Central Carbon Metabolic Pathways Research Articles

Spatially resolved characterization of tissue metabolic compartments in fasted and high-fat diet livers.

Cells adapt their metabolism to physiological stimuli, and metabolic heterogeneity exists between cell types, within tissues, and subcellular compartments. The liver plays an essential role in maintaining whole-body metabolic homeostasis and is structurally defined by metabolic zones. These zones are well-understood on the transcriptomic level, but have not been comprehensively characterized on the metabolomic level. Mass spectrometry imaging (MSI) can be used to map hundreds of metabolites directly from a tissue section, offering an important advance to investigate metabolic heterogeneity in tissues compared to extraction-based metabolomics methods that analyze tissue metabolite profiles in bulk. We established a workflow for the preparation of tissue specimens for matrix-assisted laser desorption/ionization (MALDI) MSI that can be implemented to achieve broad coverage of central carbon, nucleotide, and lipid metabolism pathways. Herein, we used this approach to visualize the effect of nutrient stress and excess on liver metabolism. Our data revealed a highly organized metabolic tissue compartmentalization in livers, which becomes disrupted under high fat diet. Fasting caused changes in the abundance of several metabolites, including increased levels of fatty acids and TCA intermediates while fatty livers had higher levels of purine and pentose phosphate-related metabolites, which generate reducing equivalents to counteract oxidative stress. This spatially conserved approach allowed the visualization of liver metabolic compartmentalization at 30 μm pixel resolution and can be applied more broadly to yield new insights into metabolic heterogeneity in vivo.

Overexpression of bacteriophage T4 and T7 endolysins differentially regulate the metabolic fingerprint of host Escherichia coli

Bioactive proteins are often overexpressed in different host systems for biotechnological/biomedical applications. Endolysins are natural bactericidal proteins that cleave the bacterial peptidoglycan membrane, and have the potential to be the next-generation enzybiotics. Therefore, the present study aims to elucidate the impact of two endolysins (T4L, T7L) overexpression on metabolic fingerprint of E. coli using NMR spectroscopy. The 1H NMR-based metabolomics analysis revealed global metabolite profiles of E. coli in response to endolysins. The study has identified nearly 75 metabolites, including organic acids, amino acids, sugars and nucleic acids. RNA Polymerase (RNAP) has been considered as reference protein for marking the specific alterations in metabolic pathways. The data suggested downregulation of central carbon metabolic pathway in both endolysins overexpression, but to a different extent. Also, the endolysin overexpression have highlighted the enhanced metabolic load and stress generation in the host cells, thus leading to the activation of osmoregulatory pathways. The overall changes in metabolic fingerprint of E. coli highlights the enhanced perturbations during the overexpression of T4L as compared to T7L. These untargeted metabolic studies shed light on the regulation of molecular pathways during the heterologous overexpression of these lytic enzymes that are lethal to the host.

Postinfection Metabolic Reprogramming of the Murine Trigeminal Ganglion Limits Herpes Simplex Virus-1 Replication.

ABSTRACTHerpes simplex virus type-1 (HSV-1) infections are known to alter the host metabolism for efficient propagation in vitro. However, in vivo metabolic perturbations upon prolonged HSV-1 infection remain poorly understood. We used high-resolution liquid chromatography coupled with mass spectrometry (LC-MS) and functional assays to determine the state of the trigeminal ganglion (TG) tissue metabolism upon prolonged corneal HSV-1 infection in a murine model. The metabolomics data indicated significant alterations in the host metabolic profile. After HSV-1 infection, the TG microenvironment assumed downregulation of central carbon metabolism and nucleotide synthesis pathways. We validated our observations using in vitro and ex vivo models through targeted inhibition of crucial metabolic polyamine pathways identified in our metabolomics screen. Our findings collectively suggested that HSV-1 infection altered the host metabolic product regulations that limit the energy and macromolecular precursors required for viral replication.

