Age-induced Changes in Ginsenoside Accumulation and Primary Metabolic Characteristics of Panax Ginseng in Transplantation Mode.

Background: Ginseng (Panax ginseng Mayer) is an important natural medicine. However, a long culture period and challenging quality control requirements limit its further use. Although artificial cultivation can yield a sustainable medicinal supply, research on the association between the transplantation and chaining of metabolic networks, especially the regulation of ginsenoside biosynthetic pathways, is limited. Methods: Herein, we performed Liquid chromatography tandem mass spectrometry based metabolomic measurements to evaluate ginsenoside accumulation and categorise differentially abundant metabolites (DAMs). Transcriptome measurements using an Illumina Platform were then conducted to probe the landscape of genetic alterations in ginseng at various ages in transplantation mode. Using pathway data and crosstalk DAMs obtained by MapMan, we constructed a metabolic profile of transplantation Ginseng. Results: Accumulation of active ingredients was not obvious during the first 4 years (in the field), but following transplantation, the ginsenoside content increased significantly from 6-8 years (in the wild). Glycerolipid metabolism and Glycerophospholipid metabolism were the most significant metabolic pathways, as Lipids and lipid-like molecule affected the yield of ginsenosides. Starch and sucrose were the most active metabolic pathways during transplantation Ginseng growth. Conclusion: This study expands our understanding of metabolic network features and the accumulation of specific compounds during different growth stages of this perennial herbaceous plant when growing in transplantation mode. The findings provide a basis for selecting the optimal transplanting time.

Deep-learning-assisted Sort-Seq enables high-throughput profiling of gene expression characteristics with high precision.

Owing to the nondeterministic and nonlinear nature of gene expression, the steady-state intracellular protein abundance of a clonal population forms a distribution. The characteristics of this distribution, including expression strength and noise, are closely related to cellular behavior. However, quantitative description of these characteristics has so far relied on arrayed methods, which are time-consuming and labor-intensive. To address this issue, we propose a deep-learning-assisted Sort-Seq approach (dSort-Seq) in this work, enabling high-throughput profiling of expression properties with high precision. We demonstrated the validity of dSort-Seq for large-scale assaying of the dose-response relationships of biosensors. In addition, we comprehensively investigated the contribution of transcription and translation to noise production in Escherichia coli, from which we found that the expression noise is strongly coupled with the mean expression level. We also found that the transcriptional interference caused by overlapping RpoD-binding sites contributes to noise production, which suggested the existence of a simple and feasible noise control strategy in E. coli.

[Qualitative and quantitative study of flavonoids in Notoginseng Radix et Rhizoma based on UPLC-Q-TOF-MS and HPLC-DAD].

The flavonoids in Panax notoginseng were qualitatively analyzed by ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry(UPLC-Q-TOF-MS), and the content of three main flavonoids in P. notoginseng of different specifications and grades collected from different habitats was determined by HPLC-DAD. Flavonoids and anthocyanins were analyzed by UPLC-Q-TOF-MS/MS in the positive and negative ion modes, respectively. Twelve flavonoid glycosides and one anthocyanin glycoside in P. notoginseng were identified, but no flavonoid aglycones were detected. Among them, 12 compounds were identified in the underground part of P. notoginseng for the first time and eight compounds were first reported in this plant. Moreover, six and four compounds were identified in the Panax genus and the Araliaceae family for the first time, respectively. A method for simultaneous determination of three flavonoids in P. notoginseng was established by HPLC-DAD. The content of flavonoids in 721 P. notoginseng samples of 124 specifications and grades collected from 20 different habitats was simultaneously determined. Among three flavonoids determined, the content of quercetin-3-O-(2″-ß-D-xylosyl)-ß-D-galactoside was the highest with the average content in the tested samples of 161.0 µg·g~(-1). The content of compounds quercetin-3-O-hexosyl-hexoside and kaempferol-3-O-pentosyl-hexoside was relatively low, with the average content of 18.5 µg·g~(-1)(calculated as quercetin-3-O-sophoroside) and 49.4 µg·g~(-1)(calculated as kaempferol-3-O-sangbu diglycoside). There were significant differences in flavonoids content of samples from different production area. The content of flavonoids in spring P. notoginseng was significantly lower than that in winter P. notoginseng when the other influencing factors such as production areas, germplasm resources, and cultivation conditions were fixed. As for P. notoginseng of different specifications, the flavonoid content in the part connecting the taproot and the aboveground stem was significantly higher than that in other parts. The results of large-scale data showed that the flavonoid content gradually increased with the increase in the number of heads. There were significant differences between the flavonoid content in most specifications and grades, especially the 20-head P. notoginseng and countless head P. notoginseng, whose content was significantly lower and significantly higher than that of other specifications and grades, respectively. This study provides a scientific basis for the study of the effective components and quality control of P. notoginseng from the perspective of flavonoids.

