Phenyllactic Acid Research Articles

Antimicrobial Effect of Honey Phenolic Compounds against E. coli-An In Vitro Study.

Growing concern over antimicrobial resistance in chronic wound patients necessitates the exploration of alternative treatments from natural sources. This study suggests that honey's phenolic compounds may offer antimicrobial benefits, warranting further investigation for therapeutic development. The main aim of this study was to investigate the antimicrobial activity of phenolic compounds and to determine the effects of their sub-inhibitory concentrations against Escherichia coli (E. coli). 3-phenyllactic acid (PLA), p-coumaric acid (PCA), and phloretin were tested against the bacterial strain of E. coli ATCC 25922. Comparison of the antimicrobial activity of honey constituents in vitro was performed using a broth culture assay. Measurement of the inhibitory properties of constituents in vitro was conducted using disc and well diffusion assays. The effects of sub-inhibitory concentrations of PCA on the susceptibility of E. coli ATCC 25922 to penicillin-streptomycin were tested. The results demonstrated that PLA was the most efficient antimicrobial agent, followed by PCA, whereas phloretin, at lower (2 mg/mL) concentrations, led to an increase in the growth of E. coli. Various modifications of the agar diffusion assay did not reveal the antibacterial properties of the studied phytochemicals. The enhancing effect of a sub-inhibitory concentration of PCA in cooperation with penicillin-streptomycin was shown. These findings might be helpful for the further investigation and development of new antimicrobial agents for the treatment of skin infections and wounds.

Polygonatum kingianum Polysaccharides Enhance the Preventive Efficacy of Heat-Inactivated Limosilactobacillus reuteri WX-94 against High-Fat-High-Sucrose-Induced Liver Injury and Gut Dysbacteriosis.

Limosilactobacillus reuteri (L. reuteri) is an efficacious probiotic that could reduce inflammation and prevent metabolic disorders. Here, we innovatively found that Polygonatum kingianum polysaccharides (PKP) promoted proliferation and increased stability of L. reuteri WX-94 (a probiotic strain showing anti-inflammation potentials) in simulated digestive fluids in vitro. PKP was composed of galactose, glucose, mannose, and arabinose. The cell-free supernatant extracted from L. reuteri cultured with PKP increased ABTS•+, DPPH•, and FRAP scavenging capacities compared with the supernatant of the medium without PKP and increased metabolites with health-promoting activities, e.g., 3-phenyllactic acid, indole-3-lactic acid, indole-3-carbinol, and propionic acid. Moreover, PKP enhanced alleviating effects of heat-inactivated L. reuteri on high-fat-high-sucrose-induced liver injury in rats via reducing inflammation and regulating expressions of protein and genes involved in fatty acid metabolism (such as HIF1-α, FAβO, CPT1, and AMPK) and fatty acid profiles in liver. Such benefits correlated with its prominent effects on enriching Lactobacillus and short-chain fatty acids while reducing Dubosiella, Fusicatenilacter, Helicobacter, and Oscillospira. Our work provides novel insights into the probiotic property of PKP and emphasizes the great potential of the inactivated L. reuteri cultured with PKP in contracting unhealthy diet-induced liver dysfunctions and gut dysbacteriosis.

Increasing the robustness of Escherichia coli for aromatic chemicals production through transcription factor engineering

Engineering microbial cell factories has been widely used to produce a variety of chemicals, including natural products, biofuels, and bulk chemicals. However, poor robustness limits microbial production on an industrial scale. Microbial robustness is essential to ensure reliable and sustainable production of targeted chemicals. In this study, we developed an approach to screen transcription factors to improve robustness using CRSPRa technology. We applied this approach to identify some transcription factors to increase the robustness of Escherichia coli to aromatic chemicals. Activation of hdfR, yldP, purR, sosS, ygeH, cueR, cra, and treR increased the robustness of E. coli to phenyllactic acid. Upregulation of some transcription factors also improved the robustness to caffeic acid (cra) or tyrosol (cra, cueR, treR, soxS, hdfR and purR). Our study demonstrated that transcription factor engineering using CRISPRa is a powerful method to increase microbial robustness. This research provides new approaches to efficiently find genes responsible for increasing microbial robustness.