In silico-guided metabolic engineering of Bacillus subtilis for efficient biosynthesis of purine nucleosides by blocking the key backflow nodes

BackgroundPurine nucleosides play essential roles in cellular physiological processes and have a wide range of applications in the fields of antitumor/antiviral drugs and food. However, microbial overproduction of purine nucleosides by de novo metabolic engineering remains a great challenge due to their strict and complex regulatory machinery involved in biosynthetic pathways.ResultsIn this study, we designed an in silico-guided strategy for overproducing purine nucleosides based on a genome-scale metabolic network model in Bacillus subtilis. The metabolic flux was analyzed to predict two key backflow nodes, Drm (purine nucleotides toward PPP) and YwjH (PPP–EMP), to resolve the competitive relationship between biomass and purine nucleotide synthesis. In terms of the purine synthesis pathway, the first backflow node Drm was inactivated to block the degradation of purine nucleotides, which greatly increased the inosine production to 13.98–14.47 g/L without affecting cell growth. Furthermore, releasing feedback inhibition of the purine operon by promoter replacement enhanced the accumulation of purine nucleotides. In terms of the central carbon metabolic pathways, the deletion of the second backflow node YwjH and overexpression of Zwf were combined to increase inosine production to 22.01 ± 1.18 g/L by enhancing the metabolic flow of PPP. By switching on the flux node of the glucose-6-phosphate to PPP or EMP, the final inosine engineered strain produced up to 25.81 ± 1.23 g/L inosine by a pgi-based metabolic switch with a yield of 0.126 mol/mol glucose, a productivity of 0.358 g/L/h and a synthesis rate of 0.088 mmol/gDW/h, representing the highest yield in de novo engineered inosine bacteria. Under the guidance of this in silico-designed strategy, a general chassis bacterium was generated, for the first time, to efficiently synthesize inosine, adenosine, guanosine, IMP and GMP, which provides sufficient precursors for the synthesis of various purine intermediates.ConclusionsOur study reveals that in silico-guided metabolic engineering successfully optimized the purine synthesis pathway by exploring efficient targets, which could be applied as a superior strategy for efficient biosynthesis of biotechnological products.

Metabolic Engineering of Shikimic Acid Biosynthesis Pathway for the Production of Shikimic Acid and Its Branched Products in Microorganisms: Advances and Prospects.

The shikimate pathway is a necessary pathway for the synthesis of aromatic compounds. The intermediate products of the shikimate pathway and its branching pathway have promising properties in many fields, especially in the pharmaceutical industry. Many important compounds, such as shikimic acid, quinic acid, chlorogenic acid, gallic acid, pyrogallol, catechol and so on, can be synthesized by the shikimate pathway. Among them, shikimic acid is the key raw material for the synthesis of GS4104 (Tamiflu®), an inhibitor of neuraminidase against avian influenza virus. Quininic acid is an important intermediate for synthesis of a variety of raw chemical materials and drugs. Gallic acid and catechol receive widespread attention as pharmaceutical intermediates. It is one of the hotspots to accumulate many kinds of target products by rationally modifying the shikimate pathway and its branches in recombinant strains by means of metabolic engineering. This review considers the effects of classical metabolic engineering methods, such as central carbon metabolism (CCM) pathway modification, key enzyme gene modification, blocking the downstream pathway on the shikimate pathway, as well as several expansion pathways and metabolic engineering strategies of the shikimate pathway, and expounds the synthetic biology in recent years in the application of the shikimate pathway and the future development direction.

