Expression of a bacterial feedback-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of the shikimate pathway in Arabidopsis elucidates potential metabolic bottlenecks between primary and secondary metabolism

The shikimate pathway of plants mediates the conversion of primary carbon metabolites via chorismate into the three aromatic amino acids and to numerous secondary metabolites derived from them. However, the regulation of the shikimate pathway is still far from being understood. We hypothesized that 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) is a key enzyme regulating flux through the shikimate pathway. To test this hypothesis, we expressed a mutant bacterial AroG gene encoding a feedback-insensitive DAHPS in transgenic Arabidopsis plants. The plants were subjected to detailed analysis of primary metabolism, using GC-MS, as well as secondary metabolism, using LC-MS. Our results exposed a major effect of bacterial AroG expression on the levels of shikimate intermediate metabolites, phenylalanine, tryptophan and broad classes of secondary metabolite, such as phenylpropanoids, glucosinolates, auxin and other hormone conjugates. We propose that DAHPS is a key regulatory enzyme of the shikimate pathway. Moreover, our results shed light on additional potential metabolic bottlenecks bridging plant primary and secondary metabolism.     

Rapid Method To Estimate the Presence of Secondary Metabolites in Microbial Extracts

Screening microbial secondary metabolites is an established method to identify novel biologically active molecules. Preparation of biological screening samples from microbial fermentation extracts requires growth conditions that promote synthesis of secondary metabolites and extraction procedures that capture the secondary metabolites produced. High-performance liquid chromatography (HPLC) analysis of fermentation extracts can be used to estimate the number of secondary metabolites produced by microorganisms under various growth conditions but is slow. In this study we report on a rapid (approximately 1 min per assay) surrogate measure of secondary metabolite production based on a metabolite productivity index computed from the electrospray mass spectra of samples injected directly into a spectrometer. This surrogate measure of productivity was shown to correlate with an HPLC measure of productivity with a coefficient of 0.78 for a test set of extracts from 43 actinomycetes. This rapid measure of secondary metabolite productivity may be used to identify improved cultivation and extraction conditions by analyzing and ranking large sets of extracts. The same methods may also be used to survey large collections of extracts to identify subsets of highly productive organisms for biological screening or additional study.     

Microalgae in the postgenomic era: a blooming reservoir for new natural products

Bacteria, fungi, algae and higher plants are the most prolific producers of natural products (secondary metabolites). Compared to macroalgae, considerably fewer natural products have been isolated from microalgae, which offer the possibility of obtaining sufficient and well-defined biological material from laboratory cultures. Interest in microalgae is reinforced by large-scale data sets from genome sequencing projects and the development of genetic tools such as transformation protocols. This review highlights what is currently known about the biosynthesis and biological role of natural products in microalgae, with examples from isoprenoids, complex polyketides, nonribosomal peptides, polyunsaturated fatty acids and oxylipins, alkaloids, and aromatic secondary metabolites. In addition, we introduce a bioinformatic analysis of available genome sequences from totally 16 microalgae, belonging to the green and red algae, heterokonts and haptophytes. The results suggest that the biosynthetic potential of microalgae is underestimated and many microalgal natural products remain to be discovered.     

Improved computational performance of MFA using elementary metabolite units and flux coupling

Extending the scope of isotope mapping models becomes increasingly important in order to analyze strains and drive improved product yields as more complex pathways are engineered into strains and as secondary metabolites are used as starting points for new products. Here we present how the elementary metabolite unit (EMU) framework and flux coupling significantly decrease the computational burden of metabolic flux analysis (MFA) when applied to large-scale metabolic models. We applied these techniques to a previously published isotope mapping model of Escherichia coli accounting for 238 reactions. We find that the combined use of EMU and flux coupling analysis leads to a ten-fold decrease in the number of variables in comparison to the original isotope distribution vector (IDV) version of the model. In addition, using OptMeas the task of identifying additional measurement choices to fully specify the flows in the metabolic network required only 2% of the computation time of the one using IDVs. The observed computational savings reveal the rapid progress in performing MFA with increasingly larger isotope models with the ultimate goal of handling genome-scale models of metabolism.     