Distinct Changes in Metabolic Profile and Sensory Quality with Different Varieties of Chrysanthemum (Juhua) Tea Measured by LC-MS-Based Untargeted Metabolomics and Electronic Tongue.

Chrysanthemum tea, a typical health tea with the same origin as medicine and food, is famous for its unique health benefits and flavor. The taste and sensory quality of chrysanthemum (Juhua) tea are mainly determined by secondary metabolites. Therefore, the present research adopted untargeted metabolomics combined with an electronic tongue system to analyze the correlation between the metabolite profiles and taste characteristics of different varieties of chrysanthemum tea. The results of sensory evaluation showed that there were significant differences in the sensory qualities of five different varieties of chrysanthemum tea, especially bitterness and astringency. The results of principal component analysis (PCA) indicated that there were significant metabolic differences among the five chrysanthemum teas. A total of 1775 metabolites were identified by using untargeted metabolomics based on UPLC-Q-TOF/MS analysis. According to the variable importance in projection (VIP) values of the orthogonal projections to latent structures discriminant analysis (OPLS-DA), 143 VIP metabolites were found to be responsible for metabolic changes between Huangju and Jinsi Huangju tea; among them, 13 metabolites were identified as the key metabolites of the differences in sensory quality between them. Kaempferol, luteolin, genistein, and some quinic acid derivatives were correlated with the "astringency" attributes. In contrast, l-(-)-3 phenyllactic acid and L-malic acid were found to be responsible for the "bitterness" and "umami" attributes in chrysanthemum tea. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the flavonoid and flavonol biosynthesis pathways had important effects on the sensory quality of chrysanthemum tea. These findings provide the theoretical basis for understanding the characteristic metabolites that contribute to the distinctive sensory qualities of chrysanthemum tea.

The Accumulation of Phenyllactic Acid Impairs Host Glutamine Metabolism and Inhibits African Swine Fever Virus Replication: A Novel Target for the Development of Anti-ASFV Drugs.

African swine fever (ASF) is a highly contagious and hemorrhagic disease caused by infection with the African swine fever virus (ASFV), resulting in a mortality rate of up to 100%. Currently, there are no effective treatments and commercially available vaccines for ASF. Therefore, it is crucial to identify biochemicals derived from host cells that can impede ASFV replication, with the aim of preventing and controlling ASF. The ASFV is an acellular organism that promotes self-replication by hijacking the metabolic machinery and biochemical resources of host cells. ASFV specifically alters the utilization of glucose and glutamine, which are the primary metabolic sources in mammalian cells. This study aimed to investigate the impact of glucose and glutamine metabolic dynamics on the rate of ASFV replication. Our findings demonstrate that ASFV infection favors using glutamine as a metabolic fuel to facilitate self-replication. ASFV replication can be substantially inhibited by blocking glutamine metabolism. The metabolomics analysis of the host cell after late-stage ASFV infection revealed a significant disruption of normal glutamine metabolic pathways due to the abundant expression of PLA (phenyllactic acid). Pretreatment with PLA also inhibited ASFV proliferation and glutamine consumption following infection. The metabolomic analysis also showed that PLA pretreatment greatly slowed down the metabolism of amino acids and nucleotides that depend on glutamine. The depletion of these building blocks directly hindered the replication of ASFV by decreasing the biosynthetic precursors produced during the replication of ASFV's progeny virus. These findings provide valuable insight into the possibility of pursuing the development of antiviral drugs against ASFV that selectively target metabolic pathways.

Reuterin, Phenyllactic Acid, and Exopolysaccharides as Main Antifungal Molecules Produced by Lactic Acid Bacteria: A Scoping Review.

The primary goal of this scoping review is to collect, analyze, and critically describe information regarding the role of the main compounds (reuterin, phenyllactic acid, and exopolysaccharides) produced by LAB that possess antifungal properties and provide some suggestions for further research. The use of lactic acid bacteria (LAB) to mitigate spoilage and extend the shelf life of foodstuffs has a long history. Recently, there has been a growing interest in the unique properties of these additions to the foodstuffs in which they are applied. In recent studies regarding biopreservation, significant attention has been given to the role of these microorganisms and their metabolites. This fascinating recent discipline aims not only to replace traditional preservation systems, but also to improve the overall quality of the final product. The biologically active by-products produced by lactic acid bacteria are synthesized under certain conditions (time, temperature, aerobiosis, acidity, water activity, etc.), which can be enacted through one of the oldest approaches to food processing: fermentation (commonly used in the dairy and bakery sectors). This study also delves into the biosynthetic pathways through which they are synthesized, with a particular emphasis on what is known about the mechanisms of action against molds in relation to the type of food.

