A dual program for CRP-mediated regulation in bacterial alarmone (p)ppGpp.

Gene expression and proper downstream cellular functions upon facing environmental shifts depend on the combined and cooperative regulation of genetic networks. Here, we identified cAMP receptor protein (CRP) as a master regulator of (p)ppGpp (guanosine tetra- and penta-phosphate) homeostasis. Via CRP-mediated direct transcriptional regulation of the (p)ppGpp synthetase/hydrolase RelA and SpoT, cAMP-CRP stimulates pervasive accumulation of (p)ppGpp under glucose-limiting conditions. Notably, CRP exerts a nonclassical property as a translational regulator through YfiQ-dependent acetylation of ribosome protein S1 at K247, which further enhances the translation of RelA, SpoT, and CRP itself. From a synthetic biology perspective, this self-activating feedback loop for (p)ppGpp synthesis highlights the function of CRP-mediated dual enhancement (CMDE) in controlling bacterial gene expression, which enables stable activation of genetic circuits. CMDE applied in synthetic circuits leads to a stable increase in p-coumaric acid, cinnamic acid, and pinosylvin production. Our findings showed that CRP-mediated dual circuits for (p)ppGpp regulation enable robust activation that could address bioproduction and other biotechnological needs.IMPORTANCETranscriptional-translational coordination is fundamental for rapid and efficient gene expression in most bacteria. Here, we uncovered the roles of cAMP-CRP in this process. We found that CRP distinctly increases RelA and SpoT transcription and translation, and that acetylation of S1 at K247 accelerates the self-activation of the leading CRP under glucose-limiting conditions. We further found that elevated (p)ppGpp significantly impedes the formation of the cAMP-CRP complex, an active form responsible for transcriptional activation. A model was created in which cAMP-CRP and (p)ppGpp cooperate to dynamically modulate the efficiency of transcriptional-translational coordination responses to stress. More broadly, productive activation in synthetic circuits was achieved through the application of CRP-mediated dual enhancement (CMDE), promising to inspire new approaches for the development of cell-based biotechnologies.

Mycobacterium tuberculosis Suppresses Inflammatory Responses in Host through Its Cholesterol Metabolites.

Cholesterol is a key carbon source for Mycobacterium tuberculosis (Mtb) survival and persistence within macrophages. However, little is known about the role of cholesterol metabolism by Mtb in host-Mtb interplay. Here, we report the immune suppression mediated by Mtb's cholesterol metabolites. Conducting the cholesterol metabolic profiling and loss-of-function experiments, we show that the cholesterol oxidation products catalyzed by a thiolase FadA5 from Mtb H37Ra, 4-androstenedione (AD), and its derivant 1,4-androstenedione (ADD) inhibit the expression of pro-inflammatory cytokines and thus promote bacterial survival in bone marrow-derived macrophages (BMDMs). Our time-resolved fluorescence resonance energy transfer (TR-FRET)-based screening further identifies the nuclear receptor LXRα as the target of ADD. Activation of LXRα via ADD impedes the nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPK) signaling and reduces cholesterol accumulation in lipid rafts upon TLR4 simulation, thereby compromising the inflammatory responses. Our findings provide the evidence that Mtb could suppress the host immunity through its cholesterol metabolic enzyme and products, which are potential targets for screening novel anti-tuberculosis (TB) agents.

Empowering probiotics with high xanthine transport for effective hyperuricemia management.

Hyperuricemia, a prevalent metabolic disorder, poses a susceptibility to various complications. The conventional pharmacotherapeutic approaches for hyperuricemia often entail notable adverse effects, posing substantial clinical challenges. Hence, the imperative lies in the development of novel, safe and effective strategies for preventing and treating hyperuricemia. Here, we developed a probiotic Escherichia coli Nissle 1917 strain, designated as YES301, which contains a rationally designed xanthine importer XanQ, enabling efficient uptake of xanthine and hypoxanthine, consequently leading to reduced serum uric acid concentrations and amelioration of renal impairments in a murine model of hyperuricemia. Importantly, YES301 exhibited a therapeutic efficacy comparable to allopurinol, a conventional uric acid-lowering agent, and manifesting fewer adverse effects and enhanced biosafety. These findings highlight the promising potential of engineered probiotics in the management of hyperuricemia through reducing intestinal purine levels.

