Cellular Metabolic Networks Research Articles

Microbial synthesis of sedoheptulose from glucose by metabolically engineered Corynebacterium glutamicum

BackgroundSeven-carbon sugars, which rarely exist in nature, are the key constitutional unit of septacidin and hygromycin B in bacteria. These sugars exhibit a potential therapeutic effect for hypoglycaemia and cancer and serve as building blocks for the synthesis of C-glycosides and novel antibiotics. However, chemical and enzymatic approaches for the synthesis of seven-carbon sugars have faced challenges, such as complex reaction steps, low overall yields and high-cost feedstock, limiting their industrial-scale production.ResultsIn this work, we propose a strain engineering approach for synthesising sedoheptulose using glucose as sole feedstock. The gene pfkA encoding 6-phosphofructokinase in Corynebacterium glutamicum was inactivated to direct the carbon flux towards the pentose phosphate pathway in the cellular metabolic network. This genetic modification successfully enabled the synthesis of sedoheptulose from glucose. Additionally, we identified key enzymes responsible for product formation through transcriptome analysis, and their corresponding genes were overexpressed, resulting in a further 20% increase in sedoheptulose production.ConclusionWe achieved a sedoheptulose concentration of 24 g/L with a yield of 0.4 g/g glucose in a 1 L fermenter, marking the highest value up to date. The produced sedoheptulose could further function as feedstock for synthesising structural seven-carbon sugars through coupling with enzymatic isomerisation, epimerisation and reduction reactions.

Transcriptome analysis reveals methanol metabolism variations for the growth damage caused by overexpression of chimeric transactivators in Pichia pastoris

Methanol is a promising substrate for sustainable biomanufacturing, and Pichia pastoris has become a commonly used yeast for methanol utilization due to its powerful methanol metabolic pathways and methanol inducible promoter. Previous reconstruction of gene circuits highly improved transcriptional activity, but excessive expression of chimeric transactivator damaged cell growth on methanol. Here we employed transcriptome analysis to investigate the effects of chimeric transactivator overexpression on cellular metabolism and regulatory networks. The results showed that strong expression of chimeric transactivator unexpectedly downregulated methanol metabolism, especially the alcohol oxidase 1 (AOX1), but without remarkable changes in expression of transcriptional factors. Meanwhile, the synthesis of peroxisomes also varied with chimeric transactivator expression. In addition, the enrichment analysis of differentially expressed genes revealed their impact on cellular metabolism. The gene expression patterns caused by different expression levels of chimeric transactivators have also been clarified. This work provides useful information to understand the transcriptional regulation of the AOX1 promoter and methanol signaling. It revealed the importance of balancing transcription factor expression for the host improvement.

The Multifaceted Role of Alpha-Lipoic Acid in Cancer Prevention, Occurrence, and Treatment.

Alpha-lipoic acid (ALA) is a naturally occurring compound synthesized by mitochondria and widely distributed in both animal and plant tissues. It primarily influences cellular metabolism and oxidative stress networks through its antioxidant properties and is an important drug for treating metabolic diseases associated with oxidative damage. Nevertheless, research indicates that the mechanism by which ALA affects cancer cells is distinct from that observed in normal cells, exhibiting pro-oxidative properties. Therefore, this review aims to describe the main chemical and biological functions of ALA in the cancer environment, including its mechanisms and effects in tumor prevention and anticancer activity, as well as its role as an adjunctive drug in cancer therapy. We specifically focus on the interactions between ALA and various carcinogenic and anti-carcinogenic pathways and discuss ALA's pro-oxidative capabilities in the unique redox environment of cancer cells. Additionally, we elaborate on ALA's roles in nanomedicine, hypoxia-inducible factors, and cancer stem cell research, proposing hypotheses and potential explanations for currently unresolved issues.

