基于比较代谢组学的理性优化方法提高阿维菌素产量

【目的】利用比较代谢组学的分析方法,研究不同发酵培养基中阿维链霉菌的胞内代谢差异,揭示合成阿维菌素的关键代谢物和代谢途径,再通过理性优化添加主要关键代谢物,提高阿维菌素产量。【方法】对M1和M2培养基中生长的菌体进行基于GC-MS的胞内代谢组学分析,通过理性添加强化前体代谢物,确定阿维菌素高产培养基。【结果】GC-MS共检测到232种物质,能够精确匹配70种胞内代谢物,通过PCA和PLS分析,最终确定了21种已知的胞内代谢物与阿维菌素的生物合成密切相关。其中乳酸、丙酮酸、琥珀酸、苏氨酸、异亮氨酸、缬氨酸和油脂类物质对阿维菌素的产量影响较为显著。通过单独或组合优化添加这些前体,阿维菌素的产量从5.36 g/L提高到了5.92 g/L,增加了10.4%。【结论】基于比较代谢组学分析的理性优化培养基的方法可有效提高阿维菌素的产量,并为提高当下生物基产品的产量提供了新思路。     

Improving methionine and ATP availability by <Emphasis Type="Italic">MET6</Emphasis> and <Emphasis Type="Italic">SAM2</Emphasis> co-expression combined with sodium citrate feeding enhanced SAM accumulation in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

S-adenosyl-l-methionine (SAM), biosynthesized from methionine and ATP, exhibited diverse pharmaceutical applications. To enhance SAM accumulation in S. cerevisiae CGMCC 2842 (wild type), improvement of methionine and ATP availability through MET6 and SAM2 co-expression combined with sodium citrate feeding was investigated here. Feeding 6 g/L methionine at 12 h into medium was found to increase SAM accumulation by 38 % in wild type strain. Based on this result, MET6, encoding methionine synthase, was overexpressed, which caused a 59 % increase of SAM. To redirect intracellular methionine into SAM, MET6 and SAM2 (encoding methionine adenosyltransferase) were co-expressed to obtain the recombinant strain YGSPM in which the SAM accumulation was 2.34-fold of wild type strain. The data obtained showed that co-expression of MET6 and SAM2 improved intracellular methionine availability and redirected the methionine to SAM biosynthesis. To elevate intracellular ATP levels, 6 g/L sodium citrate, used as an auxiliary energy substrate, was fed into the batch fermentation medium, and an additional 19 % increase of SAM was observed after sodium citrate addition. Meanwhile, it was found that addition of sodium citrate improved the isocitrate dehydrogenase activity which was associated with the intracellular ATP levels. The results demonstrated that addition of sodium citrate improved intracellular ATP levels which promoted conversion of methionine into SAM. This study presented a feasible approach with considerable potential for developing highly SAM-productive strains based on improving methionine and ATP availability.     

Effects of aeration on metabolic profiles of <Emphasis Type="Italic">Mortierella alpina</Emphasis> during the production of arachidonic acid

To investigate the metabolic regulation against oxygen supply, comparative metabolomics was performed to explore the metabolic responses of Mortierella alpina in the process of arachidonic acid (ARA) production. More than 110 metabolites involved in Embden–Meyerhof–Parnas pathway, pentose phosphate pathway, tricarboxylic acid cycle, inositol phosphate metabolism, fatty acid biosynthesis, and amino acid metabolism were identified by gas chromatography–mass spectrometry. Samples at different aeration rates were clearly distinguished by principal components analysis and partial least squares analysis, indicating that oxygen supply had a profound effect on the metabolism of M. alpina. Eleven major metabolites were identified as potential biomarkers to be primarily responsible for the difference of metabolism. Further study of metabolic changes with the relevant pathways demonstrated that the levels of several intermediate metabolites in relation to central carbon metabolism changed remarkably via both processes and citrate and malate was supposed to play vital roles in polyunsaturated acid (PUFA) synthesis. Increase of myo-inositol and sorbitol were probably for osmo-regulation and redox balance, while enhanced phosphoric acid and pyroglutamic acid were supposed to have function in the activation of signal transduction pathway for stress resistance. The present study provides a novel insight into the metabolic responses of M. alpina to aeration rates and the metabolic characteristics during the ARA fermentation.     