PLA inhibits TNF-α-induced PANoptosis of prostate cancer cells through metabolic reprogramming

Previous studies have shown that phenyllactic acid (alpha-Hydroxyhydrocinnamic acid, 2-Hydroxy-3-phenylpropionic acid, PLA), a type of organic acid metabolite, has excellent diagnostic efficacy when used to differentiate between prostate cancer, benign prostatic hyperplasia, and prostatitis. This research aims to explore the molecular mechanism by which PLA influences the PANoptosis of prostate cancer (PCa) cell lines. First, we found that PLA was detected in all prostate cancer cell lines (PC-3, PC-3 M, DU145, LNCAP). Further experiments showed that the addition of PLA to prostate cancer cells could promote ATP generation, enhance cysteine desulfurase (NFS1) expression, and reduce tumor necrosis factor alpha (TNF-α) levels, thereby inhibiting apoptosis in prostate cancer cells. Notably, overexpression of NFS1 can inhibit the binding of TNF-α to serpin mRNA binding protein 1 (SERBP1), suggesting that NFS1 competes with TNF-α for binding to SERBP1. Knockdown of SERBP1 significantly reduced the level of small ubiquity-related modifier (SUMO) modification of TNF-α. This suggests that NFS1 reduces the SUMO modification of TNF-α by competing with SERBP1, thereby reducing the expression and stability of TNF-α and ultimately inhibiting apoptosis in prostate cancer cell lines. In conclusion, PLA inhibits TNF-α induced panapoptosis of prostate cancer cells through metabolic reprogramming, providing a new idea for targeted treatment of prostate cancer.

Transcriptomic analyses of Vibrio parahaemolyticus under the phenyllactic acid stress

Phenyllactic acid (PLA) generally recognized as a natural organic acid shows against Vibrio parahaemolyticus activity. In this study, V. parahaemolyticus ATCC17802 (Vp17802) was cultured under the stress of 1/2MIC PLA, and then the antibacterial mechanisms were explored via transcriptomics. The minimum inhibitory concentration (MIC) of PLA against Vp17802 was 3.2 mg/mL, and the time-kill analysis resulted that Vp17802 was inhibited. PLA was able to destroy the bacterial membrane, leading to the leakage of intracellular substances and decline of ATP levels. The RNA-sequencing analysis results indicated that 1616 significantly differentially expressed genes were identified, among which 190 were up-regulated and 1426 were down-regulated. Down-regulation of the icd2 gene in the TCA cycle mediates blockage of tyrosine metabolic, arginine biosynthesis, and oxidative phosphorylation, causing insufficient energy supply of Vp17802. Moreover, PLA could cause amino acids, metal ions, and phosphate transporters to be blocked, affecting the acquisition of nutrients. The treatment by PLA altered the expression of genes encoding functions involved in quorum sensing, flagellar assembly, and cell chemotaxis pathway, which may be interfering with the biofilm formation in Vp17802, reducing cell motility. Overall, 1.6 mg/mL PLA inhibited the growth of Vp17802 by disrupting to uptake of nutrients, cell metabolism, and the formation of biofilms. The results suggested a new direction for exploring the activity of PLA against Vp17802 and provided a theoretical basis for bacterial pathogen control in the food industry.Key points•RNA sequencing was carried out to indicate the antibacterial mechanism of Vp17802.•The icd2 gene in the TCA cycle mediates blockage of metabolic of Vp17802.•The biofilm formation has interfered with 1.6 mg/mL PLA, which could reduce cell motility and virulence.Graphical

Characterization and stability assessment of polyphenols bound to Lycium barbarum polysaccharide: Insights from gastrointestinal digestion and colon fermentation