Enhancement of vitamin B6 production driven by omics analysis combined with fermentation optimization

BackgroundMicrobial engineering aims to enhance the ability of bacteria to produce valuable products, including vitamin B6 for various applications. Numerous microorganisms naturally produce vitamin B6, yet the metabolic pathways involved are rigorously controlled. This regulation by the accumulation of vitamin B6 poses a challenge in constructing an efficient cell factory.ResultsIn this study, we conducted transcriptome and metabolome analyses to investigate the effects of the accumulation of pyridoxine, which is the major commercial form of vitamin B6, on cellular processes in Escherichia coli. Our omics analysis revealed associations between pyridoxine and amino acids, as well as the tricarboxylic acid (TCA) cycle. Based on these findings, we identified potential targets for fermentation optimization, including succinate, amino acids, and the carbon-to-nitrogen (C/N) ratio. Through targeted modifications, we achieved pyridoxine titers of approximately 514 mg/L in shake flasks and 1.95 g/L in fed-batch fermentation.ConclusionOur results provide insights into pyridoxine biosynthesis within the cellular metabolic network for the first time. Our comprehensive analysis revealed that the fermentation process resulted in a remarkable final yield of 1.95 g/L pyridoxine, the highest reported yield to date. This work lays a foundation for the green industrial production of vitamin B6 in the future.

Self-controlled in silico gene knockdown strategies to enhance the sustainable production of heterologous terpenoid by Saccharomyces cerevisiae

Microbial bioengineering is a growing field for producing plant natural products (PNPs) in recent decades, using heterologous metabolic pathways in host cells. Once heterologous metabolic pathways have been introduced into host cells, traditional metabolic engineering techniques are employed to enhance the productivity and yield of PNP biosynthetic routes, as well as to manage competing pathways. The advent of computational biology has marked the beginning of a novel epoch in strain design through in silico methods. These methods utilize genome-scale metabolic models (GEMs) and flux optimization algorithms to facilitate rational design across the entire cellular metabolic network. However, the implementation of in silico strategies can often result in an uneven distribution of metabolic fluxes due to the rigid knocking out of endogenous genes, which can impede cell growth and ultimately impact the accumulation of target products. In this study, we creatively utilized synthetic biology to refine in silico strain design for efficient PNPs production. OptKnock simulation was performed on the GEM of Saccharomyces cerevisiae OA07, an engineered strain for oleanolic acid (OA) bioproduction that has been reported previously. The simulation predicted that the single deletion of fol1, fol2, fol3, abz1, and abz2, or a combined knockout of hfd1, ald2 and ald3 could improve its OA production. Consequently, strains EK1∼EK7 were constructed and cultivated. EK3 (OA07△fol3), EK5 (OA07△abz1), and EK6 (OA07△abz2) had significantly higher OA titers in a batch cultivation compared to the original strain OA07. However, these increases were less pronounced in the fed-batch mode, indicating that gene deletion did not support sustainable OA production. To address this, we designed a negative feedback circuit regulated by malonyl-CoA, a growth-associated intermediate whose synthesis served as a bypass to OA synthesis, at fol3, abz1, abz2, and at acetyl-CoA carboxylase-encoding gene acc1, to dynamically and autonomously regulate the expression of these genes in OA07. The constructed strains R_3A, R_5A and R_6A had significantly higher OA titers than the initial strain and the responding gene-knockout mutants in either batch or fed-batch culture modes. Among them, strain R_3A stand out with the highest OA titer reported to date. Its OA titer doubled that of the initial strain in the flask-level fed-batch cultivation, and achieved at 1.23 ± 0.04 g L−1 in 96 h in the fermenter-level fed-batch mode. This indicated that the integration of optimization algorithm and synthetic biology approaches was efficiently rational for PNP-producing strain design.

Enhancing 2-Pyrone Synthase Efficiency by High-Throughput Mass-Spectrometric Quantification and In Vitro/In Vivo Catalytic Performance Correlation.

Engineering efficient biocatalysts is essential for metabolic engineering to produce valuable bioproducts from renewable resources. However, due to the complexity of cellular metabolic networks, it is challenging to translate success in vitro into high performance in cells. To meet such a challenge, an accurate and efficient quantification method is necessary to screen a large set of mutants from complex cell culture and a careful correlation between the catalysis parameters in vitro and performance in cells is required. In this study, we employed a mass-spectrometry based high-throughput quantitative method to screen new mutants of 2-pyrone synthase (2PS) for triacetic acid lactone (TAL) biosynthesis through directed evolution in E. coli. From the process, we discovered two mutants with the highest improvement (46 fold) in titer and the fastest kcat (44 fold) over the wild type 2PS, respectively, among those reported in the literature. A careful examination of the correlation between intracellular substrate concentration, Michaelis-Menten parameters and TAL titer for these two mutants reveals that a fast reaction rate under limiting intracellular substrate concentrations is important for in-cell biocatalysis. Such properties can be tuned by protein engineering and synthetic biology to adopt these engineered proteins for the maximum activities in different intracellular environments.