Metabolic response of <Emphasis Type="Italic">Tetragenococcus halophilus</Emphasis> under salt stress

In this study, the effect of salt stress on metabolic response of Tetragenecoccus halophilus was investigated, and the metabolic alternations were analyzed using liquid chromatography-mass spectrometry according to the metabolomics approach. A total of 81 intracellular metabolites were identified, and significant differences were observed in the levels of metabolites mainly participating in central carbon metabolism, fatty acid metabolism, and amino acid metabolism. Analysis of the membrane fatty acid distribution showed that higher proportions of unsaturated fatty acid were observed in salt-treated cells. Additionally, salt-stressed cells exhibited higher amounts of compatible solutes including proline, glycine, citrulline, and N-acetyltyrptophan, and lower amounts of branched-chain amino acids. Interestingly, higher amounts of indole, salicylic acid, and coronatine, which are regarded as signaling molecule and suggested to combat osmotic stress, were detected in salt-shocked cells compared with the untreated cells. Taken together, these results suggested that increased unsaturated membrane fatty acids, accumulation of compatible solutes, and up-regulation of signaling molecule may be potential mechanisms employed by T. halophilus during salt stress.     

<Superscript>1</Superscript>H NMR analysis of <Emphasis Type="Italic">Citrus macrophylla</Emphasis> subjected to Asian citrus psyllid (<Emphasis Type="Italic">Diaphorina citri</Emphasis> Kuwayama) feeding

The Asian citrus psyllid (ACP) is a phloem-feeding insect that can host and transmit the bacterium Candidatus Liberibacter asiaticus (CLas), which is the putative causative agent of the economically important citrus disease, Huanglongbing (HLB). ACP are widespread in Florida, and are spreading in California; they are the primary mode of CLas transmission in citrus groves. To understand the effects of ACP feeding, different numbers of ACP [0 ACP (control), 5 ACP (low), 15–20 ACP (medium), and 25–30 ACP (high)] were allowed to feed on Citrus macrophylla greenhouse plants. After 7 days of feeding, leaves were collected and analyzed using ¹H NMR. Metabolite concentrations from leaves of trees with ACP feeding had higher variability than control trees. Many metabolites were higher in concentration in the low ACP feeding group relative to control; however, leaves from trees with high ACP feeding had lower concentrations of many metabolites relative to control, including many amino acids such as phenylalanine, arginine, isoleucine, valine, threonine, and leucine. These results suggest ACP density-dependent changes in primary metabolism that can be measured by ¹H NMR. The implications in plant defense are discussed.     

Intracellular Metabolic Changes of <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. LH During the Biodegradation of Diesel Oil

In recent years, some marine microbes have been used to degrade diesel oil. However, the exact mechanisms underlying the biodegradation are still poorly understood. In this study, a hypothermophilous marine strain, which can degrade diesel oil in cold seawater was isolated from Antarctic floe-ice and identified and named as Rhodococcus sp. LH. To clarify the biodegradation mechanisms, a gas chromatography-mass spectrometry (GC-MS)-based metabolomics strategy was performed to determine the diesel biodegradation process-associated intracellular biochemical changes in Rhodococcus sp. LH cells. With the aid of partial least squares-discriminant analysis (PLS-DA), 17 differential metabolites with variable importance in the projection (VIP) value greater than 1 were identified. Results indicated that the biodegradation of diesel oil by Rhodococcus sp. LH was affected by many different factors. Rhodococcus sp. LH could degrade diesel oil through terminal or sub-terminal oxidation reactions, and might also possess the ability to degrade aromatic hydrocarbons. In addition, some surfactants, especially fatty acids, which were secreted by Rhodococcus into medium could also assist the strain in dispersing and absorbing diesel oil. Lack of nitrogen in the seawater would lead to nitrogen starvation, thereby restraining the amino acid circulation in Rhodococcus sp. LH. Moreover, nitrogen starvation could also promote the conversation of relative excess carbon source to storage materials, such as 1-monolinoleoylglycerol. These results would provide a comprehensive understanding about the complex mechanisms of diesel oil biodegradation by Rhodococcus sp. LH at the systematic level.     