Spatial transcriptome uncovers rich coordination of metabolism in E. coli K12 biofilm.

Microbial communities often display region-specific properties, which give rise to complex interactions and emergent behaviors that are critical to the homeostasis and stress response of the communities. However, systems-level understanding of these properties still remains elusive. In this study, we established RAINBOW-seq and profiled the transcriptome of Escherichia coli biofilm communities with high spatial resolution and high gene coverage. We uncovered three modes of community-level coordination, including cross-regional resource allocation, local cycling and feedback signaling, which were mediated by strengthened transmembrane transport and spatially specific activation of metabolism. As a consequence of such coordination, the nutrient-limited region of the community maintained an unexpectedly high level of metabolism, enabling it to express many signaling genes and functionally unknown genes with potential sociality functions. Our work provides an extended understanding of the metabolic interplay in biofilms and presents a new approach of investigating complex interactions in bacterial communities on the systems level.

Profiling of bacterial transcriptome from ultra-low input with MiniBac-seq.

There is a lack of appropriate methods for preparing bacterial RNA-seq library with ultra-low amount of RNA. To address this issue, we developed miniBac-seq, a strand-specific method for high-quality library construction from sub-nanogram of total RNA, which is 100-fold lower than the current benchmark kit and dramatically reduces preparation cost ($28 + $15 × samples). We further demonstrated the high sensitivity of miniBac-seq via detecting more than 500 genes from amount of total RNA equivalent to that of a single bacterial cell. Finally, we profiled the transcriptome of growth-arrested bacteria in isogenic culture of Escherichia coli. This subpopulation of bacteria is generally low in abundance but is a potent reservoir of antibiotic persistence, and their gene expression has been largely unknown due to technical limitations. Using miniBac-seq, we identified potential molecular driver towards arrested growth as well as antibiotic tolerance. Our method thus expands the capacity to quantify bacterial transcriptome in situ, which is useful to the understanding of bacterial physiology and regulation in their native contexts.

Jujuboside A inhibits oxidative stress damage and enhances immunomodulatory capacity of human umbilical cord mesenchymal stem cells through up-regulating IDO expression.

Impaired immunomodulatory capacity and oxidative stress are the key factors limiting the effectiveness of mesenchymal stem cell transplantation therapy. The present study was aimed to investigate the effects of jujuboside A (JuA) on the protective effect and immunomodulatory capacity of human umbilical cord mesenchymal stem cells (hUC-MSCs). Hydrogen peroxide was used to establish an oxidative damage model of hUC-MSCs, while PBMCs isolated from rats were used to evaluate the effect of JuA pre-treatment on the immunomodulatory capacity of hUC-MSCs. Furthermore, Hoechst 33258 staining, lactate dehydrogenase test, measurement of malondialdehyde, Western blot, high-performance liquid chromatography; and flow cytometry were performed. Our results indicated that JuA (25 µmol·L-1) promoted the proliferation of hUC-MSCs, but did not affect the differentiating capability of these cells. JuA pre-treatment inhibited apoptosis, prevented oxidative damage, and up-regulated the protein expression of nuclear factor-erythroid factor 2-related factor 2 and heme oxygenase 1 in hUC-MSCs in which oxidative stress was induced with H2O2. In addition, JuA pre-treatment enhanced the inhibitory effect of hUC-MSCs against abnormally activated PBMCs, which was related to stimulation of the expression and activity of indoleamine 2,3-dioxygenase. In conclusion, our results demonstrate that JuA pre-treatment can enhance the survival and immunomodulatory ability through pathways related to oxidative stress, providing a new option for the improvement of hUC-MSCs in the clinical setting.

