生物法合成3-羟基丙酸的研究进展

从3-羟基丙酸的性质出发,介绍了生物法合成3-羟基丙酸以及它在生物体内的五种代谢途径,此外还简要介绍了3-羟基丙酸在合成生物聚酯、抗植物病虫害上的一些应用。     

遗传改造微生物代谢途径生产新型柴油燃料的研究进展

生物柴油是一种能替代柴油的可再生燃料,然而通过植物油料化学转酯化生产的第一代生物柴油在性能和生产工艺上有很多缺点。近年来随着合成生物学和代谢工程的迅速发展,通过选择合适的微生物并利用各种生物技术改造其代谢合成途径,如脂肪酸合成途径、异戊二烯合成途径,研究人员能利用微生物直接生产性能更加优越、品质更高的新型第二代生物柴油——长链烷烃。文章总结了目前遗传改造微生物代谢途径生产新型柴油的研究进展,并指出目前该领域存在的问题以及今后的发展方向。     

吃进“废气”,吐出“宝贝”——记代谢工程改造光合细胞工厂生产异戊二烯

异戊二烯主要用于生产合成橡胶,还用于生产多种精细化工品及黏合剂和润滑剂。目前异戊二烯完全由石化原料生产。随着全球气候变暖和化石资源的日益短缺,构建以廉价生物质或CO2为原料的异戊二烯生物法合成线路已引起研究者的极大关注。中国科学院上海植物生理生态研究所杨琛课题组在蓝细菌中构建异戊二烯合成途径,利用代谢流量分析和代谢组学分析指导蓝细菌中异戊二烯合成途径的设计和改造,通过循环鉴定合成途径限速步骤和解除限速步骤,逐步提高异戊二烯合成途径的代谢通量,最终经过一系列改造后获得的工程菌可将光合作用所固定的碳的40%用于异戊二烯的合成,产量高达1.26 g/L。除了高效合成异戊二烯,该研究所构建的工程菌还可以作为平台,构建光合自养细胞工厂,合成各种萜类化合物。     

引入新型异戊二烯醇利用途径促进解脂耶氏酵母中β-胡萝卜素的合成*

目的:微生物体内异戊二烯类化合物的前体物异戊烯焦磷酸酯的天然合成路径受到严格的代谢调控,因此限制了异戊二烯类化合物的高效生物合成,而新型异戊二烯醇利用途径独立于生物体内源性代谢路径,通过在微生物中引入IUP能够进行异戊烯焦磷酸酯的大量合成,从而促进异戊二烯类化合物的大量合成。方法:在油脂酵母解脂耶氏酵母中引入IUP,强化异戊烯焦磷酸酯生物合成,促进β-胡萝卜素的高效积累。结果:通过生物信息学的方法预测IUP中两个关键蛋白酿酒酵母来源的胆碱激酶ScCK和拟南芥来源的异戊烯磷酸激酶AtIPK,均为酸性亲水性蛋白,无跨膜区和信号肽,二者都具有疏松不稳定的结构特征,显著富集于磷酸类物质的合成通路中。在解脂耶氏酵母中利用同源重组技术引入外源β-胡萝卜素合成关键基因carRP和carB,强化甲羟戊酸途径的关键基因thmgR和ggs1,使工程菌株中积累2.68 mg/L β-胡萝卜素。通过Cre-loxP系统回收基因组上的ura标签,再将IUP进一步整合到工程菌株染色体上。当培养基中含有20 mM异戊二烯醇作为底物、碳氮比为4/3且发酵96 h后,重组解脂耶氏酵母中β-胡萝卜素的产量提高到410.2 mg/L,较原始工程菌的产量提高了近200倍。结论:IUP能够促进解脂耶氏酵母中β-胡萝卜素的高效积累,为利用IUP开展β-胡萝卜素和其他异戊二烯类化合物的高效生物合成提供新思路。     

辅酶Q10生物合成与生产研究进展

微生物发酵法是生产辅酶Q10的最佳工艺.辅酶Q10的生物合成途径包括异戊二烯焦磷酸合成、聚十异戊二烯焦磷酸合成、苯环修饰等过程.1-脱氧-D-木酮糖-5-磷酸合成酶、聚十异戊二烯焦磷酸合成酶、对羟基笨甲酸聚十异戊二烯焦磷酸转移酶等是Q10合成的关键酶.生产辅酶Q10的菌种可通过诱变、基因重组和支路敲除等方法获得.氧化还原电位控制、pH控制补料分批发酵、发酵萃取耦合技术等新工艺逐浙应用于辅酶Q10生产.     