Creating a bacterium that forms eukaryotic nucleosome core particles.

The nucleosome is one of the hallmarks of eukaryotes, a dynamic platform that supports many critical functions in eukaryotic cells. Here, we engineer the in vivo assembly of the nucleosome core in the model bacterium Escherichia coli. We show that bacterial chromosome DNA and eukaryotic histones can assemble in vivo to form nucleosome complexes with many features resembling those found in eukaryotes. The formation of nucleosomes in E. coli was visualized with atomic force microscopy and using tripartite split green fluorescent protein. Under a condition that moderate histones expression was induced at 1 µM IPTG, the nucleosome-forming bacterium is viable and has sustained growth for at least 110 divisions in longer-term growth experiments. It exhibits stable nucleosome formation, a consistent transcriptome across passages, and reduced growth fitness under stress conditions. In particular, the nucleosome arrays in E. coli genic regions have profiles resembling those in eukaryotic cells. The observed compatibility between the eukaryotic nucleosome and the bacterial chromosome machinery may reflect a prerequisite for bacteria-archaea union, providing insight into eukaryogenesis and the origin of the nucleosome.

The microbiota-dependent tryptophan metabolite alleviates high-fat diet-induced insulin resistance through the hepatic AhR/TSC2/mTORC1 axis.

Type 2 diabetes (T2D) is potentially linked to disordered tryptophan metabolism that attributes to the intricate interplay among diet, gut microbiota, and host physiology. However, underlying mechanisms are substantially unknown. Comparing the gut microbiome and metabolome differences in mice fed a normal diet (ND) and high-fat diet (HFD), we uncover that the gut microbiota-dependent tryptophan metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) is present at lower concentrations in mice with versus without insulin resistance. We further demonstrate that the microbial transformation of tryptophan into 5-HIAA is mediated by Burkholderia spp. Additionally, we show that the administration of 5-HIAA improves glucose intolerance and obesity in HFD-fed mice, while preserving hepatic insulin sensitivity. Mechanistically, 5-HIAA promotes hepatic insulin signaling by directly activating AhR, which stimulates TSC2 transcription and thus inhibits mTORC1 signaling. Moreover, T2D patients exhibit decreased fecal levels of 5-HIAA. Our findings identify a noncanonical pathway of microbially producing 5-HIAA from tryptophan and indicate that 5-HIAA might alleviate the pathogenesis of T2D.

Engineered Bacteria for Visualization and Localization of Gastrointestinal Bleeding: A Promising Application.

Gastrointestinal bleeding, especially obscure gastrointestinal bleeding (OGIB), is a common and serious clinical emergency with a notable incidence rate. However, the current diagnostic method, gastroscopy, is invasive and often struggles to efficiently detect microhemorrhagic lesions, leading to diagnostic challenges and potential misdiagnoses. Here, we developed an intelligently engineered bacterium utilizing synthetic biology techniques for in vivo localization detection of gastrointestinal bleeding. By constructing three gene circuit modules within E. coli Nissle 1917 for heme recognition, response, and output generation, we have successfully enabled specific heme sensing and real-time optical signal production in vivo. This innovative strategy overcomes the limitations of the existing diagnostic methods, offering a noninvasive and precise means of detecting gastrointestinal bleeding. These advancements hold promise for enhancing diagnostic accuracy and treatment efficacy in future clinical settings within the realm of gastroenterology.

Heterologous activation and metabolites identification of the pks7 gene cluster from Saccharopolyspora erythraea.