Integrated metabolomic and transcriptomic analysis reveals variation in the metabolites and genes of Platycodon grandiflorus roots from different regions.

INTRODUCTION: Platycodon grandiflorum root (PG), a popular traditional Chinese medicine, contains considerable chemical components with broad pharmacological activities. The complexity and diversity of the chemical components of PG from different origins contribute to its broad biological activities. The quality of southern PG is superior to that of northern PG, but the mechanisms underlying these differences remain unclear. OBJECTIVES: In order to study variation in the differentially accumulated metabolites (DAMs), differentially expressed genes (DEGs), as well as their interactions and signalling pathways among PG from Anhui and Liaoning. METHODS: The metabolomes based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the transcriptome based on high-throughput sequencing technology were combined to comprehensively analyse PGn and PGb. RESULTS: A total of 6515 DEGs and 83 DAMs from the comparison of PG from Anhui and Liaoning were detected. Integrated analysis of metabolomic and transcriptomic data revealed that 215 DEGs and 57 DAMs were significantly enriched in 48 pathways according to KEGG pathway enrichment analysis, and 15 DEGs and 10 DAMs significantly enriched in the main pathway sesquiterpenoid and triterpenoid and phenylpropanoid biosynthesis might play a key role in complex response or regulatory processes. CONCLUSION: Differences in PG from southern and northern China might thus stem from differences in environmental factors, such as precipitation, light duration, and humidity. The results of our study provide new insight into geographic variation in gene expression and metabolite accumulation and will enhance the utilisation of PG resources.

Role of Gut Microbiota in the Anti-Colitic Effects of Anthocyanin-Containing Potatoes.

SCOPE: Anthocyanin-containing potatoes exert anti-inflammatory activity in colitic mice. Gut bacterial dysbiosis plays a critical role in ulcerative colitis. This study examined the extent to which the anti-colitic activity of anthocyanin-containing red/purple-fleshed potatoes depends on the gut bacteria using a chemically-induced rodent model of colitis with the intact and antibiotic-ablated microbiome. METHODS AND RESULTS: Four-week-old male mice (C57BL6) are randomly assigned to the control diet or 20% purple-/red-fleshed potatoes supplemented diet group. The microbiota-ablated group received an antibiotic cocktail in drinking water. At week nine, colitis is induced by 2% dextran sulfate sodium (DSS) in drinking water for five days. Administration of antibiotics resulted in a 95% reduction in gut bacterial load and fecal SCFAs. DSS-induced elevated gut permeability and body weight loss are more pronounced in antibiotic mice compared to non-antibiotic mice. Purple- or red-fleshed potato supplementation (20% w/w) ameliorated DSS-induced reduction in colon length and mucin 2 expression levels, and increase in permeability, spleen weight, myeloperoxidase (MPO) activity, and inflammatory cytokines (IL-6, IL-17, and IL1-ß) expression levels in non-antibiotic mice, but not in gut microbiota ablated mice. CONCLUSIONS: Anthocyanin-containing potatoes are potent in alleviating colitis, and the gut microbiome is critical for the anti-colitic activity of anthocyanin-containing potatoes.

A strategy to discover selective α-glucosidase/acetylcholinesterase inhibitors from five function-similar citrus herbs through LC-Q-TOF-MS, bioassay and virtual screening.