类异戊二烯非甲羟戊酸代谢途径的分子生物学研究进展

近期发现的类异戊二烯非甲羟戊酸代谢途径是类异戊二烯化合物生成合成的另一途径。文章对该代谢途径的分子生物学研究进展进行了综述。重点介绍非甲羟戊酸代谢途径的发现和5－磷酸脱氧木糖合成酶、5－磷酸脱氧木糖还原异构醇、异戊二烯焦磷酸合成酶的分子克隆的最新进展以及非甲羟戊酸代谢途径的发现在农业和医药等领域的应用。     

提高大肠杆菌通过MVA途径合成异戊二烯

异戊二烯是橡胶合成的重要前体物质。为了提高菌株的异戊二烯产量,本实验室在研究中构建了一株异戊二烯产气的菌株BW-01,基于蛋白质预算理论的指导,理性设计通过改变质粒拷贝数、增加稀有密码子等合成生物学手段调控关键限速酶编码基因表达,从而提高大肠杆菌外源MVA代谢途径的异戊二烯产量。摇瓶发酵实验中我们构建的新产气菌株BW-07比原有的产气菌株BW-01的产量提高了73%,达到了761.1 mg/L。为后续菌株改造及进行发酵罐实验奠定了基础。     

红景天苷生物、化学和生物催化合成的分子理论及应用

红景天苷是红景天属植物的主要活性成分之一,研究红景天苷的高效合成具有重要的科研和应用价值。本文全面概述近年来合成红景天苷的研究状况,主要包括生物合成途径、化学合成途径、生物催化合成途径。对生物合成途径中红景天苷的代谢合成途径、关键酶及基因、实践现状进行了分析;总结了以Koenigs-Knorr法为理论基础的化学合成的研究进展;对具有广泛发展前景的体外生物催化合成状况进行了理论与实践的概述。通过对这些合成方法的展望,为人们了解合成红景天苷的研究现状、进一步深入研究提供参考。     

异戊二烯合成酶研究进展

异戊二烯(Isoprene)的排放具有特殊的生物学功能,对大气环境具有重要影响,另外,异戊二烯也是一种具有广泛用途的化合物。在生物体内,异戊二烯是由异戊二烯合成酶(Isoprene synthase,Isps)催化烯丙基二磷酸(Dimethylallyl diphosphate,DMAPP)脱去焦磷酸(Pyrophosphate)而生成的。因此,作为异戊二烯合成过程中的关键酶,Isps在异戊二烯的自然排放和生物合成过程都发挥着重要的作用,对Isps的研究具有非常重要的意义。到目前为止,已经在多种植物中发现了该酶,研究表明,来源于不同生物的异戊二烯合成酶具有保守的结构特征和相似的生化性质。文中就Isps的最新研究进展进行综述,包括比较分析不同生物来源Isps的生化特征和结构特征,探讨催化机制,并对该酶在生物工程领域的应用进行展望。     

生物法合成3-羟基丙酸的研究进展

3-羟基丙酸是一种重要的平台化合物,应用广泛。生物法是实现高效合成3-羟基丙酸的重要手段,近年来发展迅速。针对3-羟基丙酸的天然合成途径、工程合成途径,特别是利用葡萄糖、甘油等廉价底物合成3-羟基丙酸的代谢工程进展进行了综述和比较。同时,对生物法合成3-羟基丙酸的主要问题进行了讨论,并提出了解决相关问题的建议。另外,根据现阶段的研究进展,对3-羟基丙酸的产业化发展进行了展望。     

Isoprene emission from plants: why and how

BACKGROUND: Some, but not all, plants emit isoprene. Emission of the related monoterpenes is more universal among plants, but the amount of isoprene emitted from plants dominates the biosphere-atmosphere hydrocarbon exchange. SCOPE: The emission of isoprene from plants affects atmospheric chemistry. Isoprene reacts very rapidly with hydroxyl radicals in the atmosphere making hydroperoxides that can enhance ozone formation. Aerosol formation in the atmosphere may also be influenced by biogenic isoprene. Plants that emit isoprene are better able to tolerate sunlight-induced rapid heating of leaves (heat flecks). They also tolerate ozone and other reactive oxygen species better than non-emitting plants. Expression of the isoprene synthase gene can account for control of isoprene emission capacity as leaves expand. The emission capacity of fully expanded leaves varies through the season but the biochemical control of capacity of mature leaves appears to be at several different points in isoprene metabolism. CONCLUSIONS: The capacity for isoprene emission evolved many times in plants, probably as a mechanism for coping with heat flecks. It also confers tolerance of reactive oxygen species. It is an example of isoprenoids enhancing membrane function, although the mechanism is likely to be different from that of sterols. Understanding the regulation of isoprene emission is advancing rapidly now that the pathway that provides the substrate is known.     