Genome sequencing and oenologically relevant traits of the Uruguayan native yeast <i>Issatchenkia terricola</i>

Issatchenkia terricola 0621 is a non-Saccharomyces yeast strain isolated from Tannat grapes from Uruguayan vineyards; it stands out for its ability to produce high levels of β-glucosidase activity, which contributes to the aromatic complexity of wines. To delve into the potential oenological applications of this strain, its high-quality genome was obtained and explored, allowing the main central carbon and nitrogen metabolic pathways to be reconstructed. I. terricola is able to utilise glycerol as the sole carbon source in a way that has not previously been described for yeasts. The genes of the fermentome and those involved in stress resistance during winemaking were also identified, and differences were found when compared to S. cerevisiae, which may explain why I. terricola is unable to complete fermentation. The pathways responsible for natural aroma synthesis were also reconstructed, and the production of aromatic acids, alcohols, esters, acetates and lactones was verified experimentally. Finally, sequences encoding for β-glucosidases, in addition to the previously characterised one, were identified in the genome. The work presented here lays the groundwork for experimental research focused on the dissection of the metabolism of a native non-Saccharomyces strain and its application for oenological and biotechnological purposes.

Central Metabolism and Growth Rate Impacts on Hydrogen and Carbon Isotope Fractionation During Amino Acid Synthesis in E. coli.

Compound specific stable isotope analysis (CSIA) of amino acids from bacterial biomass is a newly emerging powerful tool for exploring central carbon metabolism pathways and fluxes. By comparing isotopic values and fractionations relative to water and growth substrate, the impact of variable flow path for metabolites through different central metabolic pathways, perturbations of these paths, and their resultant consequences on intracellular pools and resultant biomass may be elucidated. Here, we explore the effects that central carbon metabolism and growth rate can have on stable hydrogen (δ2H) and carbon (δ13C) compound specific isotopic values of amino acids, and whether diagnostic isotopic fingerprints are revealed by these paired analyses. We measured δ2H and δ13C in amino acids in the wild type Escherichia coli (MG1655) across a range of growth rates in chemostat cultures to address the unknown isotopic consequences as metabolic fluxes are shuffled between catabolic and anabolic metabolisms. Additionally, two E. coli knockout mutants, one with deficiency in glycolysis –pgi (LC1888) and another inhibiting the oxidative pentose phosphate pathway (OPPP) –zwf (LC1889), were grown on glucose and used as a comparison against the wild type E. coli (MG1655) to address the isotopic changes of amino acids produced in these perturbed metabolic pathways. Amino acid δ2H values, which collectively vary in composition by more than 400‰, are altered along with δ13C values demonstrating fundamental shifts in central metabolic pathways and/or fluxes. Within our linear discriminant analysis with a simple model organism to examine potential amino acid fingerprinting, our knockout strains and variable growth rate samples plot across a wider array of organism classification than merely within the boundaries of other bacterial data.

Characterization of two novel pentose-fermenting and GABA-producing species: Levilactobacillus tujiorum sp. nov. and Secundilactobacillus angelensis sp. nov. Isolated from a solid-state fermented zha-chili

Lactobacilli are dominant in zha-chili. This study provides a taxonomic characterization of five bacterial strains isolated from zha-chili in China. The cells were Gram-positive, facultative anaerobic, non-spore-forming, flagella-free, catalase-negative, heterofermentative, pentose-fermenting, and gamma-aminobutyric acid (GABA)-producing rods. For HBUAS51241T, HBUAS51329, and HBUAS51416, C16:0, C18:1ω9c and C19:0 iso were the predominant cellular fatty acids; diphosphatidylglycerol (DPG), phosphatidylglycerol (DP), glycolipids (GL), and glycolipids (AL) were the major phospholipids. While for HBUAS51383T and HBUAS58055, C16:0, C18:1ω9c, C19:0 cyclo ω8c were the predominant cellular fatty acids; DPG, DP, GL, and AL were the major phospholipids. Strains HBUAS51241T, HBUAS51329, and HBUAS51416 showed 98.1–99.1% 16S rRNA gene sequence similarity, 80.2–81.4% ANI, 87.7–90.0% AAI, and 23.8–32.8% digital DDH to their closest related type strains Levilactobacillus hammesii DSM 16381T, Levilactobacillus parabrevis ATCC 53295T, and Levilactobacillus fuyuanensis 244-4T. Strains HBUAS51383T and HBUAS58055 showed 98.7–99.5% 16S rRNA gene sequence similarity, 75.4–81.4% ANI, 75.5–89.1% AAI, and 19.7–24.0% digital DDH to their closest related type strains Secundilactobacillus silagincola IWT5T, Secundilactobacillus silagei JCM 19001T, Secundilactobacillus pentosiphilus IWT25T, Secundilactobacillus mixtipabuli IWT30T, Secundilactobacillus odoratitofui DSM 19909T, and Secundilactobacillus similis DSM 23365T. The central carbon metabolism pathways for the five strains were summarizeded. Based on the phenotypic, chemotaxonomic, and genomic data, we propose two novel species Levilactobacillus tujiorum sp. nov. whose type strain is HBUAS51241T (=GDMCC 1.3022T = JCM 35241T), and Secundilactobacillus angelensis sp. nov. whose type strain is HBUAS51383T (=GDMCC 1.3021T = JCM 35209T).