The lack of direct connection between traditional herbal medicines and multiple biological targets is a bottleneck in herbal research and quality evaluation. To solve this problem, a strategy for the discovery of active ingredients from function-similar herbal medicines based on multiple biological targets was proposed in this article. The technical route includes chromatographic separation, mass spectrometry analysis, enzymatic activity detection, pharmacophore analysis and molecular docking. Five citrus herbs of Citri Reticulatae Pericarpium (CRP), Citri Exocarpium Rubrum (CER), Citri Grandis Exocarpium (CGE), Aurantii Fructus Immaturus (AFI) and Aurantii Fructus (AF) were used as the research objects. A total of 136 chemical components were identified from above five herbs based on LC-Q-TOF-MS/MS and database matching. The extracts of the five herbs showed obvious inhibitory effects on α-glucosidase and acetylcholinesterase in a concentration-dependent manner. Interestingly, the different types of components in the herbs exhibited selectivity for different targets: flavanone glycosides are effective on α-glucosidase but ineffective on acetylcholinesterase; polymethoxyflavonoids are effective on acetylcholinesterase but ineffective on α-glucosidase. Furthermore, we found for the first time that the components in citrus herbs exhibit opposite structure-activity relationships on the above two targets. For example, the methoxy group can enhance the activity of compounds on acetylcholinesterase but weaken the activity of compounds on α-glucosidase. The selective action is a supplement to the "multi-components, multi-targets" system of herbal medicines. Pharmacophore analysis and molecular docking were applied to explore the interaction between active ingredients and biological targets from the perspective of ligands and receptors, respectively. By combining the above multiple technologies, a strong connection among herbal medicines, chemical components and multiple biological targets was established. This work not only helps to understand the similar function of citrus herbs for the treatment of diabetes and Alzheimer's disease, but also provides selective lead compounds for the development of related drugs. This strategy is also helpful to improve the quality evaluation of citrus herbs from the perspective of biological activity.

Anthocyanin-containing purple potatoes ameliorate DSS-induced colitis in mice.

Ulcerative colitis (UC), a major form of inflammatory bowel disease (IBD), is on the rise worldwide. Approximately three million people suffer from IBD in the United States alone, but the current therapeutic options (e.g., corticosteroids) come with adverse side effects including reduced ability to fight infections. Thus, there is a critical need for developing effective, safe and evidence-based food products with anti-inflammatory activity. This study evaluated the antiinflammatory potential of purple-fleshed potato using a dextran sodium sulfate (DSS) murine model of colitis. Mice were randomly assigned to control (AIN-93G diet), P15 (15% purple-fleshed potato diet) and P25 (25% purple-fleshed potato diet) groups. Colitis was induced by 2% DSS administration in drinking water for six days. The results indicated that purple-fleshed potato supplementation suppressed the DSS-induced reduction in body weight and colon length as well as the increase in spleen and liver weights. P15 and P25 diets suppressed the elevation in the intestinal permeability, colonic MPO activity, mRNA expression and protein levels of pro-inflammatory interleukins IL-6 and IL-17, the relative abundance of specific pathogenic bacteria such as Enterobacteriaceae, Escherichia coli (E. coli) and pks+ E. coli, and the increased flagellin levels induced by DSS treatment. P25 alone suppressed the elevated systemic MPO levels in DSS-exposed mice, and elevated the relative abundance of Akkermansia muciniphila (A. muciniphila) as well as attenuated colonic mRNA expression level of IL-17 and the protein levels of IL-6 and IL-1ß. Therefore, the purple-fleshed potato has the potential to aid in the amelioration of UC symptoms.

Guide-target mismatch effects on dCas9-sgRNA binding activity in living bacterial cells.