Abstract C100: Novel functions of ATF4 in early stages of pancreatic cancer tumorigenesis

Abstract Biological stresses that are cell-intrinsic (e.g., oncogene-induced imbalances in protein translation, lipid synthesis, mitochondrial dysfunction) or cell-extrinsic (e.g., nutrient deprivation, hypoxia, drug exposure) disrupt cellular function and viability if not mitigated. The Integrated Stress Response (ISR) consists of upstream regulatory kinases (PERK, GCN2, HRI, PKR) that respond to such stresses in part by activating the ATF4 transcription factor. ATF4 then rewires nutrient uptake, cellular metabolism, mitochondrial function, and protein chaperone networks to restore cellular homeostasis. We have shown that ATF4 is upregulated in mouse and human pancreatic ductal adenocarcinoma (PDAC), even compared to normal acinar cells where ATF4 is expressed at relatively high levels. To investigate the role of ATF4 in PDAC, we generated mice with inducible acinar-specific deletion of Atf4 using Ptf1a-CreERT to delete Atf4 following treatment of adult Atf4-floxed mice with tamoxifen. In contrast to previous studies showing germline deletion of Atf4 in mice caused premature mortality at approximately 3 weeks of age due to pancreatic insufficiency, inducible deletion of Atf4 in adult acinar cells does not cause pancreatic deficiency or animal demise. Rather, adult Ptf1a-CreERT;Atf4fl/fl (CA) mice are viable although more sensitive to cerulein-induced pancreatitis. Specifically, compared to control (WT) mice, CA mice exhibit more extensive acinar-ductal metaplasia (ADM), including more extensive expression of cytokeratin-19 (CK19), increased cell death and take longer to recover normal pancreatic architecture following in vivo cerulein challenge. Using ex vivo acinar explant assays derived from WT and CA mice, we further show that induction of ADM in WT acinar cells with TGF-a is associated with down-regulation of Atf4 and Atf4 target genes in WT acinar explants while CA explants undergo accelerated ADM. Furthermore, expression of exogenous Atf4 in acinar cells inhibited ADM. These in vivo and ex vivo findings are consistent with down-regulation of Atf4 being required for ADM. Given the tumor-promoting role of ADM in the early stages of PDAC, we next crossed the CA mice to LSL-KRasG12D mice to generate LSL-KRasG12D;Ptf1a-CreERT;Atf4fl/fl (KCA) mice to examine how loss of Atf4 affected pancreatic intraepithelial neoplasia (PanIN) and PDAC formation. Surprisingly, given the critical need to down-regulate Atf4 in ADM, we show for the first time that genetic deletion of Atf4 in pancreatic acinar cells blocks both PanIN and PDAC formation in KCA mice compared to age-matched KC mice. Rather, KCA mice developed a fatty pancreas devoid of tumor with very few CK19-positive cells forming. These results suggest that Atf4 is critical for PanIN formation and prevents PDAC. This also suggests that Atf4 plays a different role in PanIN formation than in ADM where its down-regulation was critical and indicates a dynamic role for Atf4 depending on oncogenic versus proteotoxic stress, as will be discussed. Citation Format: Leah M. Ziolkowski, Kay F. Macleod. Novel functions of ATF4 in early stages of pancreatic cancer tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C100.

The assessment of the potential hepatotoxicity of new drugs by in vitro metabolomics.

Drug hepatotoxicity assessment is a relevant issue both in the course of drug development as well as in the post marketing phase. The use of human relevant in vitro models in combination with powerful analytical methods (metabolomic analysis) is a promising approach to anticipate, as well as to understand and investigate the effects and mechanisms of drug hepatotoxicity in man. The metabolic profile analysis of biological liver models treated with hepatotoxins, as compared to that of those treated with non-hepatotoxic compounds, provides useful information for identifying disturbed cellular metabolic reactions, pathways, and networks. This can later be used to anticipate, as well to assess, the potential hepatotoxicity of new compounds. However, the applicability of the metabolomic analysis to assess the hepatotoxicity of drugs is complex and requires careful and systematic work, precise controls, wise data preprocessing and appropriate biological interpretation to make meaningful interpretations and/or predictions of drug hepatotoxicity. This review provides an updated look at recent in vitro studies which used principally mass spectrometry-based metabolomics to evaluate the hepatotoxicity of drugs. It also analyzes the principal drawbacks that still limit its general applicability in safety assessment screenings. We discuss the analytical workflow, essential factors that need to be considered and suggestions to overcome these drawbacks, as well as recent advancements made in this rapidly growing field of research.