Salmonella Central Carbon Metabolism Enhances Bactericidal Killing by Fluoroquinolone Antibiotics.

The efficacy of killing by bactericidal antibiotics has been reported to depend in large part on the ATP levels, with low levels of ATP leading to increased persistence after antibiotic challenge. Here, we show that an atp operon deletion strain of Salmonella enterica serovar Typhimurium lacking the ATP synthase was at least 10-fold more sensitive to killing by the fluoroquinolone antibiotic ciprofloxacin and yet showed either increased survival or no significant difference compared with the wild-type strain when challenged with aminoglycoside or β-lactam antibiotics, respectively. The increased cell killing and reduced bacterial survival (persistence) after fluoroquinolone challenge were found to involve metabolic compensation for the loss of the ATP synthase through central carbon metabolism reactions and increased NAD(P)H levels. We conclude that the intracellular ATP levels per se do not correlate with bactericidal antibiotic persistence to fluoroquinolone killing; rather, the central carbon metabolic pathways active at the time of challenge and the intracellular target of the antibiotic determine the efficacy of treatment.

Plasma Acylcarnitines as Metabolic Signatures of Declining Health-Related Quality of Life Measure in Community-Dwelling Older Adults: A Combined Cross-sectional and Longitudinal Pilot Study.

Health-related quality of life (HRQoL) measures are predictors of adverse health outcomes in older adults. Studies have demonstrated cross-sectional associations between HRQoL measures and blood-based biochemical markers. Acylcarnitines (ACs) are a class of metabolites generated in the mitochondria and are predictive of multiple geriatric syndromes. Changes in ACs reflect alterations in central carbon metabolic pathways. However, the prospective relationship between plasma ACs and declining HRQoL has not been examined. This study aimed to investigate both cross-sectional and longitudinal associations of baseline ACs with baseline and declining EuroQol-5 Dimension/EuroQol Visual Analogue Scale (EQ-5D/EQ-VAS) in community-dwelling older adults. One hundred and twenty community-dwelling older adults with EQ-5D/EQ-VAS measurements at baseline and follow-up were included. We quantified ACs at baseline using targeted plasma metabolomics profiling. Multivariate regressions were performed to examine cross-sectional and longitudinal associations between the measures. Cross-sectionally, ACs showed no significant associations with either EQ-5D index or EQ-VAS scores. Longitudinally, multiple baseline short-chain ACs were significantly and inversely associated with declining EQ-5D index score, explaining up to 8.5% of variance in the decline. Within a cohort of community-dwelling older adults who had high HRQoL at baseline, we showed that higher levels of short-chain ACs are longitudinally associated with declining HRQoL. These findings reveal a novel association between central carbon metabolic pathways and declining HRQoL. Notably, dysregulation in mitochondrial central carbon metabolism could be detected prior to clinically important decline in HRQoL, providing the first evidence of objective biomarkers as novel predictors to monitor HRQoL in nonpharmacological interventions and epidemiology.