Progress in the microbial production of <Emphasis Type="Italic">S</Emphasis>-adenosyl-<Emphasis Type="SmallCaps">l</Emphasis>-methionine

S-Adenosyl-l-methionine (SAM), which exists in all living organisms, serves as an activated group donor in a range of metabolic reactions, including trans-methylation, trans-sulfuration and trans-propylamine. Compared with its chemical synthesis and enzyme catalysis production, the microbial production of SAM is feasible for industrial applications. The current clinical demand for SAM is constantly increasing. Therefore, vast interest exists in engineering the SAM metabolism in cells for increasing product titers. Here, we provided an overview of updates on SAM microbial productivity improvements with an emphasis on various strategies that have been used to enhance SAM production based on increasing the precursor and co-factor availabilities in microbes. These strategies included the sections of SAM-producing microbes and their mutant screening, optimization of the fermentation process, and the metabolic engineering. The SAM-producing strains that were used extensively were Saccharomyces cerevisiae, Pichia pastoris, Candida utilis, Scheffersomyces stipitis, Kluyveromyces lactis, Kluyveromyces marxianus, Corynebacterium glutamicum, and Escherichia coli, in addition to others. The optimization of the fermentation process mainly focused on the enhancement of the methionine, ATP, and other co-factor levels through pulsed feeding as well as the optimization of nitrogen and carbon sources. Various metabolic engineering strategies using precise control of gene expression in engineered strains were also highlighted in the present review. In addition, some prospects on SAM microbial production were discussed.     

Changes in intracellular metabolism underlying the adaptation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains to ethanol stress

Saccharomyces cerevisiae is often stressed by the ethanol which accumulates during the production of bioethanol by the fermentation process. The study of ethanol-adapted S. cerevisiae strains provide an opportunity to clarify the molecular mechanism underlying the adaptation or tolerance of S. cerevisiae to ethanol stress. The aim of this study was to clarify this molecular mechanism by investigating the ethanol adaptation-associated intracellular metabolic changes in S. cerevisiae using a gas chromatography–mass spectrometry-based metabolomics strategy. A partial least-squares-discriminant analysis between the parental strain and ethanol-adapted strains identified 12 differential metabolites of variable importance with a projection value of >1. The ethanol-adapted strains had a more activated glycolysis pathway and higher energy production than the parental strain, suggesting the possibility that an increased energy production and energy requirement might be partly responsible for an increased ethanol tolerance. An increased glycine content also partly contributed to the higher ethanol tolerance of the ethanol-adapted strains. The decreased oleic acid content may be a self-protection mechanism of ethanol-adapted strains to maintain membrane integrity through decreasing membrane fluidity. We suggest that while being exposed to ethanol stress, ethanol-adapted S. cerevisiae cells may remodel their metabolic phenotype and the composition of their cell membrane to adapt to ethanol stress and acquire higher ethanol tolerance.     

LC–MS-based metabolome analysis on steroid metabolites from the starfish <Emphasis Type="Italic">Patiria</Emphasis> (=<Emphasis Type="Italic">Asterina</Emphasis>) <Emphasis Type="Italic">pectinifera</Emphasis> in conditions of active feeding and stresses

Introduction

Starfish are recognized as interesting source of natural steroid products with pharmaceutical potential. Polar steroid metabolites of starfish have unique chemical structures and exhibit various biological activities but their biological functions are controversial.

Objectives

The objective of this study was to investigate the response of polar steroid metabolome of the starfish Patiria (=Asterina) pectinifera on various environmental factors and stresses.

Methods

Here we first have applied MS-based environmental metabolomics to elucidate the metabolic changes of polar steroid metabolome of starfish. Using HPLC–ESI–Q/TOF–MS approach followed by statistical analysis including principal component analysis and partial least squares discriminant analysis for data classification and potential biomarkers selection, we investigated the changes induced by feeding, injury, variations in water temperature and salinity, and oxygen deficiency.

Results

According to multivariate and univariate statistical analysis the responses to feeding, injury and water heating were better expressed than the others and have some similarity in their action on the steroid metabolome of the starfish P. pectinifera. Most constituents of asterosaponin pool were reduced and most constituents of polyhydroxysteroid and related glycoside pool were increased at that.

Conclusion

Our results indicate that various metabolic changes in polar steroid constituents of P. pectinifera are induced by feeding and stresses. We believe that these responses are connected with biological multifunctionality of these compounds.

     

Metabolic profiling of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> metamorphosis: a new insight into the central metabolic pathways

Introduction

Metamorphosis is a complicated process in which cell proliferation, differentiation, and death are orchestrated to form the mature structures of insects. In Drosophila, this process is controlled by ecdysone, a steroid hormone responsible for tissue remodeling and organogenesis that gives rise to the adult fly.

Objective

By using a metabolomics approach, this study aimed to elucidate global changes in the central metabolic pathways in Drosophila throughout metamorphosis and then further examine the effects of temperature and origin on metabolic profiles.

Methods

Targeted and non-targeted metabolic profiling of time-course samples from Drosophila were constructed to cover a wide range of cellular metabolites during metamorphosis.