The microbial genome remains a huge treasure trove for the discovery of diverse natural products. Saccharopolyspora erythraea NRRL23338, the industry producer of erythromycin, has a dozen of biosynthetic gene clusters whose encoding products are unidentified. Heterologous expression of one of the polyketide clusters pks7 in Streptomyces albus B4 chassis resulted in the characterization of its function responsible for synthesizing both 6-methylsalicyclic acid and 6-ethylsalicyclic acid. Meanwhile, two new 6-ethylsalicyclic acid ester derivatives were isolated as shunt metabolites. Their structures were identified by comprehensive analysis of MS and NMR experiments. Putative functions of genes within the pks7 BGC were also discussed.

Metabolic engineering of the oleaginous yeast Yarrowia lipolytica for 2-phenylethanol overproduction.

The rose fragrance molecule 2-phenylethanol (2-PE) has huge market demand in the cosmetics, food and pharmaceutical industries. However, current 2-PE synthesis methods do not meet the efficiency market requirement. In this study, CRISPR-Cas9-related metabolic engineering strategies were applied to Yarrowia lipolytica for the de novo biosynthesis of 2-PE. Initially, overexpressing exogenous feedback-resistant EcAROGfbr and EcPheAfbr increased 2-PE production to 276.3 mg/L. Subsequently, the ylARO10 and ylPAR4 from endogenous genes were enhanced with the multi-copies to increase the titer to 605 mg/L. Knockout of ylTYR1 and enhancement of shikimate pathway by removing the precursor metabolic bottleneck and overexpressing the genes ylTKT, ylARO1, and ylPHA2 resulted in a significant increase of the 2-PE titer to 2.4 g/L at 84 h, with the yield of 0.06 g/gglu, which is the highest yield for de novo synthesis in yeast. This study provides a valuable precedent for the efficient biosynthesis of shikimate pathway derivatives.

A network of acetyl phosphate-dependent modification modulates c-di-AMP homeostasis in Actinobacteria.

Cyclic purine nucleotides are important signal transduction molecules across all domains of life. 3',5'-cyclic di-adenosine monophosphate (c-di-AMP) has roles in both prokaryotes and eukaryotes, while the signals that adjust intracellular c-di-AMP and the molecular machinery enabling a network-wide homeostatic response remain largely unknown. Here, we present evidence for an acetyl phosphate (AcP)-governed network responsible for c-di-AMP homeostasis through two distinct substrates, the diadenylate cyclase DNA integrity scanning protein (DisA) and its newly identified transcriptional repressor, DasR. Correspondingly, we found that AcP-induced acetylation exerts these regulatory actions by disrupting protein multimerization, thus impairing c-di-AMP synthesis via K66 acetylation of DisA. Conversely, the transcriptional inhibition of disA was relieved during DasR acetylation at K78. These findings establish a pivotal physiological role for AcP as a mediator to balance c-di-AMP homeostasis. Further studies revealed that acetylated DisA and DasR undergo conformational changes that play crucial roles in differentiation. Considering the broad distribution of AcP-induced acetylation in response to environmental stress, as well as the high conservation of the identified key sites, we propose that this unique regulation of c-di-AMP homeostasis may constitute a fundamental property of central circuits in Actinobacteria and thus the global control of cellular physiology.IMPORTANCESince the identification of c-di-AMP is required for bacterial growth and cellular physiology, a major challenge is the cell signals and stimuli that feed into the decision-making process of c-di-AMP concentration and how that information is integrated into the regulatory pathways. Using the bacterium Saccharopolyspora erythraea as a model, we established that AcP-dependent acetylation of the diadenylate cyclase DisA and its newly identified transcriptional repressor DasR is involved in coordinating environmental and intracellular signals, which are crucial for c-di-AMP homeostasis. Specifically, DisA acetylated at K66 directly inactivates its diadenylate cyclase activity, hence the production of c-di-AMP, whereas DasR acetylation at K78 leads to increased disA expression and c-di-AMP levels. Thus, AcP represents an essential molecular switch in c-di-AMP maintenance, responding to environmental changes and possibly hampering efficient development. Therefore, AcP-mediated posttranslational processes constitute a network beyond the usual and well-characterized synthetase/hydrolase governing c-di-AMP homeostasis.