The Metabolic Landscape in Osteoarthritis.

Articular cartilage function depends on the temporal and zonal distribution of coordinated metabolic regulation in chondrocytes. Emerging evidence shows the importance of cellular metabolism in the molecular control of the cartilage and its dysregulation in degenerative diseases like osteoarthritis (OA). Compared to most other tissues, chondrocytes are sparsely located in the extracellular matrix, lacking the typical proximity of neural, vascular, and lymphatic tissue. Making up under 5% of the total tissue weight of cartilage, chondrocytes have a relative deficiency of access to nutrients and oxygen, as well as limited pathways for metabolite removal. This makes cartilage a unique tissue with hypocellularity, prolonged metabolic rate, and tissue turnover. Studies in the past decade have shown that several pathways of central carbon metabolism are essential for cartilage homeostasis. Here, we summarised the literature findings on the role of cellular metabolism in determining the chondrocyte function and how this metabolic dysregulation led to cartilage aging in OA and provided an outlook on how the field may evolve in the coming years. Although the various energy metabolism pathways are inextricably linked with one another, for the purpose of this review, we initially endeavoured to examine them individually and in relative isolation. Subsequently, we comment on what is known regarding the integration and linked signalling pathways between these systems and the therapeutic opportunities for targeting OA metabolism.

Characteristics of genomic mutations and signaling pathway alterations in thymic epithelial tumors.

BackgroundTo elucidate the mechanisms of thymic epithelial tumor (TET) canceration by characterizing genomic mutations and signaling pathway alterations.MethodsPrimary tumor and blood samples were collected from 21 patients diagnosed with TETs (thymoma and thymic cancer), 15 of whom were screened by nucleic acid extraction and whole exon sequencing. Bioinformatics was used to comprehensively analyze the sequencing data for these samples, including gene mutation information and the difference of tumor mutation burden (TMB) between thymoma and thymic carcinoma groups. We performed signaling pathway and functional enrichment analysis using the WebGestalt 2017 toolkit.ResultsZNF429 (36%) was the gene with the highest mutation frequency in thymic carcinoma. Mutations in BAP1 (14%), ABI1 (7%), BCL9L (7%), and CHEK2 (7%) were exclusively detected in thymic carcinoma, whereas ZNF721 mutations (14%) and PABPC1 (14%) were found exclusively in thymoma. The mean TMB values for thymic carcinoma and thymoma were 0.722 and 0.663 mutations per megabase (Mb), respectively, and these differences were not statistically significant. The ErbB signaling pathway was enriched in the thymoma and intersection groups, and pathways of central carbon metabolism in cancer, longevity regulating and MAPK signaling were only found in the thymoma group, while pathways in cancer (hsa05200) was found in the thymoma and thymic carcinoma groups.ConclusionsMultiple differences in somatic genes and pathways have been identified. Our findings provide insights into differences between thymoma and thymic carcinoma that could aid in designing personalized clinical therapeutic strategies.

Comparative Proteomics Reveals the Effect of the Transcriptional Regulator Sp13016 on Butenyl-Spinosyn Biosynthesis in Saccharopolyspora pogona.

Butenyl-spinosyn is a highly effective and broad-spectrum biopesticide produced by Saccharopolyspora pogona. However, the yield of this compound is difficult to increase because the regulatory mechanism of secondary metabolism is still unknown. Here, the transcriptional regulator Sp13016 was discovered to be highly associated with butenyl-spinosyn synthesis and bacterial growth. Overexpression of sp13016 improved butenyl-spinosyn production to a level that was 2.84-fold that of the original strain, while deletion of sp13016 resulted in a significant decrease in yield and growth inhibition. Comparative proteomics revealed that these phenotypic changes were attributed to the influence of Sp13016 on the central carbon metabolism pathway to regulate the supply of precursors. Our research helps to reveal the regulatory mechanism of butenyl-spinosyn biosynthesis and provides a reference for increasing the yield of natural products of Actinomycetes.