合成支架在代谢工程中的研究进展

代谢工程作为通过引入外源合成途径或改造优化代谢网络,进行高附加值的天然代谢产物生物合成的技术,已经得到广泛应用。但随着目标合成产物的结构日渐复杂,构建多基因的从头合成途径造成宿主生物代谢失衡与中间产物对宿主细胞产生毒害作用等一系列问题发生的可能性也随之增加。为解决这些问题合成支架策略应运而生,合成支架将途径酶共定位以提高局部酶和代谢物的浓度,来增强代谢通量并限制中间产物与宿主细胞环境间的相互作用,成为生物催化和合成生物学研究的热点之一。尽管由核酸、蛋白质构成的合成支架策略已经应用于多种代谢物的异源合成,并取得了不同程度的成功,但合成支架的精确组装仍然是一项艰巨的任务。文中详细介绍了合成支架技术的研究现状,详细阐述了合成支架技术的原理和实例,并初步探讨了其应用前景。     

次生代谢产物与植物抗病防御反应

次生代谢产物是由植物次生代谢产生的许多结构不同的小分子有机化合物,它们广泛参与植物的生长、发育、防御等生理过程。次生代谢产物在植物的抗病防御反应中发挥着重要作用,可以作为生化壁垒防御病原物侵染,还可以作为信号物质参与植物的抗病反应;在植物与病原物互作中,植物合成新的抗菌物质植保素,原有的抗菌物质也会增加。植物次生代谢产物的积累受到病原物、发育,环境等多种因素的调节。本文重点介绍次生代谢产物在植物抗病防御中的相关作用以及影响其合成的各种因素。     

Fungal secondary metabolism - from biochemistry to genomics

Much of natural product chemistry concerns a group of compounds known as secondary metabolites. These low-molecular-weight metabolites often have potent physiological activities. Digitalis, morphine and quinine are plant secondary metabolites, whereas penicillin, cephalosporin, ergotrate and the statins are equally well known fungal secondary metabolites. Although chemically diverse, all secondary metabolites are produced by a few common biosynthetic pathways, often in conjunction with morphological development. Recent advances in molecular biology, bioinformatics and comparative genomics have revealed that the genes encoding specific fungal secondary metabolites are clustered and often located near telomeres. In this review, we address some important questions, including which evolutionary pressures led to gene clustering, why closely related species produce different profiles of secondary metabolites, and whether fungal genomics will accelerate the discovery of new pharmacologically active natural products.     

Flux Imbalance Analysis and the Sensitivity of Cellular Growth to Changes in Metabolite Pools

Stoichiometric models of metabolism, such as flux balance analysis (FBA), are classically applied to predicting steady state rates - or fluxes - of metabolic reactions in genome-scale metabolic networks. Here we revisit the central assumption of FBA, i.e. that intracellular metabolites are at steady state, and show that deviations from flux balance (i.e. flux imbalances) are informative of some features of in vivo metabolite concentrations. Mathematically, the sensitivity of FBA to these flux imbalances is captured by a native feature of linear optimization, the dual problem, and its corresponding variables, known as shadow prices. First, using recently published data on chemostat growth of Saccharomyces cerevisae under different nutrient limitations, we show that shadow prices anticorrelate with experimentally measured degrees of growth limitation of intracellular metabolites. We next hypothesize that metabolites which are limiting for growth (and thus have very negative shadow price) cannot vary dramatically in an uncontrolled way, and must respond rapidly to perturbations. Using a collection of published datasets monitoring the time-dependent metabolomic response of Escherichia coli to carbon and nitrogen perturbations, we test this hypothesis and find that metabolites with negative shadow price indeed show lower temporal variation following a perturbation than metabolites with zero shadow price. Finally, we illustrate the broader applicability of flux imbalance analysis to other constraint-based methods. In particular, we explore the biological significance of shadow prices in a constraint-based method for integrating gene expression data with a stoichiometric model. In this case, shadow prices point to metabolites that should rise or drop in concentration in order to increase consistency between flux predictions and gene expression data. In general, these results suggest that the sensitivity of metabolic optima to violations of the steady state constraints carries biologically significant information on the processes that control intracellular metabolites in the cell.     