As an effective programmable DNA targeting tool, CRISPR-Cas9 system has been adopted in varieties of biotechnological applications. However, the off-target effects, derived from the tolerance towards guide-target mismatches, are regarded as the major problems in engineering CRISPR systems. To understand this, we constructed two sgRNA libraries carrying saturated single- and double-nucleotide mismatches in living bacteria cells, and profiled the comprehensive landscape of in vivo binding affinity of dCas9 toward DNA target guided by each individual sgRNA with particular mismatches. We observed a synergistic effect in seed, where combinatorial double mutations caused more severe activity loss compared with the two corresponding single mutations. Moreover, we found that a particular mismatch type, dDrG (D = A, T, G), only showed moderate impairment on binding. To quantitatively understand the causal relationship between mismatch and binding behaviour of dCas9, we further established a biophysical model, and found that the thermodynamic properties of base-pairing coupled with strand invasion process, to a large extent, can account for the observed mismatch-activity landscape. Finally, we repurposed this model, together with a convolutional neural network constructed based on the same mechanism, as a predictive tool to guide the rational design of sgRNA in bacterial CRISPR interference.

Establishment of CRISPR interference in Methylorubrum extorquens and application of rapidly mining a new phytoene desaturase involved in carotenoid biosynthesis.

The methylotrophic bacterium Methylorubrum extorquens AM1 holds a great potential of a microbial cell factory in producing high value chemicals with methanol as the sole carbon and energy source. However, many gene functions remain unknown, hampering further rewiring of metabolic networks. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been demonstrated to be a robust tool for gene knockdown in diverse organisms. In this study, we developed an efficient CRISPRi system through optimizing the promoter strength of Streptococcus pyogenes-derived deactivated cas9 (dcas9). When the dcas9 and sgRNA were respectively controlled by medium PR/tetO and strong PmxaF-g promoters, dynamic repression efficacy of cell growth through disturbing a central metabolism gene glyA was achieved from 41.9 to 96.6% dependent on the sgRNA targeting sites. Furthermore, the optimized CRISPRi system was shown to effectively decrease the abundance of exogenous fluorescent protein gene mCherry over 50% and to reduce the expression of phytoene desaturase gene crtI by 97.7%. We then used CRISPRi technology combined with 26 sgRNAs pool to rapidly discover a new phytoene desaturase gene META1_3670 from 2470 recombinant mutants. The gene function was further verified through gene deletion and complementation as well as phylogenetic tree analysis. In addition, we applied CRISPRi to repress the transcriptional level of squalene-hopene cyclase gene shc involved in hopanoid biosynthesis by 64.9%, which resulted in enhancing 1.9-fold higher of carotenoid production without defective cell growth. Thus, the CRISPRi system developed here provides a useful tool in mining functional gene of M. extorquens as well as in biotechnology for producing high-valued chemicals from methanol. KEY POINTS: Developing an efficient CRISPRi to knockdown gene expression in C1-utilizing bacteria CRISPRi combined with sgRNAs pool to rapidly discover a new phytoene desaturase gene Improvement of carotenoid production by repressing a competitive pathway.

Dynamics of transcription-translation coordination tune bacterial indole signaling.

Indole signaling is an important cross-species communication pathway in the mammalian gut. In bacteria, upon induction by tryptophan, the molecular sensor (tnaC) controls indole biosynthesis by precisely coordinating dynamics of the corresponding macromolecular machineries during its transcription and translation. Our understanding of this regulatory program is still limited owing to its rapid dynamic nature. To address this shortcoming, we adopted a massively parallel profiling method to quantify the responses of 1,450 synthetic tnaC variants in the presence of three concentrations of tryptophan in living bacterial cells. The resultant dataset enabled us to comprehensively probe the key intermediate states of macromolecular machineries during the transcription and translation of tnaC. We also used modeling to provide a systems-level understanding of how these critical states collectively shape the output of this regulatory program quantitatively. A similar methodology will likely apply to other poorly understood dynamics-dependent cis-regulatory elements.

Genome-wide screening identifies promiscuous phosphatases impairing terpenoid biosynthesis in Escherichia coli.

