MultiMetEval: Comparative and Multi-Objective Analysis of Genome-Scale Metabolic Models

Comparative metabolic modelling is emerging as a novel field, supported by the development of reliable and standardized approaches for constructing genome-scale metabolic models in high throughput. New software solutions are needed to allow efficient comparative analysis of multiple models in the context of multiple cellular objectives. Here, we present the user-friendly software framework Multi-Metabolic Evaluator (MultiMetEval), built upon SurreyFBA, which allows the user to compose collections of metabolic models that together can be subjected to flux balance analysis. Additionally, MultiMetEval implements functionalities for multi-objective analysis by calculating the Pareto front between two cellular objectives. Using a previously generated dataset of 38 actinobacterial genome-scale metabolic models, we show how these approaches can lead to exciting novel insights. Firstly, after incorporating several pathways for the biosynthesis of natural products into each of these models, comparative flux balance analysis predicted that species like Streptomyces that harbour the highest diversity of secondary metabolite biosynthetic gene clusters in their genomes do not necessarily have the metabolic network topology most suitable for compound overproduction. Secondly, multi-objective analysis of biomass production and natural product biosynthesis in these actinobacteria shows that the well-studied occurrence of discrete metabolic switches during the change of cellular objectives is inherent to their metabolic network architecture. Comparative and multi-objective modelling can lead to insights that could not be obtained by normal flux balance analyses. MultiMetEval provides a powerful platform that makes these analyses straightforward for biologists. Sources and binaries of MultiMetEval are freely available from https://github.com/PiotrZakrzewski/MetEval/downloads.     

Consistency Analysis of Genome-Scale Models of Bacterial Metabolism: A Metamodel Approach

Genome-scale metabolic models usually contain inconsistencies that manifest as blocked reactions and gap metabolites. With the purpose to detect recurrent inconsistencies in metabolic models, a large-scale analysis was performed using a previously published dataset of 130 genome-scale models. The results showed that a large number of reactions (~22%) are blocked in all the models where they are present. To unravel the nature of such inconsistencies a metamodel was construed by joining the 130 models in a single network. This metamodel was manually curated using the unconnected modules approach, and then, it was used as a reference network to perform a gap-filling on each individual genome-scale model. Finally, a set of 36 models that had not been considered during the construction of the metamodel was used, as a proof of concept, to extend the metamodel with new biochemical information, and to assess its impact on gap-filling results. The analysis performed on the metamodel allowed to conclude: 1) the recurrent inconsistencies found in the models were already present in the metabolic database used during the reconstructions process; 2) the presence of inconsistencies in a metabolic database can be propagated to the reconstructed models; 3) there are reactions not manifested as blocked which are active as a consequence of some classes of artifacts, and; 4) the results of an automatic gap-filling are highly dependent on the consistency and completeness of the metamodel or metabolic database used as the reference network. In conclusion the consistency analysis should be applied to metabolic databases in order to detect and fill gaps as well as to detect and remove artifacts and redundant information.     

Multiscale Metabolic Modeling of C4 Plants: Connecting Nonlinear Genome-Scale Models to Leaf-Scale Metabolism in Developing Maize Leaves

C4 plants, such as maize, concentrate carbon dioxide in a specialized compartment surrounding the veins of their leaves to improve the efficiency of carbon dioxide assimilation. Nonlinear relationships between carbon dioxide and oxygen levels and reaction rates are key to their physiology but cannot be handled with standard techniques of constraint-based metabolic modeling. We demonstrate that incorporating these relationships as constraints on reaction rates and solving the resulting nonlinear optimization problem yields realistic predictions of the response of C4 systems to environmental and biochemical perturbations. Using a new genome-scale reconstruction of maize metabolism, we build an 18000-reaction, nonlinearly constrained model describing mesophyll and bundle sheath cells in 15 segments of the developing maize leaf, interacting via metabolite exchange, and use RNA-seq and enzyme activity measurements to predict spatial variation in metabolic state by a novel method that optimizes correlation between fluxes and expression data. Though such correlations are known to be weak in general, we suggest that developmental gradients may be particularly suited to the inference of metabolic fluxes from expression data, and we demonstrate that our method predicts fluxes that achieve high correlation with the data, successfully capture the experimentally observed base-to-tip transition between carbon-importing tissue and carbon-exporting tissue, and include a nonzero growth rate, in contrast to prior results from similar methods in other systems.     