Polysaccharides and polyphenols are biologically active components that coexist in Lycium barbarum fruit, and there may be interactions between them that affect the release of each other. In this study, polyphenols bound to L. barbarum polysaccharide (LBP) were characterized, and the stability of bound phenolics (BP) was assessed by gastrointestinal digestion and colon fermentation. The results showed that a total of 65 phytochemicals such as flavonoids, phenolic acids, and coumarins were identified by UPLC-MS/MS. Quantitative analysis revealed that the major phenolic constituents were rutin, p-coumaric acid, catechin, ferulic acid, protocatechuic acid, and gallic acid, and their contents were 58.72, 24.03, 14.24, 13.28, 10.39, and 6.7 mg GAE/100 g DW, respectively. The release of BP by gastric digestion and gastrointestinal digestion was 9.67 % and 19.39 %, respectively. Most polyphenols were greatly affected by gastric digestion, while rutin was released in small intestine. The BP were fully released (49.77 %) and metabolized by gut microorganisms, and a considerable number of intermediates and end-products were detected, such as phloroglucinol, phenylacetic acid, and phenyllactic acid. Microbiomics data emphasized the positive impact of LBP on gut bacteria of Bacteroides, Parabacteroides, and Clostridioides. These findings could deepen our understanding of the bioavailability and biological fate of BP and also provide reference data for nutrient release and utilization of L. barbarum as a whole.

Enhancement of disease resistance against Alternaria alternata in winter jujube fruit by phenyllactic acid through regulating Ca2+ signaling transduction and mitogen-activated protein kinase cascades

Alternaria rot caused by Alternaria alternata is one of destructive fungal diseases of postharvest winter jujubes. This study performed to evaluate the potential mechanisms participated in the enhancement disease resistance of winter jujubes by phenyllactic acid (PLA). Results demonstrated that PLA reduced the lesion diameter of A. alternata-inoculated winter jujubes. Additionally, PLA treatment led to an up-regulation in the transcription levels of ZjCML23/28, ZjCIPK11/12/20, ZjCDPK1/3/5, ZjMAPKKK18, ZjMAPKK6, and ZjMAPK7/19 as well as ZjNPR1 and ZjWRKY14/22/44/55/64 in fruit. Conversely, the transcription levels of ZjMAPK15 and ZjMAPKKK17 were down-regulated. Simultaneously, PLA enhanced the gene transcription levels and enzymatic activities of peroxidase, β-1,3-glucanase, and chitinase in winter jujubes, while facilitating the deposition of lignin, flavonoids, and total phenolics. Overall, these findings suggest that PLA enhances the resistance of winter jujube fruit via positively modulating the transcription of key Ca2+ receptor protein, activating the mitogen-activated protein kinase cascades, and potentially involving NPR1 and WRKY transcription factors.

Anti-phototoxicity and anti-melanogenesis activities of eelgrass Zostera marina and its phenolic constituents

The eelgrass Zostera marina L. has several economic roles, from its earlier usage in the insulation industry to protecting the earth from global warming. In this study, we aimed to discover the cosmetic potential of Z. marina. A methanolic extract of Z. marina showed anti-phototoxicity and anti-melanogenesis activity with an IC50 of 17.5 μM, followed by a phytochemical analysis of its phenolic constituents. Ten compounds (1−10) were isolated by several chromatographic techniques and identified by means of nuclear magnetic resonance spectroscopy (NMR) as well as high-resolution mass spectrometry (HR/MS). The identified compounds are caffeic acid (1), 3,4-dihydroxybenzoic acid (protocatechuic acid) (2), luteolin (3), diosmetin (4), 4-coumaroyl-4′-hydroxyl phenyllactic acid (5), rosmarinic acid (6), caffeoyl-4′-hydroxy-phenyllactic acid (isorinic acid) (7), apigenin 7-O-β-D-glucopyranoside (8), luteolin 7-O-β-D-glucopyranoside (9), and luteolin 7-sulfate (10). This is the first report to identify compounds 5 and 7 from the family Zosteraceae. The isolated compounds were assessed for their anti-aging abilities and were found to exhibit good anti-phototoxicity and anti-melanogenesis activities by increasing the viability of UVB-irradiated HaCaT cells by 6% to 34% and by inhibiting melanin synthesis in B16 melanoma cells by 44% to 65%.