Terpenoids are a large family of natural compounds that are important for both biotechnological applications and basic microorganism physiology. Inspired by the current literature, we hypothesized that recently deciphered phosphatase promiscuity may be an unexplored factor that negatively affects terpenoid biosynthesis by redirecting carbon flux away from the pathway via unrecognized catalytic activities on the phosphorylated intermediates. We used lycopene as a proof-of-concept to test this hypothesis. Based on an extensive bioinformatics analysis, we selected 56 phosphatase-encoding genes in Escherichia coli and constructed a knockdown library for these genes in a lycopene overproducer via CRISPR interference (CRISPRi). We screened this phosphatase knockdown library and observed enrichment (28 of 56) for genes that impair lycopene biosynthesis. Further scaled-up cultivation, combinatorial knockdown, and knockout assays in strains that overproduce lycopene or another terpenoid (ß-carotene) confirmed the proposed relationship between promiscuous phosphatases and impaired terpenoid biosynthesis. This study hence suggests the necessity of reconsidering the interactions of promiscuous phosphatases with ubiquitous phosphorylated components of metabolic networks with respect to engineering metabolism.

Improved sgRNA design in bacteria via genome-wide activity profiling.

CRISPR/Cas9 is a promising tool in prokaryotic genome engineering, but its success is limited by the widely varying on-target activity of single guide RNAs (sgRNAs). Based on the association of CRISPR/Cas9-induced DNA cleavage with cellular lethality, we systematically profiled sgRNA activity by co-expressing a genome-scale library (∼70 000 sgRNAs) with Cas9 or its specificity-improved mutant in Escherichia coli. Based on this large-scale dataset, we constructed a comprehensive and high-density sgRNA activity map, which enables selecting highly active sgRNAs for any locus across the genome in this model organism. We also identified 'resistant' genomic loci with respect to CRISPR/Cas9 activity, notwithstanding the highly accessible DNA in bacterial cells. Moreover, we found that previous sgRNA activity prediction models that were trained on mammalian cell datasets were inadequate when coping with our results, highlighting the key limitations and biases of previous models. We hence developed an integrated algorithm to accurately predict highly effective sgRNAs, aiming to facilitate CRISPR/Cas9-based genome engineering, screenings and antimicrobials design in bacteria. We also isolated the important sgRNA features that contribute to DNA cleavage and characterized their key differences among wild type Cas9 and its mutant, shedding light on the biophysical mechanisms of the CRISPR/Cas9 system.

Pooled CRISPR interference screening enables genome-scale functional genomics study in bacteria with superior performance.

To fully exploit the microbial genome resources, a high-throughput experimental platform is needed to associate genes with phenotypes at the genome level. We present here a novel method that enables investigation of the cellular consequences of repressing individual transcripts based on the CRISPR interference (CRISPRi) pooled screening in bacteria. We identify rules for guide RNA library design to handle the unique structure of prokaryotic genomes by tiling screening and construct an E. coli genome-scale guide RNA library (~60,000 members) accordingly. We show that CRISPRi outperforms transposon sequencing, the benchmark method in the microbial functional genomics field, when similar library sizes are used or gene length is short. This tool is also effective for mapping phenotypes to non-coding RNAs (ncRNAs), as elucidated by a comprehensive tRNA-fitness map constructed here. Our results establish CRISPRi pooled screening as a powerful tool for mapping complex prokaryotic genetic networks in a precise and high-throughput manner.

A rapid and specific colorimetric method for free tryptophan quantification.

Tryptophan is one of the eight essential amino acids and plays an important role in many biological processes. For its interaction with human health, environment and relevant commercial interest in biotechnology-based production, rapid and specific quantification method for this molecule accessible to common laboratories is badly needed. We herein reported a simple colorimetric method for free tryptophan quantification with 96-well-plate-level throughput. Our protocol firstly converted tryptophan to indole enzymatically by purified tryptophanases and then used reactivity of indole with hydroxylamine to form pink product with absorption peak at 530nm, enabling the quantification of tryptophan with simple spectrometry in just two hours. We presented that this method exhibited a linear detection range from 100µM to 600µM (R2 = 0.9969) with no detection towards other naturally occurring tryptophan analogs or tryptophan residues in proteins. It was very robust in complicated biological samples, as demonstrated by quantifying the titer of 36 mutated tryptophan-producing strains with Pearson correlation coefficient of 0.93 in contrast to that measured by high performance liquid chromatography (HPLC). Our method should be potent for routine free tryptophan quantification in a high-throughput manner, facilitating studies in medicine, microbiology, food chemistry, metabolic engineering, etc.