Metabolism of halogenated compounds in the white rot fungus Bjerkandera adusta studied by membrane inlet mass spectrometry and tandem mass spectrometry

Membrane inlet mass spectrometry has been used for the characterization of halogenated organic compounds produced by the fungus Bjerkandera adusta. Using this technique we obtained electron impact-, chemical ionization-, electron capture negative chemical ionization-mass spectra and tandem mass spectra directly from the growth medium. Through this direct analysis of the samples we identified novel bioconversion products and confirmed recently published data on the production of both chlorinated and brominated methoxybenzaldehyde metabolites. Growth profiles of the culture grown on a defined medium showed that the production of secondary metabolites starts after approximately 6 days and reaches maximal concentrations of 25-250 muM after 15-20 days. Although delayed, the production of secondary metabolites paralleled a depletion of glucose from the medium and stopped shortly after all glucose had been consumed. Experiments in which fluoro- and bromo-labeled 4-methoxybenzaldehydes were added to the medium at day 8 showed biotransformation of these compounds into chloro-3-fluoro-4-methoxy-benzaldehyde and chloro-3-bromo-4-methoxybenzaldehyde, respectively. No dichlorinated products were observed, suggesting that halogenation takes place only at the meta position on the 4-methoxybenzaldehydes. These experiments are the first to bring direct evidence of a halogenation mechanism, where the enzymatic attack takes place directly on the 4-methoxybenzaldehyde intermediates. (c) John Wiley & Sons, Inc.     

Relationship between secondary metabolism and fungal development.

Filamentous fungi are unique organisms-rivaled only by actinomycetes and plants-in producing a wide range of natural products called secondary metabolites. These compounds are very diverse in structure and perform functions that are not always known. However, most secondary metabolites are produced after the fungus has completed its initial growth phase and is beginning a stage of development represented by the formation of spores. In this review, we describe secondary metabolites produced by fungi that act as sporogenic factors to influence fungal development, are required for spore viability, or are produced at a time in the life cycle that coincides with development. We describe environmental and genetic factors that can influence the production of secondary metabolites. In the case of the filamentous fungus Aspergillus nidulans, we review the only described work that genetically links the sporulation of this fungus to the production of the mycotoxin sterigmatocystin through a shared G-protein signaling pathway.     

植物次生代谢及其与环境的关系

人类对植物次生代谢产物（天然产物）的早期研究源于它们的应用价值,近些年来人们越来越认识到植物次生代谢产物广泛的生物学效应,开始重新评价这些化合物在植物生命活动以及生态系统中可能扮演的角色。植物的次生代谢是植物在长期进化中与环境（生物的和非生物的）相互作用的结果,次生代谢产物在植物提高自身保护和生存竞争能力、协调与环境关系上充当着重要的角色。介绍了植物次生代谢及其产物的特点,概述了植物次生代谢与温度、水分、光照、养分、CO2浓度、UV-B辐射、环境污染等非生物环境以及与化学防御、化感作用、菌根共生、微生物病害的关系。研究植物次生代谢与环境的关系,可以从更深的层次发掘植物与环境的内在联系,为全面、深入认识植物与环境的相互关系提供新的研究途径,同时也有利于人类更有效、合理地利用植物的次生代谢产物。     

Discovering the hidden secondary metabolome of Myxococcus xanthus: a study of intraspecific diversity

As a monophyletic group, the myxobacteria are known to produce a broad spectrum of secondary metabolites. However, the degree of metabolic diversity that can be found within a single species remains unexplored. The model species Myxococcus xanthus produces several metabolites also present in other myxobacterial species, but only one compound unique to M. xanthus has been found to date. Here, we compare the metabolite profiles of 98 M. xanthus strains that originate from 78 locations worldwide and include 20 centimeter-scale isolates from one location. This screen reveals a strikingly high level of intraspecific diversity in the M. xanthus secondary metabolome. The identification of 37 nonubiquitous candidate compounds greatly exceeds the small number of secondary metabolites previously known to derive from this species. These results suggest that M. xanthus may be a promising source of future natural products and that thorough intraspecific screens of other species could reveal many new compounds of interest.     