Development of phenyllactic acid ionic liquids and evaluation of cytotoxicity to human cervical epithelial cells.

Phenyllactic acid (PLA), is a naturally produced, broad-spectrum antimicrobial compound with activity against bacteria and fungi. PLA can be produced by a variety of lactic acid bacteria, including vaginal Lactobacillus species, which are healthy constituents of the vaginal microbiome with a protective role against invading pathogenic bacteria and/or fungi. Additionally, PLA has been shown to exhibit anti-inflammatory and immunomodulatory properties, overall indicating its therapeutic potential as an intravaginally delivered compound for modulation of the vaginal microbiome. However, PLA has low kinetic solubility in water. Hence, strategies to improve the solubility of PLA are necessary to facilitate its intravaginal delivery. Using biocompatible cations, choline and carnitine, we successfully transformed both d- and l-enantiomers of crystalline PLA into amorphous low-melting ionic liquids (ILs) with high water solubility. We further evaluated the in vitro cytotoxicity of PLA ILs to human cervical epithelial cells. Microscopic visualisation of cellular morphology using crystal violet staining and MTT cell proliferation assay revealed that PLA ILs result in minimal morphological changes and low cytotoxicity to human cervical epithelial cells. Overall, we successfully demonstrated that transforming PLA into ILs efficiently enhances its solubility in water and these formulations are not toxic to human epithelial cells. This investigation lays the groundwork for future testing of PLA ILs for their antimicrobial properties and metabolic activity within the cervicovaginal microenvironment.

Efficient production of phenyllactic acid in Escherichia coli via metabolic engineering and fermentation optimization strategies.

Phenyllactic acid (PhLA), an important natural organic acid, can be used as a biopreservative, monomer of the novel polymeric material poly (phenyllactic acid), and raw material for various medicines. Herein, we achieved a high-level production of PhLA in Escherichia coli through the application of metabolic engineering and fermentation optimization strategies. First, the PhLA biosynthetic pathway was established in E. coli CGSC4510, and the phenylalanine biosynthetic pathway was disrupted to improve the carbon flux toward PhLA biosynthesis. Then, we increased the copy number of the key genes involved in the synthesis of the PhLA precursor phenylpyruvic acid. Concurrently, we disrupted the tryptophan biosynthetic pathway and enhanced the availability of phosphoenolpyruvate and erythrose 4-phosphate, thereby constructing the genetically engineered strain MG-P10. This strain was capable of producing 1.42 ± 0.02 g/L PhLA through shake flask fermentation. Furthermore, after optimizing the dissolved oxygen feedback feeding process and other conditions, the PhLA yield reached 52.89 ± 0.25 g/L in a 6 L fermenter. This study successfully utilized metabolic engineering and fermentation optimization strategies to lay a foundation for efficient PhLA production in E. coli as an industrial application.

Investigation of Urine Organic Acid Profile in Coronavirus Disease (COVID-19) Patients.

Coronavirus disease 2019 (COVID-19) is a viral infection caused by severe acute respiratory syn-drome coronavirus 2 (SARS-CoV-2). The high mortality and morbidity rate and its course with a wide variety of clinical symptoms indicate that the disease affects many metabolic pathways. The aim of our study was to investi-gate the metabolic effects of SARS-CoV-2 by evaluating the urinary organic acid profile in patients with SARS-CoV-2 infection. Can metabolites in urine guide the diagnosis, follow-up, treatment, and prognosis of COVID-19? Forty-two patients, including 30 SARS-CoV-2 RT-PCR positive patients and 12 SARS-CoV-2 RT-PCR negative controls, were studied. SARS-COV-2 RT-PCR of nasopharyngeal swab samples was studied. Urines were evaluated in the GCMS-QP2010 SE Gas Chromatograph Mass Spectrometer (SHIMADZU) device for organic acid profile in the metabolism laboratory. Urine organic acid profile was evaluated by studying 117 organic acids in each patient. Tiglylglycine, 2-hydroxybutyric acid, 4-hydroxyphenylpyruvic acid, 3-hydroxypropionic acid, erythro-4,5-dihydroxyhexanoic acid lactone, 2-hydroxyphenylacetic acid, N-acetyltyrosine, 3-phenyllactic acid, 5-hydroxyindolacetic acid, 3-hydroxysebacic acid, palmitic acid, 3-methylglutaconic acid, 3-methylglutaric acid, lactic acid, pyruvic acid-oxime, 3-hydroxysobutyric acid, and organic acids were found to be increased, compared to the control group. Tiglylglycine has been specifically identified as a potential biomarker of respiratory chain disorders. The deterioration in lipid metabolism and pyruvate pathway in COVID-19 patients was evaluated as remarkable.