Engineering Streptomyces albus B4 for Enhanced Production of (R)-Mellein: A High-Titer Heterologous Biosynthesis Approach.

The natural compound (R)-(-)-mellein exhibits antiseptic and fungicidal activities. We investigated its biosynthesis using the polyketide synthase encoded by SACE_5532 (pks8) from Saccharopolyspora erythraea heterologously expressed in Streptomyces albus B4, a chassis chosen for its fast growth, genetic manipulability, and ample large short-chain acyl-CoA precursor supply. High-level heterologous (R)-(-)-mellein yield was achieved by pks8 overexpression and duplication. The precursor supply pathways were strengthened by overexpression of SACE_0028 (encoding acetyl-CoA carboxylase) and four genes involved in ß-oxidation (fadD, fadE, fadB, and fadA). Cell growth inhibition by (R)-(-)-mellein production at high concentration was relieved by in situ adsorption using Amberlite XAD16 resin. The final strain, B4mel12, produced (R)-(-)-mellein at 6395.2 mg/L in shake-flask fermentation. Overall, this is the first report of heterologous (R)-(-)-mellein synthesis in microorganism with a high titer. (R)-(-)-mellein prototype in this study opens a possibility for the overproduction of valuable melleins in S. albus B4.

Revealing Differential RNA Editing Specificity of Human ADAR1 and ADAR2 in Schizosaccharomyces pombe.

Adenosine-to-inosine (A-to-I) RNA editing is an important post-transcriptional modification mediated by the adenosine deaminases acting on RNA (ADAR) family of enzymes, expanding the transcriptome by altering selected nucleotides A to I in RNA molecules. Recently, A-to-I editing has been explored for correcting disease-causing mutations in RNA using therapeutic guide oligonucleotides to direct ADAR editing at specific sites. Humans have two active ADARs whose preferences and specificities are not well understood. To investigate their substrate specificity, we introduced hADAR1 and hADAR2, respectively, into Schizosaccharomyces pombe (S. pombe), which lacks endogenous ADARs, and evaluated their editing activities in vivo. Using transcriptome sequencing of S. pombe cultured at optimal growth temperature (30 °C), we identified 483 A-to-I high-confident editing sites for hADAR1 and 404 for hADAR2, compared with the non-editing wild-type control strain. However, these sites were mostly divergent between hADAR1 and hADAR2-expressing strains, sharing 33 common sites that are less than 9% for each strain. Their differential specificity for substrates was attributed to their differential preference for neighboring sequences of editing sites. We found that at the -3-position relative to the editing site, hADAR1 exhibits a tendency toward T, whereas hADAR2 leans toward A. Additionally, when varying the growth temperature for hADAR1- and hADAR2-expressing strains, we observed increased editing sites for them at both 20 and 35 °C, compared with them growing at 30 °C. However, we did not observe a significant shift in hADAR1 and hADAR2's preference for neighboring sequences across three temperatures. The vast changes in RNA editing sites at lower and higher temperatures were also observed for hADAR2 previously in budding yeast, which was likely due to the influence of RNA folding at these different temperatures, among many other factors. We noticed examples of longer lengths of dsRNA around the editing sites that induced editing at 20 or 35 °C but were absent at the other two temperature conditions. We found genes' functions can be greatly affected by editing of their transcripts, for which over 50% of RNA editing sites for both hADAR1 and hADAR2 in S. pombe were in coding sequences (CDS), with more than 60% of them resulting in amino acid changes in protein products. This study revealed the extensive differences in substrate selectivity between the two active human ADARS, i.e., ADAR1 and ADAR2, and provided novel insight when utilizing the two different enzymes for in vivo treatment of human genetic diseases using the RNA editing approach.

Engineering of a TetR family transcriptional regulator BkdR enhances heterologous spinosad production in Streptomyces albus B4 chassis.