Online Monitoring of Penicillin Manufacture Based on Production Variables and Metabolic Fluxes

The existing data-based monitoring and diagnosis methods for microbial manufacturing processes only consider macroscopic production variables and ignore microscopic metabolic mechanisms and flux changes. This paper proposed a fault monitoring strategy based on metabolic flux variables (intracellular and extracellular exchange fluxes) by introducing microbial cellular metabolic network information. First, the metabolic network of the microbial manufacturing process was analyzed and the dynamic metabolic flux analysis (DMFA) was used to obtain the dynamic distribution of metabolic fluxes inside microbial cells based on measurable extracellular metabolites concentration. Then, production variables and flux variables were combined to constitute a sample dataset of the microbial cell growth process. On this basis, a multiway principal component analysis (MPCA) method was used to establish a fault diagnosis model to monitor the process. Finally, the method’s effectiveness was verified using the fault monitoring strategy proposed in this paper for online monitoring of the penicillin manufacturing process.

Inter-tissue communication of mitochondrial stress and metabolic health.

Mitochondria function as a hub of the cellular metabolic network. Mitochondrial stress is closely associated with aging and a variety of diseases, including neurodegeneration and cancer. Cells autonomously elicit specific stress responses to cope with mitochondrial stress to maintain mitochondrial homeostasis. Interestingly, mitochondrial stress responses may also be induced in a non-autonomous manner in cells or tissues that are not directly experiencing such stress. Such non-autonomous mitochondrial stress responses are mediated by secreted molecules called mitokines. Due to their significant translational potential in improving human metabolic health, there has been a surge in mitokine-focused research. In this review, we summarize the findings regarding inter-tissue communication of mitochondrial stress in animal models. In addition, we discuss the possibility of mitokine-mediated intercellular mitochondrial communication originating from bacterial quorum sensing.

SESN1, as a potential target for postoperative cognitive dysfunction, attenuates sevoflurane-induced neuronal cell damage in the hippocampus

Postoperative cognitive dysfunction (POCD) is a devastating complication with long-term consequences, and new therapeutic targets and drugs are still needed for the treatment of POCD. Sestrin are a family of stress-inducing proteins that regulate cellular metabolic networks. However, the possible effects of Sestrin on POCD were still unclear. This study aimed to investigate the effects of Sestrin 1 (SESN1) in postoperative cognitive dysfunction (POCD) cell model and reveal its mechanism. We constructed an in vitro model of POCD by treating primary rat hippocampal neurons with sevoflurane. Herein, we noticed SESN1 enhanced cell viability induced by sevoflurane. Further, SESN1 improved sevoflurane-induced cell inflammation. We further found that SESN1 improved sevoflurane induced reactive oxygen species (ROS) production and inhibited apoptosis. Mechanically, SESN1 restrained NOD-like receptor thermal protein domain 3 (NLRP3) inflammasome activation and therefore suppressed POCD. In conclusion, SESN1, as a potential target for postoperative cognitive dysfunction, attenuates sevoflurane-induced neuronal cell damage in the hippocampus. These findings will provide guidance for the mechanism study of POCD and future drug development for treatment of POCD.

Single-Cell RNA Sequencing Reveals Molecular Features of Heterogeneity in the Murine Retinal Pigment Epithelium.