Immune-enhancing effect of Weizmannia coagulans BCG44 and its supernatant on cyclophosphamide-induced immunosuppressed mice and RAW264.7 cells via the modulation of the gut microbiota.

We established a model of cyclophosphamide (CTX)-induced immunosuppressed mice and RAW264.7 cells to assess the effectiveness of W. coagulans BCG44 and its supernatant in enhancing immune function and modulating the gut microbiota. W. coagulans BCG44 and its supernatant restored Th17/Treg balance and alleviated gut inflammation by elevating the expression of interleukin-10 (IL-10) and decreasing IL-6 and toll-like receptor 4 (TLR4). Meanwhile, W. coagulans BCG44 and its supernatant downregulated the levels of lipopolysaccharide and D-lactic acid while increasing the expression of tight junction proteins (ZO-1 and occludin) and goblet cells/crypts to ameliorate mucosal damage. W. coagulans BCG44 and its supernatant may restore the gut microbiota in the immunosuppressed mice by regulating keystone species (Lactobacillus and Lachnospiraceae). PICRUSt2 function prediction and BugBase analysis showed thatW. coagulansBCG44 and its supernatant significantly down-regulated American trypanosomiasis and potentially_pathogenic. In addition, under normal versus inflamed culture conditions, stimulation of RAW246.7 cells with W. coagulans BCG44 supernatant activated immune response with increasing proliferation ability and the gene expression of IL-10 while decreasing TLR4. Metabolites in the W. coagulans BCG44 supernatant included arginine, tyrosine, solamargine, tryptophan, D-mannose, phenyllactic acid, and arachidonic acid. Collectively, these findings suggested that W. coagulans BCG44 and its supernatant possess potential immunomodulatory activity and modulate gut microbiota dysbiosis in the CTX-induced immunosuppressed mice.

Sepsis-Associated Metabolites and Their Biotransformation by Intestinal Microbiota

High concentration of sepsis-associated aromatic microbial metabolites (AMM) stands as a prognostically unfavorable factor, indicating the progression of multiple organ dysfunction and an increased risk of death in patients with sepsis and septic shock. This study is based on a hypothesis that excess of sepsis-associated AMM in patients with sepsis is caused by metabolic alterations (dysfunction) in the intestinal microbiota.The aim of this study was to compare the potential of normobiota and pathobiota to bio-transform sepsis-associated metabolites of aromatic amino acids tyrosine and phenylalanine, such as phenyllactic acid(PhLA) and 4-hydroxyphenyllactic acid (4-HPhLA).Materials and methods. Samples of intestinal contents of patients with septic shock (N=10, pathobiota) and healthy volunteers (N=9, normobiota) were placed in test tubes with the omnipurpose thioglycol medium. The clinical model of excessive inflow of sepsis-associated AMM into the intestine (for example, from blood or sites of inflammation) was reproduced in the in vitro experiment by adding PhLA or 4-HPhLA in clinically significant concentrations (25 mkM) into each test tube with pathobiota and normobiota. After incubation in a thermostat (37°, 24 hours), AMМ concentrations were measured in the samples with pathobiota and normobiota using GC-MS analysis.Results. Concentration of AMM decreased within 24 hours in the tubes with normobiota after PhLA or 4-HPhLA were added. In the tubes with pathobiota, no decrease in AMM concentrations was documented after loading with PhLA or 4-HPhLA. Concentrations of PhLA (P=0.002) and 4-HPhLA (P0.001) were statistically significantly higher in pathobiota samples compared to normobiota.Conclusion. The in vitro experiment demonstrates that after excessive load with sepsis-associated metabolites (PhLA, 4-HPhLA), the microbiota of healthy people is capable to bio-transform such metabolites to the end products of microbial metabolism, while pathobiota of septic patients exhibits altered biotransformational potential. This data demonstrate that microbiota dysfunction may contribute to the pathogenesis of sepsis.