Results

This was the first wide-scale metabolomics study of Drosophila metamorphosis focusing on central metabolism. The abundance of detected metabolites changed drastically and correlated strongly with the development of Drosophila pupae. In non-stress conditions, temperature affected the developmental time, but the metabolic state at a certain stage of metamorphosis remained stable. Between D. melanogaster Canton S and Oregon R, similar metabolic profiles throughout metamorphosis was observed. However, there were still differences in purine and pyrimidine metabolism at an early stage in the pupal period, which was matched by differences in ecdysteroid levels.

Conclusion

This study supported the strength of metabolomics in the field of developmental biology. The results provided a general view on the metabolic profile of Drosophila during metamorphosis, which provides basic 3 knowledge for future metabolomics studies using Drosophila.

     

A metabolomics and proteomics study of the <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> in the grass carp fermentation

Background

Lactobacillus plantarum, a versatile lactic acid-fermenting bacterium, isolated from the traditional pickles in Ningbo of China, was chosen for grass carp fermentation, which could also improve the flavor of grass carp. We here explored the central metabolic pathways of L. plantarum by using metabolomic approach, and further proved the potential for metabolomics combined with proteomics approaches for the basic research on the changes of metabolites and the corresponding fermentation mechanism of L. plantarum fermentation.

Results

This study provides a cellular material footprinting of more than 77 metabolites and 27 proteins in L. plantarum during the grass carp fermentation. Compared to control group, cells displayed higher levels of proteins associated with glycolysis and nucleotide synthesis, whereas increased levels of serine, ornithine, aspartic acid, 2-piperidinecarboxylic acid, and fumarate, along with decreased levels of alanine, glycine, threonine, tryptophan, and lysine.

Conclusions

Our results may provide a deeper understanding of L. plantarum fermentation mechanism based on metabolomics and proteomic analysis and facilitate future investigations into the characterization of L. plantarum during the grass carp fermentation.

     

NMR-based metabolomics reveals the metabolite profiles of <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> under ferric iron stimulation

Vibrio parahaemolyticus is a halophilic bacterium endemic to coastal areas, and its pathogenicity has caused widespread seafood poisoning. In our previous research, the protein expression of V. parahaemolyticus in Fe³⁺ medium was determined using isobaric tags for relative and absolute quantitation (iTRAQ). Here, nuclear magnetic resonance (NMR) was used to detect changes in the V. parahaemolyticus metabolome. NMR spectra were obtained using methanol-water extracts of intracellular metabolites from V. parahaemolyticus under various culture conditions, and 62 metabolites were identified, including serine, arginine, alanine, ornithine, tryptophan, glutamine, malate, NAD⁺, NADP⁺, oxypurinol, xanthosine, dCTP, uracil, thymine, hypoxanthine, and betaine. Among these, 21 metabolites were up-regulated after the stimulation of the cells by ferric iron, and 9 metabolites were down-regulated. These metabolites are involved in amino acid and protein synthesis, energy metabolism, DNA and RNA synthesis and osmolality. Based on these results, we conclude that Fe³⁺ influences the metabolite profiles of V. parahaemolyticus.     

Combining metabolomics and network analysis to improve tacrolimus production in <Emphasis Type="Italic">Streptomyces tsukubaensis</Emphasis> using different exogenous feedings

Tacrolimus is widely used as an immunosuppressant in the treatment of various autoimmune diseases. However, the low fermentation yield of tacrolimus has thus far restricted its industrial applications. To solve this problem, the time-series response mechanisms of the intracellular metabolism that were highly correlated with tacrolimus biosynthesis were investigated using different exogenous feeding strategies in S. tsukubaensis. The metabolomic datasets, which contained 93 metabolites, were subjected to weighted correlation network analysis (WGCNA), and eight distinct metabolic modules and seven hub metabolites were identified to be specifically associated with tacrolimus biosynthesis. The analysis of metabolites within each metabolic module suggested that the pentose phosphate pathway (PPP), shikimate and aspartate pathway might be the main limiting factors in the rapid synthesis phase of tacrolimus accumulation. Subsequently, all possible key-limiting steps in the above metabolic pathways were further screened using a genome-scale metabolic network model (GSMM) of S. tsukubaensis. Based on the prediction results, two newly identified targets (aroC and dapA) were overexpressed experimentally, and both of the engineered strains showed higher tacrolimus production. Moreover, the best strain, HT-aroC/dapA, that was engineered to simultaneously enhanced chorismate and lysine biosynthesis was able to produce 128.19 mg/L tacrolimus, 1.64-fold higher than control (78.26 mg/L). These findings represent a valuable addition to our understanding of tacrolimus accumulation in S. tsukubaensis, and pave the way to further production improvements.     