Transcriptomic analysis of the mammalian retinal pigment epithelium (RPE) aims to identify cellular networks that influence ocular development, maintenance, function, and disease. However, available evidence points to RPE cell heterogeneity within native tissue, which adds complexity to global transcriptomic analysis. Here, to assess cell heterogeneity, we performed single-cell RNA sequencing of RPE cells from two young adult male C57BL/6J mice. Following quality control to ensure robust transcript identification limited to cell singlets, we detected 13,858 transcripts among 2667 and 2846 RPE cells. Dimensional reduction by principal component analysis and uniform manifold approximation and projection revealed six distinct cell populations. All clusters expressed transcripts typical of RPE cells; the smallest (C1, containing 1–2% of total cells) exhibited the hallmarks of stem and/or progenitor (SP) cells. Placing C1–6 along a pseudotime axis suggested a relative decrease in melanogenesis and SP gene expression and a corresponding increase in visual cycle gene expression upon RPE maturation. K-means clustering of all detected transcripts identified additional expression patterns that may advance the understanding of RPE SP cell maintenance and the evolution of cellular metabolic networks during development. This work provides new insights into the transcriptome of the mouse RPE and a baseline for identifying experimentally induced transcriptional changes in future studies of this tissue.

Effect of Expression of Nuclear-Encoded Cytochrome C Oxidase Subunit 4 Isoforms on Metabolic Profiles of Glioma Cells.

Although often effective at treating newly diagnosed glioblastoma (GBM), increasing evidence suggests that chemo- and radiotherapy-induced alterations in tumor metabolism promote GBM recurrence and aggressiveness, as well as treatment resistance. Recent studies have demonstrated that alterations in glioma cell metabolism, induced by a switch in the isoform expression of cytochrome c oxidase subunit 4 (COX4), a key regulatory subunit of mammalian cytochrome c oxidase, could promote these effects. To understand how the two COX4 isoforms (COX4-1 and COX4-2) differentially affect glioma metabolism, glioma samples harvested from COX4-1- or COX4-2-overexpressing U251 cells were profiled using Gas chromatography–mass spectrometry GC-MS and Liquid Chromatography - Tandem Mass Spectrometry LC-MS/MS metabolomics platforms. The concentration of 362 metabolites differed significantly in the two cell types. The two most significantly upregulated pathways associated with COX4-1 overexpression were purine and glutathione metabolism; the two most significantly downregulated metabolic pathways associated with COX4-1 expression were glycolysis and fatty acid metabolism. Our study provides new insights into how Cytochrome c oxidase (CcO) regulatory subunits affect cellular metabolic networks in GBM and identifies potential targets that may be exploited for therapeutic benefit.

Clinical and biochemical footprints of inherited metabolic diseases. VIII. Neoplasias.

Cancer, caused by multiple cumulative pathogenic variants in tumor suppressor genes and proto-oncogenes, is a leading cause of mortality worldwide. The uncontrolled and rapid cell growth of the tumors requires a reprogramming of the complex cellular metabolic network to favor anabolism. Adequate management and treatment of certain inherited metabolic diseases might prevent the development of certain neoplasias, such as hepatocellular carcinoma in tyrosinemia type 1 or hepatocellular adenomas in glycogen storage disorder type 1a. We reviewed and updated the list of known metabolic etiologies associated with various types of benign and malignant neoplasias, finding 64 relevant inborn errors of metabolism. This is the eighth article of the series attempting to create a comprehensive list of clinical and metabolic differential diagnosis by system involvement.

The relationship between amino acid and lipid metabolism in oleaginous eukaryotic microorganism.

Amino acids are the building blocks of protein, promoting the balance between growth and lipid synthesis. However, the accumulation of microbial lipids involves multiple pathways, which requires the analysis of the global cellular metabolic network in which amino acid metabolism is involved. This review illustrates the dependence patterns of intracellular amino acids and lipids of oleaginous eukaryotic microorganisms in different environments and points out the contribution of amino acid metabolic precursors to the de novo synthesis of fatty acids. We emphasized the key role of amino acid metabolism in lipid remodeling and autophagy behavior and highlighted the regulatory effects of amino acids and their secondary metabolites as signal factors for microbial lipid synthesis. The application prospects of omics technology and genetic engineering technology in the field of microbial lipids are described. KEY POINTS: • Overview of microbial lipid synthesis mediated by amino acid metabolism • Insight into metabolic mechanisms founding multiple regulatory networks is provided • Description of microbial lipid homeostasis mediated by amino acid excitation signal.

Protein O-GlcNAcylation Regulates Innate Immune Cell Function.