An evolved pyrrolysyl-tRNA synthetase with polysubstrate specificity expands the toolbox for engineering enzymes with incorporation of noncanonical amino acids

Aminoacyl-tRNA synthetase (aaRS) is a core component for genetic code expansion (GCE), a powerful technique that enables the incorporation of noncanonical amino acids (ncAAs) into a protein. The aaRS with polyspecificity can be exploited in incorporating additional ncAAs into a protein without the evolution of new, orthogonal aaRS/tRNA pair, which hence provides a useful tool for probing the enzyme mechanism or expanding protein function. A variant (N346A/C348A) of pyrrolysyl-tRNA synthetase from Methanosarcina mazei (MmPylRS) exhibited a wide substrate scope of accepting over 40 phenylalanine derivatives. However, for most of the substrates, the incorporation efficiency was low. Here, a MbPylRS (N311A/C313A) variant was constructed that showed higher ncAA incorporation efficiency than its homologous MmPylRS (N346A/C348A). Next, N-terminal of MbPylRS (N311A/C313A) was engineered by a greedy combination of single variants identified previously, resulting in an IPE (N311A/C313A/V31I/T56P/A100E) variant with significantly improved activity against various ncAAs. Activity of IPE was then tested toward 43 novel ncAAs, and 16 of them were identified to be accepted by the variant. The variant hence could incorporate nearly 60 ncAAs in total into proteins. With the utility of this variant, eight various ncAAs were then incorporated into a lanthanide-dependent alcohol dehydrogenase PedH. Incorporation of phenyllactic acid improved the catalytic efficiency of PedH toward methanol by 1.8-fold, indicating the role of modifying protein main chain in enzyme engineering. Incorporation of O-tert-Butyl-L-tyrosine modified the enantioselectivity of PedH by influencing the interactions between substrate and protein. Enzymatic characterization and molecular dynamics simulations revealed the mechanism of ncAAs affecting PedH catalysis. This study provides a PylRS variant with high activity and substrate promiscuity, which increases the utility of GCE in enzyme mechanism illustration and engineering.Graphical

Intestinal Geographical Pattern of Gut Microbiome and Metabolites Identifies Novel Regulators of Graft-Versus-Host Disease

Introduction: The severity of T cell-mediated gastrointestinal graft-versus-host disease (GI-GVHD) correlates with a decrease in the gut microbiome diversity, loss of obligate anaerobic bacteria and alterations in levels of salutary metabolites like short chain fatty acids (SCFAs). However, it is unknown whether the intestinal anatomic geography of the microbiome and its metabolites impacts GI GVHD severity. Herein, we investigate the pattern of the gut microbiome and metabolites from four distinct anatomical sites of intestines after allogeneic stem cell transplant (allo-SCT) via shotgun metagenomics and untargeted mass-spectrometry. Methods: To determine the composition of the gut microbiome and gut metabolites of mice after allo-SCT, we utilized contemporaneous analyses of shotgun metagenomic sequencing and untargeted mass spectrometry on the gut contents of mice 7 or 21 days after major histocompatibility complex (MHC)-disparate BALB/c→C57BL/6 (B6) allogeneic SCT. We investigated the microbial community composition and gut metabolites from four distinct gut locations (terminal ileum, cecum, transverse colon, descending colon) and performed integrative analysis to assess for novel pathways that may regulate GVHD biology. For shotgun metagenomics analyses, reads were depth normalized with BBnorm v.38.96 and assembled per-sample with MEGAHIT v.1.2.9. Prodigal v.2.6.3 was used to find genes in each bin. KofamScan v.1.3.0 was used to assign KEGG ortholog IDs (KO) to translated genes predicted by Prodigal. Community composition profiles were produced with Kraken2 v.2.1.2 and Bracken v.2.6.1. Functional profiles for each sample were determined with the Kraken community composition profiles and Humann3 v.3.0.0, with a GTDB database built with Struo2. Results: Consistent with previous reports from direct stool analyses, our data demonstrated that after allo-SCT, gut microbial diversity is reduced, relative abundance of obligate anaerobes decreases, and relative abundance of facultative anaerobes increases at all the intestinal anatomic sites, but the difference was maximal in ileo-cecal contents. Furthermore, metabolomic analyses reveal a decrease in previously described metabolites such as, SCFAs, bile acids and indoles at these sites. The distinct patterns of change between control and allogeneic samples depended not only on the gut location probed but also correlated with timepoint after transplant (7 days or 21 days) highlighting the importance of probing different gut locations and timepoints. The largest driver of differences between sample groups was (1) gut location (2) time after transplant (3) transplant type. Integrative analyses demonstrated loss of microbial functional genes related to butyrate processing following allo-SCT complementing previous studies that demonstrated reduction in butyrate levels. Further bioinformatic analyses identified novel microbial functional genes and metabolites that may have biological implications. Analyses revealed that after allo-SCT, phenyllactic acid (PLA) levels increased in the transverse colon and distal colon (fig.1). PLA is an anti-microbial compound and has been shown to have immune modulatory properties. Administration of PLA in drinking water of mice after allo-SCT worsened GVHD severity and increased mortality indicating PLA may play a role in inducing dysbiosis and worsening GVHD severity. Conclusion: Contemporaneous analyses of shotgun metagenomic and untargeted mass spectrometry of gut contents after allo-SCT reveal changes in microbial composition and metabolites consistent with previous data from analyses from stool. However, our analysis demonstrates distinct patterns of key metabolites and microbes in between syngeneic and allogeneic depending on gut location and time after transplant. Furthermore, our analyses have identified novel functional genes and metabolites that are implicated in GVHD severity.