KSHV Reprogramming of Host Energy Metabolism for Pathogenesis.

Reprogramming of energy metabolism is a key for cancer development. Kaposi’s sarcoma-associated herpesvirus (KSHV), a human oncogenic herpesvirus, is tightly associated with several human malignancies by infecting B-lymphocyte or endothelial cells. Cancer cell energy metabolism is mainly dominated by three pathways of central carbon metabolism, including aerobic glycolysis, glutaminolysis, and fatty acid synthesis. Increasing evidence has shown that KSHV infection can alter central carbon metabolic pathways to produce biomass for viral replication, as well as the survival and proliferation of infected cells. In this review, we summarize recent studies exploring how KSHV manipulates host cell metabolism to promote viral pathogenesis, which provides the potential therapeutic targets and strategies for KSHV-associated cancers.

Genomics, Exometabolomics, and Metabolic Probing Reveal Conserved Proteolytic Metabolism of Thermoflexus hugenholtzii and Three Candidate Species From China and Japan

Thermoflexus hugenholtzii JAD2T, the only cultured representative of the Chloroflexota order Thermoflexales, is abundant in Great Boiling Spring (GBS), NV, United States, and close relatives inhabit geothermal systems globally. However, no defined medium exists for T. hugenholtzii JAD2T and no single carbon source is known to support its growth, leaving key knowledge gaps in its metabolism and nutritional needs. Here, we report comparative genomic analysis of the draft genome of T. hugenholtzii JAD2T and eight closely related metagenome-assembled genomes (MAGs) from geothermal sites in China, Japan, and the United States, representing “Candidatus Thermoflexus japonica,” “Candidatus Thermoflexus tengchongensis,” and “Candidatus Thermoflexus sinensis.” Genomics was integrated with targeted exometabolomics and 13C metabolic probing of T. hugenholtzii. The Thermoflexus genomes each code for complete central carbon metabolic pathways and an unusually high abundance and diversity of peptidases, particularly Metallo- and Serine peptidase families, along with ABC transporters for peptides and some amino acids. The T. hugenholtzii JAD2T exometabolome provided evidence of extracellular proteolytic activity based on the accumulation of free amino acids. However, several neutral and polar amino acids appear not to be utilized, based on their accumulation in the medium and the lack of annotated transporters. Adenine and adenosine were scavenged, and thymine and nicotinic acid were released, suggesting interdependency with other organisms in situ. Metabolic probing of T. hugenholtzii JAD2T using 13C-labeled compounds provided evidence of oxidation of glucose, pyruvate, cysteine, and citrate, and functioning glycolytic, tricarboxylic acid (TCA), and oxidative pentose-phosphate pathways (PPPs). However, differential use of position-specific 13C-labeled compounds showed that glycolysis and the TCA cycle were uncoupled. Thus, despite the high abundance of Thermoflexus in sediments of some geothermal systems, they appear to be highly focused on chemoorganotrophy, particularly protein degradation, and may interact extensively with other microorganisms in situ.

The Exciting Journey of A Metabolite ‐ A Visual Online Lesson on Metabolic Flux for Undergraduates

Metabolism tends to be a challenging and discouraging topic in biochemistry for undergraduate students, but understanding metabolic pathways and their misregulation has become increasingly important. With input from educators, we have used advanced animation and graphics tools to build a visually engaging online lesson on the fundamental concept of metabolic flux. The lesson consists of five short modules that initially introduce the student to individual metabolic pathways, and also provide an overall view of all the central carbon metabolism pathways together so they intuitively understand the interconnectedness. The later modules reveal how flow of metabolites through those pathways change under different conditions such as high altitude or diseases such as cancer, and also briefly explains how metabolic flux is measured experimentally by tracing isotopes of elements specifically carbon. This lesson contains instruction on use for educators and provides example cases that can be used for group discussions after every module. The individual modules can be integrated with regular undergraduate biochemistry instruction at different points in the curriculum. We believe this visualization will help illustrate the critical interplay between catabolism and anabolism, and develop in students thinking at a systems level to better understand metabolic regulation and the dynamic nature of flux. We hope to assess these outcomes in classrooms in the Fall of 2021.