Metabolic derangements identified through untargeted metabolomics in a cross-sectional study of Nigerian children with severe acute malnutrition

Introduction

Severe acute malnutrition (SAM) is a major cause of child mortality worldwide, however the pathogenesis of SAM remains poorly understood. Recent studies have uncovered an altered gut microbiota composition in children with SAM, suggesting a role for microbes in the pathogenesis of malnutrition.

Objectives

To elucidate the metabolic consequences of SAM and whether these changes are associated with changes in gut microbiota composition.

Methods

We applied an untargeted multi-platform metabolomics approach [gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS)] to stool and plasma samples from 47 Nigerian children with SAM and 11 control children. The composition of the stool microbiota was assessed by 16S rRNA gene sequencing.

Results

The plasma metabolome discriminated children with SAM from controls, while no significant differences were observed in the microbial or small molecule composition of stool. The abundance of 585 features in plasma were significantly altered in malnourished children (Wilcoxon test, FDR corrected P?<?0.1), representing approximately 15% of the metabolome. Consistent with previous studies, children with SAM exhibited a marked reduction in amino acids/dipeptides and phospholipids, and an increase in acylcarnitines. We also identified numerous metabolic perturbations which have not been reported previously, including increased disaccharides, truncated fibrinopeptides, angiotensin I, dihydroxybutyrate, lactate, and heme, and decreased bioactive lipids belonging to the eicosanoid and docosanoid family.

Conclusion

Our findings provide a deeper understanding of the metabolic consequences of malnutrition. Further research is required to determine if specific metabolites may guide improved management, and/or act as novel biomarkers for assessing response to treatment.

     

NMR spectroscopy and mass spectrometry in metabolomics analysis of <Emphasis Type="Italic">Salvia</Emphasis>

The interest in using the ‘-omics’ approach for nutrition, agriculture, food science and human health have seen an explosive growth in the last years. Particularly, metabolomics analysis is becoming an integral part of a system biological approach for investigating organisms. In this review, the limitations and advantages of NMR spectroscopy and mass spectrometry were discussed in details using the study reported in the literature on different Salvia species (S. hispanica, S. miltiorrhiza, S. officinalis, S. runcinata and S. stenophylla). Both approaches identify and quantify several classes of compounds but not the complete metabolite profile of the plant. A combined approach of these two powerful techniques provides better results allowing to determine both primary and secondary metabolites.     

Comparative metabolomics of the interaction between rice and the brown planthopper

Introduction

The interactions between plants and insect herbivores are complex and multifaceted. Rice and its specialist insect pest the brown planthopper (BPH), Nilaparvata lugens Stål (Hemiptera: Delphacidae) constitute an ideal system for studying plant–insect interactions.

Objectives

Combined metabolomics analyses of rice plant and BPH were conducted to understand the mechanism of host rice plant defense and BPH insect response.

Methods

Metabolite dynamics in rice leaf sheath and BPH honeydew was investigated using the gas chromatography–mass spectrometry (GC–MS) method. The GC–MS data were analyzed by principal component analysis and partial least squares-discriminant analysis.

Results

Twenty-six metabolites were detected in the leaf sheath extracts. Rice leaf sheath metabolomics analysis results show that BPH feeding induces distinct changes in the metabolite profiles of YHY15 and TN1 plants. These results suggest that BPH infestation enhance fatty acid oxidation, the glyoxylate cycle, gluconeogenesis and the GABA shunt in TN1 plants, and glycolysis and the shikimate pathway in YHY15. We propose that the BPH15 gene mediates a resistance reaction that increases the synthesis of secondary metabolites through the shikimate pathway. Thirty-three metabolites were identified in BPH honeydew. Honeydew metabolomics analysis results show that when BPH insects were fed on resistant YHY15 plants, most of the amino acids in honeydew were significantly decreased compared to those of BPH fed on TN1 plants. Based on metabolomics results, we propose that BPH feeding on resistant YHY15 plants would enhance amino acid absorption. At the same time, urea was significantly increased in BPH fed on YHY15.

Conclusion

Metabolomics study is valuable in understanding the complex and multifaceted interaction between plants and insect herbivores and provide essential clue for development of novel control BPH strategies.