Phenyllactic acid treatment for controlling anthracnose disease (Colletotrichum musae) and preserving banana fruit quality during storage

Banana is considered climacteric fruit that undergoes rapid post-harvest senescence, making it highly susceptible to diseases such as anthracnose, leading to a decline in quality. To address this issue, researchers investigated the effects of phenyllactic acid (PLA), a novel natural bio-preservative with broad-spectrum antibacterial properties, on banana fruit after harvesting. The study revealed that PLA treatment resulted in improved attributes of postharvest quality, including reduced changes in peel color, soluble solids concentration, and firmness. Additionally, respiratory rate, ethylene release, and disease index were significantly decreased, indicating that the fruit's physiological processes were better regulated. Furthermore, PLA treatment enhanced the activity of disease-resistant enzymes β-1,3-glucanase and chitinase, and upregulated the expression of pathogenesis-related genes. Importantly, PLA demonstrated its efficacy in suppressing the growth of anthracnose-causing fungi (Colletotrichum musae) and reducing disease severity on harvested fruit. In conclusion, PLA treatment shows promise as a new approach to maintain the freshness and quality of harvested banana fruit by reducing disease development, regulating physiological processes, and enhancing disease resistance mechanisms.

Elimination of Microcystis aeruginosa through Leuconostoc mesenteroides DH and its underlying mechanism

Microcystis aeruginosa is ubiquitously found in various water bodies and can produce microcystins (MCs), which threaten the health of aquatic animals and human beings. The elimination of excessive M. aeruginosa is beneficial for the protection of the ecosystems and public health. In this regard, algae-lysing bacteria have been extensively studied as an effective measure for their eradication. However, the active substances generated by algae-lysing bacteria are limited. For this study, we reveal that the phenyllactic acid (PLA) produced by Leuconostoc mesenteroides DH exhibits high efficacy for the removal of M. aeruginosa, and explore the elimination mechanism of strain DH on M. aeruginosa. It was found that a cell-free supernatant of strain DH possessed high removal activities against M. aeruginosa. Abundant reactive oxygen species were induced in algal cells following exposure to strain DH supernatant, as well as superoxide dismutase and catalase responses. Furthermore, the integrity of algal cell membranes and photosynthesis was seriously damaged. Interestingly, added exogenous eugenol significantly inhibited the synthesis of active substance produced by strain DH, which further identified that PLA is one of the active substances that contribute to the eradication of M. aeruginosa on the basis of metabolomics analysis. Our finding demonstrated, for the first time, that PLA (as an anti-cyanobacterial compound) can be used for the removal of M. aeruginosa, which provides a theoretical basis for the control of M. aeruginosa.