Metabolite-mediated protein posttranslational modifications (PTM) represent highly evolutionarily conserved mechanisms by which metabolic networks participate in fine-tuning diverse cellular biological activities. Modification of proteins with the metabolite UDP-N-acetylglucosamine (UDP-GlcNAc), known as protein O-GlcNAcylation, is one well-defined form of PTM that is catalyzed by a single pair of enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Previous studies have discovered critical roles of protein O-GlcNAcylation in many fundamental biological activities via modifying numerous nuclear and cytoplasmic proteins. A common mechanism by which O-GlcNAc affects protein function is through the cross-regulation between protein O-GlcNAcylation and phosphorylation. This is of particular importance to innate immune cell functions due to the essential role of protein phosphorylation in regulating many aspects of innate immune signaling. Indeed, as an integral component of cellular metabolic network, profound alteration in protein O-GlcNAcylation has been documented following the activation of innate immune cells. Accumulating evidence suggests that O-GlcNAcylation of proteins involved in the NF-κB pathway and other inflammation-associated signaling pathways plays an essential role in regulating the functionality of innate immune cells. Here, we summarize recent studies focusing on the role of protein O-GlcNAcylation in regulating the NF-κB pathway, other innate immune signaling responses and its disease relevance.

Engineering a Controllable Targeted Protein Degradation System and a Derived OR-GATE-Type Inducible Gene Expression System in Synechococcus elongatus PCC 7942.

Cyanobacteria are important model organisms for exploring the mechanisms of photosynthesis and are considered as promising microbial platforms for photosynthetic biomanufacturing. The development of efficient cyanobacteria cell factories requires efficient and convenient tools to dynamically regulate and manipulate target proteins, modules, and pathways. Targeted protein degradation is important to achieve rapid responses of cellular metabolic networks to artificial or environmental signals, and there are currently limited approaches to induce protein degradation in cyanobacteria. In this work, we developed an Escherichia coli sourced ssrA-tagging system in an important cyanobacteria strain, Synechococcus elongatus PCC 7942, to achieve inducible degradation of target proteins. A modified version of the E. coli ssrA tag (ssrADAS) proved to be immune to the native ClpXP system in Synechococcus elongatus PCC 7942, while induced expression of the E. coli sourced adaptor SspB and ClpXP resulted in effective degradation of the tagged proteins. Compared to the previously developed down-regulation approaches, the inducible ssrADAS-SspB-ClpXPEc system facilitated the smart and rapid degradation of target proteins in PCC7942 cells at different growth stages. Furthermore, when used to regulate the degradation of LacI, the repressor element of LacO-LacI transcription regulation system, an efficient and stringent inducible gene expression system was obtained based on an OR-GATE type genetic circuit design. The tools developed in this work expanded the cyanobacteria synthetic biology toolbox and will facilitate the success of future dynamic metabolic engineering.

Systems biomarkers for papillary thyroid cancer prognosis and treatment through multi-omics networks

The identification of biomolecules associated with papillary thyroid cancer (PTC) has upmost importance for the elucidation of the disease mechanism and the development of effective diagnostic and treatment strategies. Despite particular findings in this regard, a holistic analysis encompassing molecular data from different biological levels has been lacking. In the present study, a meta-analysis of four transcriptome datasets was performed to identify gene expression signatures in PTC, and reporter molecules were determined by mapping gene expression data onto three major cellular networks, i.e., transcriptional regulatory, protein-protein interaction, and metabolic networks. We identified 282 common genes that were differentially expressed in all PTC datasets. In addition, six proteins (FYN, JUN, LYN, PML, SIN3A, and RARA), two Erb-B2 receptors (ERBB2 and ERBB4), two cyclin-dependent receptors (CDK1 and CDK2), and three histone deacetylase receptors (HDAC1, HDAC2, and HDAC3) came into prominence as proteomic signatures in addition to several metabolites including lactaldehyde and proline at the metabolome level. Significant associations with calcium and MAPK signaling pathways and transcriptional and post-transcriptional activities of 12 TFs and 110 miRNAs were also observed at the regulatory level. Among them, six miRNAs (miR-30b-3p, miR-15b-5p, let-7a-5p, miR-130b-3p, miR-424-5p, and miR-193b-3p) were associated with PTC for the first time in the literature, and the expression levels of miR-30b-3p, miR-15b-5p, and let-7a-5p were found to be predictive of disease prognosis. Drug repositioning and molecular docking simulations revealed that 5 drugs (prochlorperazine, meclizine, rottlerin, cephaeline, and tretinoin) may be useful in the treatment of PTC. Consequently, we report here biomolecule candidates that may be considered as prognostic biomarkers or potential therapeutic targets for further experimental and clinical trials for PTC.