Dynamic response of Aspergillus niger to periodical glucose pulse stimuli in chemostat cultures.

In industrial large-scale bioreactors, microorganisms encounter heterogeneous substrate concentration conditions, which can impact growth or product formation. Here we carried out an extended (12 h) experiment of repeated glucose pulsing with a 10-minperiod to simulate fluctuating glucose concentrations with Aspergillus niger producing glucoamylase, and investigated its dynamic response by rapid sampling and quantitative metabolomics. The 10-minperiod represents worst-case conditions, as in industrial bioreactors the average cycling duration is usually in the order of 1 min. We found that cell growth and the glucoamylase productivity were not significantly affected, despite striking metabolomic dynamics. Periodical dynamic responses were found across all central carbon metabolism pathways, with different time scales, and the frequently reported ATP paradox was confirmed for this A. niger strain under the dynamic conditions. A thermodynamics analysis revealed that several reactions of the central carbon metabolism remained in equilibrium even under periodical dynamic conditions. The dynamic response profiles of the intracellular metabolites did not change during the pulse exposure, showing no significant adaptation of the strain to the more than 60 perturbation cycles applied. The apparent high tolerance of the glucoamylase producing A. niger strain for extreme variations in the glucose availability presents valuable information for the design of robust industrial microbial hosts.

Engineering a Central Carbon Metabolism Pathway to Increase the Intracellular Acetyl-CoA Pool in Synechocystis sp. PCC 6803 Grown under Photomixotrophic Conditions.

In cyanobacteria, photomixotrophic growth is considered as a promising strategy to achieve both high cell density and product accumulation. However, the conversion of glucose to acetyl coenzyme A (acetyl-CoA) in the native glycolytic pathway is insufficient, which decreases the carbon utilization and productivity of engineered cyanobacteria under photomixotrophic conditions. To increase the carbon flux from glucose to key intracellular precursor acetyl-CoA in Synechocystis sp. PCC 6803 (hereafter, Synechocystis 6803) under photomixotrophic conditions, a synthetic nonoxidative cyclic glycolysis (NOG) pathway was introduced into the wild type strain, which successfully increased the intracellular pool of acetyl-CoA by approximately 1-fold. To minimize the competition for glucose, the native Embden-Meyerhof-Parnas (EMP) and Entner-Doudoroff (ED) pathways were knocked out, respectively. Notably, eliminating the native ED pathway in the engineered strain carrying the NOG pathway further increased the intracellular pool of acetyl-CoA up to 2.8-fold. Another carbon consuming pathway in Synechocystis 6803, the glycogen biosynthesis pathway, was additionally knocked out in the above-mentioned engineered strain, which enabled an increase of the intracellular acetyl-CoA pool by up to 3.5-fold when compared with the wild type strain. Finally, the content of intracellular lipids was analyzed as an index of the productive capacity of the engineered Synechocystis 6803 cell factory under photomixotrophic conditions. The results showed the total lipids yield increased about 26% compared to the wild type (from 15.71% to 34.12%, g/g glucose), demonstrating that this integrated approach could represent a general strategy not only for the improvement of the intracellular concentration of acetyl-CoA, but also for the production of value-added chemicals that require acetyl-CoA as a key precursor in cyanobacteria.