Reconstruction and Validation of a Genome-Scale Metabolic Model for the Filamentous Fungus Neurospora crassa Using FARM

The filamentous fungus Neurospora crassa played a central role in the development of twentieth-century genetics, biochemistry and molecular biology, and continues to serve as a model organism for eukaryotic biology. Here, we have reconstructed a genome-scale model of its metabolism. This model consists of 836 metabolic genes, 257 pathways, 6 cellular compartments, and is supported by extensive manual curation of 491 literature citations. To aid our reconstruction, we developed three optimization-based algorithms, which together comprise Fast Automated Reconstruction of Metabolism (FARM). These algorithms are: LInear MEtabolite Dilution Flux Balance Analysis (limed-FBA), which predicts flux while linearly accounting for metabolite dilution; One-step functional Pruning (OnePrune), which removes blocked reactions with a single compact linear program; and Consistent Reproduction Of growth/no-growth Phenotype (CROP), which reconciles differences between in silico and experimental gene essentiality faster than previous approaches. Against an independent test set of more than 300 essential/non-essential genes that were not used to train the model, the model displays 93% sensitivity and specificity. We also used the model to simulate the biochemical genetics experiments originally performed on Neurospora by comprehensively predicting nutrient rescue of essential genes and synthetic lethal interactions, and we provide detailed pathway-based mechanistic explanations of our predictions. Our model provides a reliable computational framework for the integration and interpretation of ongoing experimental efforts in Neurospora, and we anticipate that our methods will substantially reduce the manual effort required to develop high-quality genome-scale metabolic models for other organisms.     

Computational Analysis of Reciprocal Association of Metabolism and Epigenetics in the Budding Yeast: A Genome-Scale Metabolic Model (GSMM) Approach

Metaboloepigenetics is a newly coined term in biological sciences that investigates the crosstalk between epigenetic modifications and metabolism. The reciprocal relation between biochemical transformations and gene expression regulation has been experimentally demonstrated in cancers and metabolic syndromes. In this study, we explored the metabolism-histone modifications crosstalk by topological analysis and constraint-based modeling approaches in the budding yeast. We constructed nine models through the integration of gene expression data of four mutated histone tails into a genome-scale metabolic model of yeast. Accordingly, we defined the centrality indices of the lowly expressed enzymes in the undirected enzyme-centric network of yeast by CytoHubba plug-in in Cytoscape. To determine the global effects of histone modifications on the yeast metabolism, the growth rate and the range of possible flux values of reactions, we used constraint-based modeling approach. Centrality analysis shows that the lowly expressed enzymes could affect and control the yeast metabolic network. Besides, constraint-based modeling results are in a good agreement with the experimental findings, confirming that the mutations in histone tails lead to non-lethal alterations in the yeast, but have diverse effects on the growth rate and reveal the functional redundancy.     

SMAD4在嗜甲醇酵母中的表达及鉴定

Ｓｍａｄ４是新近发现的一种肿瘤抑制基因。在５０％的胰腺癌和３０％的结肠癌中,此基因发生突变或缺失。利用Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ酵母表达系统,以胞外分泌的方法表达了ＳＭＡＤ４蛋白,并对ＳＭＡＤ４蛋白的生化性质进行了分析。结果显示表达蛋白的分子量约６７ｋＤ,经Ｗｅｓｔｅｒｎ印迹鉴定,与ＳＭＡＤ４抗全有特异的结合反应,Ｎ末端１３个氨基酸的序列与Ｓｍａｄ４ ｃＤＮＡ推断的序列完全一致。     