A promoter engineering-based strategy enhances polyhydroxyalkanoate production in Pseudomonas putida KT2440

Polyhydroxyalkanoate (PHA), a class of biopolyester synthesized by various bacteria, is considered as an alternative to petroleum-based plastics because of its excellent physochemical and material properties. Pseudomonas putida KT2440 can produce medium-chain-length PHA (mcl-PHA) from glucose, fatty acid and glycerol, and its whole-genome sequences and cellular metabolic networks have been intensively researched. In this study, we aim to improve the PHA yield of P. putida KT2440 using a novel promoter engineering-based strategy. Unlike previous studies, endogenous strong promoters screening from P. putida KT2440 instead of synthetic or exogenous promoters was applied to the optimization of PHA biosynthesis pathway. Based on RNA-seq and promoter prediction, 30 putative strong promoters from P. putida KT2440 were identified. Subsequently, the strengths of these promoters were characterized by reporter gene assays. Furthermore, each of 10 strong promoters screened by transcriptional level and GFP fluorescence was independently inserted into upstream of PHA synthase gene (phaC1) on chromosome. As a result, the transcriptional levels of the phaC1 and phaC2 genes in almost all of the promoter-substituted strains were improved, and the relative PHA yields of the three promoter-substituted strains KTU-P1C1, KTU-P46C1 and KTU-P51C1 were improved obviously, reaching 30.62 wt%, 33.24 wt% and 33.29 wt% [the ratio of PHA weight to cell dry weight (CDW)], respectively. By further deletion of the glucose dehydrogenase gene in KTU-P1C1, KTU-P46C1 and KTU-P51C1, the relative PHA yield of the resulting mutant strain KTU-P46C1-∆gcd increased by 5.29% from 33.24% to 38.53%. Finally, by inserting P46 into upstream of pyruvate dehydrogenase gene in the genome of KTU-P46C1-∆gcd, the relative PHA yield and CDW of the resulting strain KTU-P46C1A-∆gcd reached nearly 42 wt% and 4.06 g/l, respectively, which increased by 90% and 40%, respectively, compared with the starting strain KTU. In particular, the absolute PHA yield of KTU-P46C1A-∆gcd reached 1.7 g/l, with a 165% improvement compared with the strain KTU. Herein, we report the highest PHA yield obtained by P. putida KT2440 in shake-flask fermentation to date. We demonstrate for the first time the effectiveness of endogenous strong promoters for improving the PHA yield and biomass of P. putida KT2440. More importantly, our findings highlight great potential of this strategy for enhanced production of secondary metabolites and heterologous proteins in P. putida KT2440.

Transcriptomes of an oceanic diatom reveal the initial and final stages of acclimation to copper deficiency.

Copper (Cu) concentration is greatly reduced in the open sea so that phytoplankton must adjust their uptake systems and acclimate to sustain growth. Acclimation to low Cu involves changes to the photosynthetic apparatus and specific biochemical reactions that use Cu, but little is known how Cu affects cellular metabolic networks. Here we report results of whole transcriptome analysis of a plastocyanin-containing diatom, Thalassiosira oceanica 1005, during its initial stages of acclimation and after long-term adaptation in Cu-deficient seawater. Gene expression profiles, used to identify Cu-regulated metabolic pathways, show downregulation of anabolic and energy-yielding reactions in Cu-limited cells. These include the light reactions of photosynthesis, carbon fixation, nitrogen assimilation and glycolysis. Reduction of these pathways is consistent with reduced growth requirements for C and N caused by slower rates of photosynthetic electron transport. Upregulation of oxidative stress defence systems persists in adapted cells, suggesting cellular damage by increased reactive oxygen species (ROS) occurs even after acclimation. Copper deficiency also alters fatty acid metabolism, possibly in response to an increase in lipid peroxidation and membrane damage driven by ROS. During the initial stages of Cu-limitation the majority of differentially regulated genes are associated with photosynthetic metabolism, highlighting the chloroplast as the primary target of low Cu availability. The results provide insights into the mechanisms of acclimation and adaptation of T. oceanica to Cu deficiency.