Identification of Polar Constituents in the Decoction of Juglans mandshurica and in the Medicated Egg Prepared with the Decoction by HPLC-Q-TOF MS².

As a folk medicinal plant, Juglans mandshurica has been used for the treatment of cancer in China and Korea. Traditionally, J. mandshurica is decocted together with chicken eggs. Both the decoction and medicated eggs possess anti-tumor properties. Clarifying the constituents of the decoction and absorbed by the medicated eggs is essential for the investigation of the active principles of J. mandshurica. Herein, the medicated eggs were prepared by decocting raw chicken eggs, having unbroken shells, with the decoction of J. mandshurica. A systematic investigation of the chemical profile of the J. mandshurica decoction and the medicated egg extraction was conducted by HPLC-Q-TOF-MS². In total, 93 peaks, including 45 tannins, 14 naphthalene derivatives, 17 organic acids, 3 diarylheptanoids, 4 lignans, 3 anthraquinones, 1 flavonoid glycoside, 3 amino acids, and 3 nitrogenous compounds, were tentatively identified in the decoction. In the medicated egg extraction, 44 peaks including 11 organic acids, 3 amino acids, 3 nitrogenous compounds, 8 naphthalene derivatives, 3 diarylheptanoids, 15 tannins, and 1 lignan were tentatively identified. The chemical profile presented provided a detailed overview of the polar chemical constituents in J. mandshurica and useful information for the research of bioactive compounds of this plant.

Maltose Utilization as a Novel Selection Strategy for Continuous Evolution of Microbes with Enhanced Metabolite Production.

We have developed a novel selection circuit based on carbon source utilization that establishes and sustains growth-production coupling over several generations in a medium with maltose as the sole carbon source. In contrast to traditional antibiotic resistance-based circuits, we first proved that coupling of cell fitness to metabolite production by our circuit was more robust with a much lower escape risk even after many rounds of selection. We then applied the selection circuit to the optimization of L-tryptophan (l-Trp) production. We demonstrated that it enriched for specific mutants with increased l-Trp productivity regardless of whether it was applied to a small and defined mutational library or a relatively large and undefined one. From the latter, we identified four novel mutations with enhanced l-Trp output. Finally, we used it to select for several high l-Trp producers with randomly generated genome-wide mutations and obtained strains with up to 65% increased l-Trp production. This selection circuit provides new perspectives for the optimization of microbial cell factories for diverse metabolite production and the discovery of novel genotype-phenotype associations at the single-gene and whole-genome levels.

Multiconstituent identification in root, branch, and leaf extracts of Juglans mandshurica using ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry.

As a traditional medicinal plant, Juglans mandshurica has been used for the treatment of cancer. Different organs of this plant showed anti-tumor activity in clinic and laboratory. Comparative identification of constituents in different plant organs is essential for investigation of the relationship between chemical constituents and pharmacological activities. For this aim, the roots, branches, and leaves of J. mandshurica were extracted with 50% v/v methanol and then subjected to ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry analysis conducted under low and high energy. As a result, we have to date identified 111 compounds consisting of 56 tannins, 29 flavonoids, 13 organic acids, 8 naphthalene derivatives, and 5 anthracenes. Five compounds, namely, diquercetin trihydroxy-truxinoyl-glucoside, two quercetin kaempferol dihydroxy-truxinoyl-glucosides, syringoyl-tri-galloyl-O-glucose, and dihydroxy-naphthalene syringoyl-glucoside, were tentatively identified as new compounds. Of the compounds identified, 76 were found in the root extract, 67 in the branch extract, and 37 in the leaf extract. Only six compounds including four organic acids and two tannins were found in all three extracts. We developed a rapid and sensitive ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry approach to identify multiple constituents of complex extracts without separation and ion selection. The results presented provide useful information on further research of the bioactive compounds of J. mandshurica.