Evolution of the isoprene biosynthetic pathway in kudzu

Isoprene synthase converts dimethylallyl diphosphate, derived from the methylerythritol 4-phosphate (MEP) pathway, to isoprene. Isoprene is made by some plants in substantial amounts, which affects atmospheric chemistry, while other plants make no isoprene. As part of our long-term study of isoprene synthesis, the genetics of the isoprene biosynthetic pathway of the isoprene emitter, kudzu (Pueraria montana), was compared with similar genes in Arabidopsis (Arabidopsis thaliana), which does not make isoprene. The MEP pathway genes in kudzu were similar to the corresponding Arabidopsis genes. Isoprene synthase genes of kudzu and aspen (Populus tremuloides) were cloned to compare their divergence with the divergence seen in MEP pathway genes. Phylogenetic analysis of the terpene synthase gene family indicated that isoprene synthases are either within the monoterpene synthase clade or sister to it. In Arabidopsis, the gene most similar to isoprene synthase is a myrcene/ocimene (acyclic monoterpenes) synthase. Two phenylalanine residues found exclusively in isoprene synthases make the active site smaller than other terpene synthase enzymes, possibly conferring specificity for the five-carbon substrate rather than precursors of the larger isoprenoids. Expression of the kudzu isoprene synthase gene in Arabidopsis caused Arabidopsis to emit isoprene, indicating that whether or not a plant emits isoprene depends on whether or not it has a terpene synthase capable of using dimethylallyl diphosphate.     

Isoprene synthesis in plants: lessons from a transgenic tobacco model

Isoprene is a highly reactive gas, and is emitted in such large quantities from the biosphere that it substantially affects the oxidizing potential of the atmosphere. Relatively little is known about the control of isoprene emission at the molecular level. Using transgenic tobacco lines harbouring a poplar isoprene synthase gene, we examined control of isoprene emission. Isoprene synthase required chloroplastic localization for catalytic activity, and isoprene was produced via the methyl erythritol (MEP) pathway from recently assimilated carbon. Emission patterns in transgenic tobacco plants were remarkably similar to naturally emitting plants under a wide variety of conditions. Emissions correlated with photosynthetic rates in developing and mature leaves, and with the amount of isoprene synthase protein in mature leaves. Isoprene synthase protein levels did not change under short-term increase in heat/light, despite an increase in emissions under these conditions. A robust circadian pattern could be observed in emissions from long-day plants. The data support the idea that substrate supply and changes in enzyme kinetics (rather than changes in isoprene synthase levels or post-translational regulation of activity) are the primary controls on isoprene emission in mature transgenic tobacco leaves.     

Isoprene emission from aspen leaves : influence of environment and relation to photosynthesis and photorespiration

Isoprene emission rates from quaking aspen (Populus tremuloides Michx.) leaves were measured simultaneously with photosynthesis rate, stomatal conductance, and intercellular CO₂ partial pressure. Isoprene emission required the presence of CO₂ or O₂, but not both. The light response of isoprene emission rate paralleled that of photosynthesis. Isoprene emission was inhibited by decreasing ambient O₂ from 21% to 2%, only when there was oxygen insensitive photosynthesis. Mannose (10 millimolar) fed through cut stems resulted in strong inhibition of isoprene emission rate and is interpreted as evidence that isoprene biosynthesis requires either the export of triose phosphates from the chloroplast, or the continued synthesis of ATP. Light response experiments suggest that photosynthetically generated reductant or ATP is required for isoprene biosynthesis. Isoprene biosynthesis and emission are not directly linked to glycolate production through photorespiration, contrary to previous reports. Isoprene emission rate was inhibited by above-ambient CO₂ partial pressures (640 microbar outside and 425 microbar inside the leaf). The inhibition was not due to stomatal closure. This was established by varying ambient humidity at normal and elevated CO₂ partial pressures to measure isoprene emission rates over a range of stomatal conductances. Isoprene emission rates were inhibited at elevated CO₂ despite no change in stomatal conductance. Addition of abscisic acid to the transpiration stream dramatically inhibited stomatal conductance and photosynthesis rate, with a slight increase in isoprene emission rate. Thus, isoprene emission is independent of stomatal conductance, and may occur through the cuticle. Temperature had an influence on isoprene emission rate, with the Q₁₀ being 1.8 to 2.4 between 35 and 45°C. At these high temperatures the amount of carbon lost through isoprene emission was between 2.5 and 8% of that assimilated through photosynthesis. This represents a significant carbon cost that should be taken into account in determining midsummer carbon budgets for plants that are isoprene emitters.     