Precursor supply plays a significant role in the production of secondary metabolites. In Streptomyces bacteria, propionyl-, malonyl-, and methylmalonyl-CoA are the most common precursors used for polyketide biosynthesis. Although propionyl-CoA synthetases participate in the propionate assimilation pathway and directly convert propionate into propionyl-CoA, malonyl- and methylmalonyl-CoA cannot be formed using common acyl-CoA synthetases. Therefore, both acetyl- and propionyl-CoA carboxylation, catalyzed by acyl-CoA carboxylases, should be considered when engineering a microorganism chassis to increase polyketide production. In this study, we identified a transcriptional regulator of the TetR family, BkdR, in Streptomyces albus B4, which binds directly to the promoter region of the neighboring pccAB operon. This operon encodes acetyl/propionyl-CoA carboxylase and negatively regulates its transcription. In addition to acetate and propionate, the binding of BkdR to pccAB is disrupted by acetyl- and propionyl-CoA ligands. We identified a 16-nucleotide palindromic BkdR-binding motif (GTTAg/CGGTCg/TTAAC) in the intergenic region between pccAB and bkdR. When bkdR was deleted, we found an enhanced supply of malonyl- and methylmalonyl-CoA precursors in S. albus B4. In this study, spinosad production was detected in the recombinant strain after introducing the entire artificial biosynthesized gene cluster into S. albus B4. When supplemented with propionate to provide propionyl-CoA, the novel bkdR-deleted strain produced 29.4% more spinosad than the initial strain in trypticase soy broth (TSB) medium. IMPORTANCE: In this study, we describe a pccAB operon involved in short-chain acyl-CoA carboxylation in S. albus B4 chassis. The TetR family regulator, BkdR, represses this operon. Our results show that BkdR regulates the precursor supply needed for heterologous spinosad biosynthesis by controlling acetyl- and propionyl-CoA assimilation. The deletion of the BkdR-encoding gene exerts an increase in heterologous spinosad yield. Our research reveals a regulatory mechanism in short-chain acyl-CoA metabolism and suggests new possibilities for S. albus chassis engineering to enhance heterologous polyketide yield.

Dual engineered bacteria improve inflammatory bowel disease in mice.

Currently, there are many different therapies available for inflammatory bowel disease (IBD), including engineered live bacterial therapeutics. However, most of these studies focus on producing a single therapeutic drug using individual bacteria, which may cause inefficacy. The use of dual drugs can enhance therapeutic effects. However, expressing multiple therapeutic drugs in one bacterial chassis increases the burden on the bacterium and hinders good secretion and expression. Therefore, a dual-bacterial, dual-drug expression system allows for the introduction of two probiotic chassis and enhances both therapeutic and probiotic effects. In this study, we constructed a dual bacterial system to simultaneously neutralize pro-inflammatory factors and enhance the anti-inflammatory pathway. These bacteria for therapy consist of Escherichia coli Nissle 1917 that expressed and secreted anti-TNF-α nanobody and IL-10, respectively. The oral administration of genetically engineered bacteria led to a decrease in inflammatory cell infiltration in colon and a reduction in the levels of pro-inflammatory cytokines. Additionally, the administration of engineered bacteria did not markedly aggravate gut fibrosis and had a moderating effect on intestinal microbes. This system proposes a dual-engineered bacterial drug combination treatment therapy for inflammatory bowel disease, which provides a new approach to intervene and treat IBD. KEY POINTS: • The paper discusses the effects of using dual engineered bacteria on IBD • Prospects of engineered bacteria in the clinical treatment of IBD.