ITRAQ-based proteomic analysis reveals the molecule mechanism of reducing higher alcohols in Chinese rice wine by nitrogen compensation

PurposeHigher alcohol is a by-product of the fermentation of wine, and its content is one of the most important parameters that affect and are used to appraise the final quality of Chinese rice wine. Ammonium compensation is an efficient and convenient method to reduce the content of higher alcohols, but the molecule mechanism is poorly understood. Therefore, an iTRAQ-based proteomic analysis was designed to reveal the proteomic changes of Saccharomyces cerevisiae to elucidate the molecular mechanism of ammonium compensation in reducing the content of higher alcohols.MethodsThe iTRAQ proteomic analysis method was used to analyze a blank group and an experimental group with an exogenous addition of 200 mg/L (NH4)2HPO4 during inoculation. The extracted intracellular proteins were processed by liquid chromatography-mass spectrometry and identified using bioinformatics tools. Real-time quantitative polymerase chain reaction was used to verify the gene expression of differentially expressed proteins.ResultsAbout 4062 proteins, including 123 upregulated and 88 downregulated proteins, were identified by iTRAQ-based proteomic analysis. GO and KEGG analysis uncovered that significant proteins were concentrated during carbohydrate metabolism, such as carbon metabolism, glyoxylate, and dicarboxylate metabolism, pyruvate metabolism, and the nitrogen metabolism, such as amino acid synthesis and catabolism pathway. In accordance with the trend of differential protein regulation in the central carbon metabolism pathway and the analysis of carbon metabolic flux, a possible regulatory model was proposed and verified, in which ammonium compensation facilitated glucose consumption, regulated metabolic flow direction into tricarboxylic acid, and further led to a decrease in higher alcohols. The results of RT-qPCR confirmed the authenticity of the proteomic analysis results at the level of gene.ConclusionAmmonium assimilation promoted by ammonium compensation regulated the intracellular carbon metabolism of S. cerevisiae and affected the distribution of metabolic flux. The carbon flow that should have gone to the synthesis pathway of higher alcohols was reversed to the TCA cycle, thereby decreasing the content of higher alcohols. These findings may contribute to an improved understanding of the molecular mechanism for the decrease in higher alcohol content through ammonium compensation.

The effects of illumination and trophic strategy on gene expression in Chlamydomonas reinhardtii

Microalgae are of substantial biotechnological interest due their polyphyletic nature which grants them access to a wide array of high-value metabolites. The inherent genetic diversity of microalgae combined with their metabolic plasticity when grown using different trophic and illumination strategies necessitate the establishment of a reference knowledge base. In the present study we present a detailed characterisation of the combined effects of wavelength selection and trophic strategy on the growth kinetics and gene expression profile of the model microalgae Chlamydomonas reinhardtii grown under moderate to high light intensity (400 μmolph m−2·s−1). The aim is twofold: (a) to establish a list of reliable housekeeping genes valid for quantitative comparisons across several combinations of different wavelengths and trophic strategies and (b) to enable the investigation of the response of central carbon metabolic pathways under these process conditions. White, blue and red light emitting diodes (LEDs) were used to grow pH controlled photo- and mixo-trophic cultures over a period of 136 h in batch mode. A panel of 10 candidate genes, along with biomass growth rate and pigment content were dynamically monitored across all conditions. Statistical analysis identified genes (acetyl-CoA carboxylase subunit α and photosystem I reaction centre subunit II) with less variability observed in their expression levels across the entirety of conditions evaluated compared to housekeeping genes often referred to in literature (receptor of activated protein kinase C and ribosomal protein (large subunit) 19). Further analysis of gene expression profiles revealed substantial differences in response to changes in both wavelength selection (upregulation of ribulose bisphosphate carboxylase small subunit under red phototrophic growth) and trophic strategy (upregulation of malate synthase from phototrophic to mixotrophic conditions). The systematic approach used to establish reliable reference genes presented herein enables robust comparisons of cellular responses across different conditions to better understand algal metabolism and improve process performance.