结核杆菌热休克蛋白70在毕赤酵母中的分泌表达与鉴定

获得结核杆菌热休克蛋白70在毕赤酵母中的分泌表达。构建了酵母表达质粒pPIC9K-hsp70,并将其线性化后用电穿孔法导入Pichia pastoris GS115,经PCR方法筛选出阳性菌落,在0.5%甲醇诱导下分泌表达。所得产物经离心收集上清、超滤浓缩脱盐、亲和层析后,分别用 SDSPAGE、Western blot和动物免疫实验对上清中的重组Hsp70进行鉴定,并考察产物对DC的作用。经SDSPAGE、Western blot分析表明表达的Hsp70表观分子量为70kD并能特异性地与抗Mt.Hsp70单抗结合,动物实验表明重组的Hsp70能在体诱导免疫应答。重组Hsp70能够诱导DC成熟并释放Th1型细胞因子。摇瓶发酵表达量达120mg/L,占培养上清30%以上。这为研究结核杆菌热休克蛋白70的生理功能提供了必要的物质条件。     

人源类溶菌酶蛋白6在毕赤酵母中的重组表达及活性分析

利用毕赤酵母(Pichia pastoris)重组表达人源类溶菌酶蛋白6(human lysozyme-like protein6,h Lyzl6),对其酶学性质进行分析。根据毕赤酵母密码子偏爱性设计并人工合成h Lyzl6基因,将其连接至含有乙醇氧化酶启动子(AOX1)的p PIC9K质粒构建重组表达载体p PIC9K-hlyzl6;重组表达载体经线性化后电转化入毕赤酵母GS115感受态细胞,经G418筛选获得高拷贝重组菌株后进行甲醇诱导表达。经甲醇诱导72 h后发酵液上清中酶活性达到最高值,发酵液上清经SDSPAGE检测在14.8 k Da处有重组h Lyzl6蛋白条带,分子量符合预期,通过甲壳素亲和层析可对其进行纯化;采用比浊法测定h Lyzl6酶学活性,结果表明h Lyzl6对溶壁微球菌(Micrococcus lysodeikticus)有较好的杀灭作用,最适反应温度为40℃,最适p H为5.5,其酶活力为54 700U/mg,Cu2+对其活性有明显抑制,EC50为30.2799 mg/L。采用基因工程方法首次在毕赤酵母GS115成功表达了重组h Lyzl6,证实其在体外具有杀菌活性,初步揭示h Lyzl6在男性生殖系统先天性免疫中发挥了一定作用,为进一步研究h Lyzl6的功能和应用开发奠定了基础。     

The Energy Landscape,Folding Pathways and the Kinetics of a Knotted Protein

The folding pathway and rate coefficients of the folding of a knotted protein are calculated for a potential energy function with minimal energetic frustration. A kinetic transition network is constructed using the discrete path sampling approach, and the resulting potential energy surface is visualized by constructing disconnectivity graphs. Owing to topological constraints, the low-lying portion of the landscape consists of three distinct regions, corresponding to the native knotted state and to configurations where either the N or C terminus is not yet folded into the knot. The fastest folding pathways from denatured states exhibit early formation of the N terminus portion of the knot and a rate-determining step where the C terminus is incorporated. The low-lying minima with the N terminus knotted and the C terminus free therefore constitute an off-pathway intermediate for this model. The insertion of both the N and C termini into the knot occurs late in the folding process, creating large energy barriers that are the rate limiting steps in the folding process. When compared to other protein folding proteins of a similar length, this system folds over six orders of magnitude more slowly.     

人骨保护素在毕赤酵母中的分泌表达及表达产物的生物活性分析(英文)

为了在毕赤酵母表达系统中分泌表达人骨保护素 (osteoprotegerin ,OPG) ,以人骨肉瘤细胞系MG6 3的mRNA为模板 ,采用RT PCR法得到人OPG编码区cDNA ,克隆入毕赤酵母表达载体pPICZ B ,电转化毕赤酵母GS115 (Mut+) ,经 3%甲醇诱导分泌表达人OPG与组氨酸的融合蛋白 .SDS PAGE及Western印迹分析表明 ,有分子量约 6 6kD的目的蛋白表达 .纯化后的表达产物加入体外培养的小鼠骨髓细胞培养基中 ,当浓度为 10 0ng ml时 ,象牙片上骨吸收陷窝的数量及玻片上的TRAP阳性多核细胞的数量均减少 (P <0 0 5 ) .而同时加入人OPG的多克隆抗体后 ,这一抑制作用可被拮抗 ,在浓度为 5 0ng ml时则无此作用 .人OPG蛋白在酵母系统的成功表达 ,为该蛋白的进一步应用研究提供了依据 .     