Allosteric regulation by c-di-AMP modulates a complete N-acetylglucosamine signaling cascade in Saccharopolyspora erythraea.

c-di-AMP is an essential and widespread nucleotide second messenger in bacterial signaling. For most c-di-AMP synthesizing organisms, c-di-AMP homeostasis and the molecular mechanisms pertaining to its signal transduction are of great concern. Here we show that c-di-AMP binds the N-acetylglucosamine (GlcNAc)-sensing regulator DasR, indicating a direct link between c-di-AMP and GlcNAc signaling. Beyond its foundational role in cell-surface structure, GlcNAc is attractive as a major nutrient and messenger molecule regulating multiple cellular processes from bacteria to humans. We show that increased c-di-AMP levels allosterically activate DasR as a master repressor of GlcNAc utilization, causing the shutdown of the DasR-mediated GlcNAc signaling cascade and leading to a consistent enhancement in the developmental transition and antibiotic production in Saccharopolyspora erythraea. The expression of disA, encoding diadenylate cyclase, is directly repressed by the regulator DasR in response to GlcNAc signaling, thus forming a self-sustaining transcriptional feedback loop for c-di-AMP synthesis. These findings shed light on the allosteric regulation by c-di-AMP, which appears to play a prominent role in global signal integration and c-di-AMP homeostasis in bacteria and is likely widespread in streptomycetes that produce c-di-AMP.

An Orthogonal CRISPR/dCas12a System for RNA Imaging in Live Cells.

CRISPR/Cas technology has made great progress in the field of live-cell imaging beyond genome editing. However, effective and easy-to-use CRISPR systems for labeling multiple RNAs of interest are still needed. Here, we engineered a CRISPR/dCas12a system that enables the specific recognition of the target RNA under the guidance of a PAM-presenting oligonucleotide (PAMmer) to mimic the PAM recognition mechanism for DNA substrates. We demonstrated the feasibility and specificity of this system for specifically visualizing endogenous mRNA. By leveraging dCas12a-mediated precursor CRISPR RNA (pre-crRNA) processing and the orthogonality of dCas12a from different bacteria, we further demonstrated the proposed system as a simple and versatile molecular toolkit for multiplexed imaging of different types of RNA transcripts in live cells with high specificity. This programmable dCas12a system not only broadens the RNA imaging toolbox but also facilitates diverse applications for RNA manipulation.

MicroRNA-144-3p Inhibits Host Lipid Catabolism and Autophagy by Targeting PPARα and ABCA1 During Mycobacterium Tuberculosis Infection.

MicroRNA-mediated metabolic reprogramming recently has been identified as an important strategy for Mycobacterium tuberculosis (Mtb) to evade host immune responses. However, it is unknown what role microRNA-144-3p (miR-144-3p) plays in cellular metabolism during Mtb infection. Here, we report the meaning of miR-144-3p-mediated lipid accumulation for Mtb-macrophage interplay. Mtb infection was shown to upregulate the expression of miR-144-3p in macrophages. By targeting peroxisome proliferator-activated receptor α (PPARα) and ATP-binding cassette transporter A1 (ABCA1), miR-144-3p overexpression promoted lipid accumulation and bacterial survival in Mtb-infected macrophages, while miR-144-3p inhibition had the opposite effect. Furthermore, reprogramming of host lipid metabolism by miR-144-3p suppressed autophagy in response to Mtb infection. Our findings uncover that miR-144-3p regulates host metabolism and immune responses to Mtb by targeting PPARα and ABCA1, suggesting a potential host-directed tuberculosis therapy by targeting the interface of miRNA and lipid metabolism.

Characterization of diverse lysine acylations in Bacillus thuringiensis: Substrate profiling and functional exploration.

Lysine acylation has been extensively investigated due to its regulatory role in a diverse range of biological functions across prokaryotic and eukaryotic species. In-depth acylomic profiles have the potential to enhance comprehension of the biological implications of organisms. However, the extent of research on global acylation profiles in microorganisms is limited. Here, four lysine acylomes were conducted in Bacillus thuringiensis by using the LC-MS/MS based proteomics combined with antibody-enrichment strategies, and a total of 3438 acetylated sites, 5797 propionylated sites, 1705 succinylated sites, and 925 malonylated sites were identified. The motif analysis of these modified proteins revealed a high conservation of glutamate in acetylation and propionylation, whereas such conservation was not observed in succinylation and malonylation modifications. Besides, conservation analysis showed that homologous acylated proteins in Bacillus subtilis and Escherichia coli were connected with ribosome and aminoacyl-tRNA biosynthesis. Further biological experiments showed that lysine acylation lowered the RNA binding ability of CodY and impaired the in vivo protein activity of MetK. In conclusion, our study expanded the current understanding of the global acylation in Bacillus, and the comparative analysis demonstrated that shared acylation proteins could play important roles in regulating both metabolism and RNA transcription progression.

