

Aspergillus as a versatile cell factory for organic acid production

Filamentous fungi from the genus Aspergillus are of high importance for biobased organic acid production. So far, a number of Aspergillus strains belonging to phylogenetically distantly related species have been successfully applied in industrial production of organic acids due to their excellent capabilities of secreting high amounts of desired organic acids. For the past decades, numerous efforts have been made to reveal the mechanisms of organic acid biosynthesis in several Aspergillus species and to improve the production of desired organic acids via genetic engineering. This review summarizes the recent breakthroughs in the fundamental understanding of physiological aspects of organic acid accumulation by fungal biocatalysts and highlights the progress in genetic engineering of aspergilli for organic acid production. The challenges for the future applications of aspergilli as commercial cell factories for organic acid production are also discussed.     

Microbial production of organic acids: expanding the markets

Microbial production of organic acids is a promising approach for obtaining building-block chemicals from renewable carbon sources. Although some acids have been produced for some time and in-depth knowledge of these microbial production processes has been gained, further microbial production processes seem to be feasible, but large-scale production has not yet been possible. Citric, lactic and succinic acid production exemplify three processes in different stages of industrial development. Although the questions being addressed by current research on these processes are diverging, a comparison is helpful for understanding microbial organic acid production in general. In this article, through analysis of the current advances in production of these acids, we present guidelines for future developments in this fast-moving field.     

Contribution of organic acids to the acidification of the rhizosphere of maize seedlings

The participation of organic acids in the process of soil acidification was related to other H⁺ pumping processes. The ratio between efflux of organic acids and proton secretion of maize roots was determined with the use of a pH-stat combined with a collecting system for organic acids. Changes in the composition of carboxylic acids influenced by nitrogen supply were monitored by HPLC and via enzymatic conversion. The following substances were found to be secreted by maize roots: glycolate, glyoxylate, fumarate, 2-oxoglutarate and oxalate. Malate, however, could not be detected. There is no organic acid dominantly secreted by the roots, but changes are observed during aging which might result from deficiencies of nutrients e.g. P. Fertilization of N-deficient plants with urea leads to a significant change in the composition of acids secreted. In this case, oxalate was additionally detected with a concomitant increase in glyoxylate, indicating important changes in metabolism. Acidification of the rhizosphere is predominantly maintained by secretion of protons, not by efflux of organic acids, which contributed 0.2 to 0.3% to this process only. The role of organic acids in nutrient uptake is discussed.     

13C-assisted metabolomics analysis reveals the positive correlation between specific erythromycin production rate and intracellular propionyl-CoA pool size in Saccharopolyspora erythraea

Metabolomics analysis is extremely essential to explore the metabolism characteristics of Saccharopolyspora erythraea. The lack of suitable methods for the determination of intracellular metabolites, however, hinders the application of metabolomics analysis for S. erythraea. Acyl-CoAs are important precursors of erythromycin; phosphorylated sugars are intermediate metabolites in EMP pathway or PPP pathway; organic acids are intermediate metabolites in TCA cycle. Reliable determination methods for intracellular acyl-CoAs, phosphorylated sugars, and organic acids of S. erythraea were designed and validated in this study. Using the optimized determination methods, the pool sizes of intracellular metabolites during an erythromycin fermentation process were precisely quantified by isotope dilution mass spectroscopy method. The quantification results showed that the specific erythromycin production rate was positively correlated with the pool sizes of propionyl-CoA as well as many other intracellular metabolites. The experiment under the condition without propanol, which is a precursor of propionyl-CoA and an important substrate in industrial erythromycin production process, also corroborated the correlation between specific erythromycin production rate and intracellular propionyl-CoA pool size. As far as we know, this is the first paper to conduct the metabolomics analysis of S. erythraea, which makes the metabolomics analysis of S. erythraea in the industrial erythromycin production process possible.

     

Anaerobic degradation of organic waste: An experience in mathematical modeling

Some key aspects of organic waste degradation were analyzed by means of mathematical models using data obtained in laboratory reactors. It was shown that an essential condition of effective methane production is the balance between sequential and parallel stages not resulting in accumulation of intermediate products that are potential inhibitors of the process. Decreased initial concentration of organic matter (dilution) and the introduction of seed culture (a methanogenic microbial community) favor the balancing of the process. Decomposition of easily degradable organic substances may lead to excessive accumulation of volatile fatty acids and acidification of the medium, which, in turn, blocks the degradation of difficult-to-degrade compounds. If the process is unbalanced, agitation eliminates the initiation centers for methanogenesis by averaging the reagent concentrations, which results in complete cessation of methane production. Such centers may be multicellular aggregates of Methanosarcina sp.     

暗发酵-光发酵两阶段联合生物制氢技术研究进展

随着能源紧缺的日益加剧,以及化石燃料燃烧引起的环境问题逐渐突显,氢能作为一种清洁可再生能源越来越受到青睐。生物制氢与热化学及电化学制氢相比其反应条件温和、低耗、绿色,是一项非常有应用前景的技术。生物制氢从广义上可以分为暗发酵和光发酵产氢两种,其中暗发酵微生物可以利用有机废弃物产生氢气以及有机酸等副产物,光合细菌在光照和固氮酶的作用下可以将暗发酵产生的有机酸继续用于产氢,因此两种发酵产氢方式相结合可以提高有机废物的资源化效率。将近年来暗发酵-光发酵两阶段生物制氢技术进行整理分析,从其产氢机理、主要影响因素、暗发酵-光发酵产氢结合方式(两步法、混合培养产氢)几个方面进行阐述,最后指出该技术面临的挑战。     

两步发酵过程中有机酸对产1，3-丙二醇的影响

考察了基因工程菌发酵生产1.3 丙二醇过程中,有机酸对发酵过程的影响,并选用了不同的离子交换树脂对甘油发酵液进行处理。发现有机酸、特别是乳酸对1.3丙二醇生产的抑制作用最明显。在使用离子交换树脂处理有机酸的过程中,确定了使用005号离子交换树脂处理效果最好,005号离子交换树脂可除去大部分的有机酸,处理后的发酵液发酵产1.3丙二醇产量比未处理的发酵液产量提高166%,转化率提高34%。     

Aerobiosis–anaerobiosis transition has a significant impact on organic acid production by Corynebacterium glutamicum

Corynebacterium glutamicum is known to produce organic acids under anaerobic culture conditions, in particular, lactic, succinic, and acetic acids. Our study is focused on acetic and succinic acid production using a lactate dehydrogenase-deficient strain of C. glutamicum. Usually, with this bacterium, the organic acid production process is based on an initial aerobic growth phase, followed by a rapid deoxygenation and an anaerobic production phase. In our study, we demonstrated that this strategy was unfavorable for the production of organic acids. Conversely, we showed that applying the best transition strategy based on progressive deoxygenation significantly increased the concentration of organic acids up to 640%. This was observed either by applying controlled dissolved oxygen concentrations or by decreasing the steps of gas flow rates. Our results also showed that applying constant oxygen transfer flux throughout the culture, and thus in the absence of the anaerobic phase, promoted constant production yields (approximately 0.5 mol of succinate or acetate per mole of glucose). In this case, acetate production (120 mM) was favored over succinate production (132 mM), resulting in a decrease in the molar ratio of products (succinate/acetate) from 4.8 to 1.1 between progressive deoxygenation and constant OTR cultures.