Deciphering Protein Glycosylation by Computational Integration of On-chip Profiling,Glycan-array Data,and Mass Spectrometry

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Highlights

• •Method to probe the isomeric variants of the glycans attached to purified proteins.
• •Uses multiple rounds of glycosidase cleavage and lectin profiling.
• •Computation integration of lectin-binding, glycan-array, and mass spectrometry data.
• •Applied to microspots for compatibility with analyzing low-abundance proteins.

     

分子伴侣及信号肽对毕赤酵母中分泌蛋白表达水平的影响

目的：探索定位于细胞质、内质网膜及内质网腔中的分子伴侣及其组合对于带有不同信号肽的胞外β-1,3-葡聚糖酶（EXGl）在巴斯德毕赤酵母GS200中表达水平的影响。方法：通过融合PCR技术分别构建带有酵母a交配因子引导肽序列（仅MF）、酵母仅交配因子信号肽序列（ccPre）和重链结合蛋白（Bip）信号肽序列的报告蛋白EXGl的表达质粒pPIC9-EXG1,同时构建分子伴侣基因及其组合的表达质粒pBLArg-IV,然后将2种重组质粒共转化至毕赤酵母宿主菌GS200,转化子经筛选获得共表达菌株,通过测定EXG1酶活来评价分子伴侣与信号肽对其表达水平的影响。结果：细胞质及内质网膜上的分子伴侣Sec61a、Sec61B及胞质中的分子伴侣Ydjl、Ssal、Hsp104及其组合对各种信号肽引导的报告蛋白EXG1的表达水平没有显著影响。然而,内质网腔中的分子伴侣Bip、EroI、PDI与HacI组合能显著提高报告蛋白EXG1的表达水平,其中,以aMF或ctPre作为信号肽引导的报告蛋白EXG1的表达水平分别提高了2．6倍和3．8倍,以Bip信号肽引导的报告蛋白EXGl的表达水平提高了20％～45％,而对于以EXG1自身信号肽引导的报告蛋白EXG1的表达水平没有显著影响。结论：在酵母表达体系中,内质网腔中的分子伴侣是报告蛋白EXG1表达水平的重要影响因素．但分子伴侣对于信号肽的选择性还须进一步证明。     

Cloning, expression, and purification of lipoprotein-associated phospholipase A2 in Pichia pastoris

Lipoprotein-associated phospholipase A₂ (Lp-PLA₂) has been shown to play a crucial role in atherosclerosis, and has been proposed as a promising target for drug discovery. Here, we cloned the Lp-PLA ₂ gene from differentiated THP-1 cells, and inserted a carboxy-terminal His₆-tagged version of the gene into the pPIC9 Pichia expression vector. The Lp-PLA₂ fusion protein was successfully expressed in Pichia pastoris expression system and could be rapidly purified to apparent homogeneity using a single-step purification method. The activity of our recombinant Lp-PLA₂ was strong when [³H] PAF was used as a substrate, and the Lp-PLA₂ inhibitor SB435495 exhibited an inhibitory curve against the recombinant Lp-PLA₂ (IC₅₀=15.93±1 μM). This novel recombinant Lp-PLA₂ could prove useful as a screening model for Lp-PLA₂ inhibitors, and may facilitate further investigation of this protein in atherosclerosis.     