Functional analysis of genes involved in the biosynthesis of isoprene in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

In comparison to other bacteria Bacillus subtilis emits the volatile compound isoprene in high concentrations. Isoprene is the smallest representative of the natural product group of terpenoids. A search in the genome of B. subtilis resulted in a set of genes with yet unknown function, but putatively involved in the methylerythritol phosphate (MEP) pathway to isoprene. Further identification of these genes would give the possibility to engineer B. subtilis as a host cell for the production of terpenoids like the valuable plant-produced drugs artemisinin and paclitaxel. Conditional knock-out strains of putative genes were analyzed for the amount of isoprene emitted. Differences in isoprene emission were used to identify the function of the enzymes and of the corresponding selected genes in the MEP pathway. We give proof on a biochemical level that several of these selected genes from this species are involved in isoprene biosynthesis. This opens the possibilities to investigate the physiological function of isoprene emission and to increase the endogenous flux to the terpenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate, for the heterologous production of more complex terpenoids in B. subtilis.     

Production of isoprene by leaf tissue

Isoprene production by Hamamelis virginiana L. and Quercus borealis Michx. leaves was studied. When ambient CO(2) concentrations were maintained with bicarbonate buffers, the rate of isoprene production at 125 microliters per liter of CO(2) was approximately four times that at 250 microliters per liter of CO(2). Isoprene production was drastically inhibited by 97% O(2). Dichlorodimethylphenylurea (0.1 mm), NaHSO(3) (10 mm), and alpha-hydroxy-2-pyridinemethanesulfonic acid (10 mm) inhibited isoprene production but increased the compensation point of the tissue. Isonicotinic acid hydrazide neither inhibited isoprene emission nor increased the compensation point of the tissue significantly. Inhibition of isoprene production does not seem to correlate with stomatal resistance. Isoprene was labeled by intermediates of the glycolate pathway, and similarities are noted between the biosynthesis of isoprene and that of beta-carotene.     

Light-Dependent Isoprene Emission (Characterization of a Thylakoid-Bound Isoprene Synthase in Salix discolor Chloroplasts)

Isoprene synthase is an enzyme that is responsible for the production of the volatile C5 hydrocarbon, isoprene, in plant leaves. Isoprene formation in numerous C3 plants is interesting because (a) large quantities of isoprene are emitted, 5 x 1014 g of C annually, (b) a plant may release 1 to 8% of its fixed C as isoprene, and (c) the function of plant isoprene production is unknown. Because of the dependence of foliar isoprene emission on light, the existence of a plastidic isoprene synthase has been postulated. To pursue this idea, a method to isolate chloroplasts from Salix discolor was developed and shows a plastidic isoprene synthase that is tightly bound to the thylakoid membrane and accessible to trypsin inactivation. The thylakoid-bound isoprene synthase has catalytic properties similar to known soluble isoprene synthases; however, the relationship between these enzymes is unknown. The discovery of a thylakoid-bound isoprene synthase with a stromal-facing domain places it in the chloroplast, where it may be subject to numerous direct and indirect light-mediated effects. Implications for the light-dependent regulation of foliar isoprene production and its function are presented.     

The Effect of Isoprene on the Properties of Spinach Thylakoids and Phosphatidyicholine Liposomes

Abstract: Isoprene is emitted from the leaves of some plants. It was recently reported that exogenous isoprene delays the onset of leaf damage during controlled increases in leaf temperature (Singsaas et al. Plant Physiology 115: 1413–1420 [1997^[17). Thylakoid membranes are presumed to be the site of action based upon isoprene's hydrophobicity, production in chloroplasts, and effect upon chlorophyll fluorescence at high temperatures. In an attempt to discern the mechanistic basis for isoprene's thermoprotective role, we studied the effect of exogenous isoprene on the peroxidation, permeability, and stability of spinach thylakoids and phosphatidylcholine liposomes. Isoprene, supplied at either 18 or 21 μ1 L¹, had no effect upon the rate of liposome peroxidation in the presence of a hydroxyl radical-generating system. Isoprene also did not affect liposome peroxidation at high temperatures. Neither the proton permeability of thylakoids nor the leakage of a fluorescent probe from liposomes was influenced by exogenous isoprene, when measured at several temperatures. Isoprene did not affect the stability of thylakoid membrane proteins during a temperature increase, as shown by differential scanning calorimetry. Therefore, despite the use of a variety of techniques to investigate fundamental membrane parameters, we were unable to demonstrate an effect of isoprene.