Relationship between Secondary Metabolism and Fungal Development

Filamentous fungi are unique organisms—rivaled only by actinomycetes and plants—in producing a wide range of natural products called secondary metabolites. These compounds are very diverse in structure and perform functions that are not always known. However, most secondary metabolites are produced after the fungus has completed its initial growth phase and is beginning a stage of development represented by the formation of spores. In this review, we describe secondary metabolites produced by fungi that act as sporogenic factors to influence fungal development, are required for spore viability, or are produced at a time in the life cycle that coincides with development. We describe environmental and genetic factors that can influence the production of secondary metabolites. In the case of the filamentous fungus Aspergillus nidulans, we review the only described work that genetically links the sporulation of this fungus to the production of the mycotoxin sterigmatocystin through a shared G-protein signaling pathway.     

Visualizing the spatial distribution of secondary metabolites produced by marine cyanobacteria and sponges via MALDI-TOF imaging

Marine cyanobacteria and sponges are prolific sources of natural products with therapeutic applications. In this paper we introduce a mass spectrometry based approach to characterize the spatial distribution of these natural products from intact organisms of differing complexities. The natural product MALDI-TOF-imaging (npMALDI-I) approach readily identified a number of metabolites from the cyanobacteria Lyngbya majuscula 3L and JHB, Oscillatoria nigro-viridis, Lyngbya bouillonii, and a Phormidium species, even when they were present as mixtures. For example, jamaicamide B, a well established natural product from the cyanobacterium Lyngbya majuscula JHB, was readily detected as were the ions that correspond to the natural products curacin A and curazole from Lyngbya majuscula 3L. In addition to these known natural products, a large number of unknown ions co-localized with the different cyanobacteria, providing an indication that this method can be used for dereplication and drug discovery strategies. Finally, npMALDI-I was used to observe the secondary metabolites found within the sponge Dysidea herbacea. From these sponge data, more than 40 ions were shown to be co-localized, many of which were halogenated. The npMALDI-I data on the sponge indicates that, based on the differential distribution of secondary metabolites, sponges have differential chemical micro-environments within their tissues. Our data demonstrate that npMALDI-I can be used to provide spatial distribution of natural products, from single strands of cyanobacteria to the very complex marine assemblage of a sponge.     

放线菌与枯草芽孢杆菌的共培养及其对活性次生代谢产物的影响

为探讨共培养对放线菌产生活性次生代谢产物的影响,结合抗菌活性测定及HPLC-PDA分析,研究了22株放线菌的单培养及其与枯草芽孢杆菌的共培养发酵代谢产物的差异,并选取抗菌活性较强的链霉菌FXJ2.014进一步研究其代谢产物。发现FXJ2.014、FXJ1.296、AS4.1252三株菌与枯草芽孢杆菌共培养时产生其在相同条件下单培养时没有的物质,其中链霉菌FXJ2.014单培养时主要产生醌霉素A,共培养时产物中增加了醌霉素结构类似物FXJ2.014-HB。进一步的抗菌、抗肿瘤活性测定结果表明,两者的生物活性有较显著的差异,且FXJ2.014-HB对多种肿瘤细胞系的抑制活性普遍弱于高毒性的醌霉素A,为有潜力的细胞毒性较小的抗生素。共培养是一条很有希望的发掘放线菌活性次生代谢产物的新途径。     

植物细胞培养生产次生代谢物的途径

利用植物细胞培养生产次生代谢产物是一种快速、高效获取天然产物的重要方法。本文从培养方法、培养技术、生物转化及基因工程应用三个方面,综述了近年来国内外应用于植物细胞培养生产次生代谢产物的途径及研究进展,论述了次生代谢产物的主要类型及合成途径,列举了应用实例和次生代谢物种类以及相应的培养条件,以期对快速选择提高目的次生代谢物的培养条件起到了一定指导作用。     

链霉菌次生代谢中双因子调控系统的研究进展

链霉菌次生代谢产物的生物合成受到严格和复杂的调控,而双因子调控系统是其中重要的一类调控因子,在链霉菌中广泛存在,且存在作用方式的多样性和作用机制的复杂性。就近些年研究较多的参与链霉菌次生代谢的两类双因子调控系统(真核型和原核型)的研究状况做了综述,重点阐明其作用机制,并对其研究趋势以及在药物代谢工程中的应用前景进行了展望。