Recent Advances in Microbial Metabolic Engineering for Production of Natural Phenolic Acids.

Phenolic acids are important natural bioactive compounds with varied physiological functions. They are extensively used in food, pharmaceutical, cosmetic, and other chemical industries and have attractive market prospects. Compared to plant extraction and chemical synthesis, microbial fermentation for phenolic acid production from renewable carbon sources has significant advantages. This review focuses on the structural information, physiological functions, current applications, and biosynthesis pathways of phenolic acids, especially advances in the development of metabolically engineered microbes for the production of phenolic acids. This review provides useful insights concerning phenolic acid production through metabolic engineering of microbial cell factories.

Screening potential biomarkers associated with insulin resistance in high-fat diet-fed mice by integrating metagenomics and untargeted metabolomics.

Insulin resistance is the primary pathophysiological basis for metabolic syndrome and type 2 diabetes. Gut microbiota and microbiota-derived metabolites are pivotal in insulin resistance. However, identifying the specific microbes and key metabolites with causal roles is a challenging task, and the underlying mechanisms require further exploration. Here, we successfully constructed a model of insulin resistance in mice induced by a high-fat diet (HFD) and screened potential biomarkers associated with insulin resistance by integrating metagenomics and untargeted metabolomics. Our findings showed a significant increase in the abundance of 30 species of Alistipes in HFD mice compared to normal diet (ND) mice, while the abundance of Desulfovibrio and Candidatus Amulumruptor was significantly lower in HFD mice than in ND mice. Non-targeted metabolomics analysis identified 21 insulin resistance-associated metabolites, originating from the microbiota or co-metabolized by both the microbiota and the host. These metabolites were primarily enriched in aromatic amino acid metabolism (tryptophan metabolism, tyrosine metabolism, and phenylalanine metabolism) and arginine biosynthesis. Further analysis revealed a significant association between the three distinct genera and 21 differentiated metabolites in the HFD and ND mice. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of representative genomes from 12 species of the three distinct genera further revealed the functional potential in aromatic amino acid metabolism and arginine biosynthesis. This study lays the groundwork for future investigations into the mechanisms through which the gut microbiota and its metabolites impact insulin resistance. IMPORTANCE: In this study, we aim to identify the microbes and metabolites linked to insulin resistance, some of which have not been previously reported in insulin resistance-related studies. This adds a complementary dimension to existing research. Furthermore, we establish a correlation between alterations in the gut microbiota and metabolite levels. These findings serve as a foundation for identifying the causal bacterial species and metabolites. They also offer insights that guide further exploration into the mechanisms through which these factors influence host insulin resistance.

Metabolic Engineering of Pseudomonas putida KT2440 for De Novo Biosynthesis of Vanillic Acid.

Vanillic acid (VA), as a plant-derived phenolic acid compound, has widespread applications and good market prospects. However, the traditional production process cannot meet market demand. In this study, Pseudomonas putida KT2440 was used for de novo biosynthesis of VA. Multiple metabolic engineering strategies were applied to construct these P. putida-based cell factories, including the introduction of a Hs-OMTopt, engineering the cofactor S-adenosylmethionine supply pathway through the overexpression of metX and metH, reforming solubility of Hs-OMTopt, increasing a second copy of Hs-OMTopt, and the optimization of the fermentation medium. The resulting strain, XCS17, de novo biosynthesized 5.4 g/L VA from glucose in a fed-batch fermentation system; this is the highest VA production titer reported up to recently. This study showed that P. putida KT2440 is a robust platform for achieving the effective production of phenolic acids.