HBcAg基因在甲醇型酵母中的表达、产物纯化及其性质的初步研究

为研究乙肝核心抗原蛋白(HBcAg)在甲醇型酵母(PichiaPastoris)中的表达和性质,用PCR方法将HBcAg基因(L)克隆到P.Pastoris胞内表达载体pPIC3k中,并利用电击和同源重组法,将重组质粒pPIC3kL转化感受态的甲醇型GS115酵母菌株。经过筛选得到阳性P.Pastoris重组子。重组菌株经甲醇诱导培养,表达产物的Western印迹结果表明,HBcAg蛋白能在甲醇型酵母(PichiaPastoris)中诱导表达,产物为一215kDa的蛋白。经蔗糖密度梯度超离心和CsCl密度梯度超离心纯化后,ELISA和密度测定结果表明重组HBcAg蛋白主要分布在密度为12576gml和13013gml的2个峰值处。电镜观察表明,该重组HBcAg蛋白能自主装配成大小不同的2种颗粒(即核心颗粒),大颗粒直径约34nm左右,小颗粒直径约30nm左右。同时,我们还观察到,该核心蛋白颗粒在体外可发生集聚现象。     

CALA在毕赤酵母中的表达、纯化及酶学特性研究

将南极假丝酵母脂肪酶A(cala)基因克隆至组成型表达载体pGAPZαA中,电激转入X-33,获得高效表达的CALA酵母工程菌株.发酵液上清经超滤浓缩、硫酸铵沉淀和阴离子交换层析等步骤,获得纯化的重组CALA,其比酶活达384.90 U/mg.该酶最适温度为70℃,最适pH值为8.0.经50℃保温2 h,仍含有60%水解酶活力;在pH7.0和8.0溶液中比较稳定.经DMSO处理1 h,仍保持90%的活性;非离子型表面活性剂能提高CALA的酶活,金属离子在不同程度上抑制CALA的酶活.     

Protein expression and purification of human Zbtb7A in Pichia pastoris via gene codon optimization and synthesis

Human Zbtb7A was proved to be an important molecular switch in oncogenesis. However, it is difficult to obtain its protein expression in prokaryotic system, due to high G+C content and rare codons in zbtb7a gene. Therefore, to further research the function and application of this protein, we optimized its coding sequence according to the codon bias of Pichia pastoris, synthesized the sequence with two-step PCR and confirmed the accuracy by DNA sequencing. The assembled fragment was introduced into P. pastoris expression vector pPIC9K and the resultant plasmid pPIC9K-zbtb7a-his(6) was transformed into the P. pastoris strain GS115 by electroporation. The products of the transformants induced by methanol were analyzed by 10% SDS-PAGE and identified by Western Blot assay. The expression conditions of the selected transformant were optimized. Additionally, a two-step purification protocol was applied to purify the recombinant protein. The results showed that the synthetic coding sequence of human Zbtb7A was successfully obtained and inserted into pPIC9K vector. Human Zbtb7A protein was expressed in P. pastoris and identified by western blot. The optimal conditions for its expression in P. pastoris were under a final concentration of 1% methanol and a time-course of 4d. Through the two-step purification, Zbtb7A protein was purified in high purity and its production reached up to as high as 18 mg/L. These results indicated that an effective procedure for expressing and purifying human Zbtb7A in P. pastoris was established.     

SARS冠状病毒核衣壳蛋白在酵母中的表达与活性检测

用PCR扩增SARS冠状病毒N蛋白全长cDNA,克隆到酵母表达载体pPIC3.5K,构建pPIC3.5K-SCoVN酵母表达质粒。表达质粒线性化后电转化到毕赤酵母GS115中,经G418-RDB, MM/MD平板与PCR扩增筛选获得His⁺ Mut⁺ 重组菌株。比较研究了不同的培养基、溶解氧以及甲醇浓度对菌株生长与重组蛋白表达的影响。结果表明：FBS培养基最适宜重组菌的生长与表达,溶氧对菌体的生长与表达有显著的影响,甲醇诱导最佳终浓度为1％（V/V）,SDS-PAGE分析重组蛋白的表达量,发现重组N 蛋白表达量占细胞总蛋白的6％,每升培养基可以生产410mg重组N蛋白,生物量达45OD600。Western　blotting结果表明,重组N 蛋白对鼠源单克隆抗体以及SARS病人恢复期血清具有较强的特异性反应。对摇瓶培养条件进行了发酵罐放大实验,结果生物量达到348OD600,表达量达到 2.5g/L,分别为摇瓶表达的7.7倍和6.1倍,为SARS早期血清学诊断研究以及为N蛋白在病毒复制以及致病机理的研究奠定了一定的基础。