L-pipecolic Acid Research Articles

Inside-Out Rational Design of Ornithine Cyclodeaminase RlOCD from Rhizobium leguminosarum by a Multiregion Synergy Strategy for Efficient Synthesis of l-Pipecolic Acid.

Lysine cyclodeaminase (LCD)-mediated synthesis of l-pipecolic acid (l-PA) from l-lysine (l-Lys) is a promising approach. However, only one LCD has been reported, and its inadequate activity limits industrial applications. To address this problem, a substrate analogue-guided enzyme mining strategy was employed. A novel ornithine cyclodeaminase (OCD) from Rhizobium leguminosarum (RlOCD) was identified in combination with directed macrogenomic approaches. RlOCD displayed a conversion rate of 28% at a substrate loading as high as 1000 mM. A multiregion synergy strategy consisting of pocket reshaping, dynamical cross-correlation matrix-guided coevolutionary design, and surface modification was used to design RlOCD from the inside-out. A quadruple mutant (V93C/L119C/I170T/R90L) designated Mu4 with significantly increased activity was obtained, which showed a 28.46-fold increase in the catalytic efficiency. The conversion of Mu4 was 91% within 10 h at 1000 mM (146.19 g L-1) loading. The space-time yield of 282.1 g L-1 d-1 is the highest level ever reported. Molecular dynamics simulations and interaction analyses revealed that efficient pocket expansion and unique conformational rearrangements increased the affinity for the substrate, resulting in a more catalytically active conformation. This study expands the toolbox for the production of l-PA and demonstrates the effectiveness and potential of Mu4 for its production.

Metabolic Engineering of Saccharomyces cerevisiae for High-Level Production of l-Pipecolic Acid from Glucose.

l-Pipecolic acid (L-PA), an essential chiral cyclic nonprotein amino acid, is gaining prominence in the food and pharmaceutical sectors due to its wide-ranging biological and pharmacological properties. Historically, L-PA has been synthesized chemically for commercial purposes. This study introduces a novel and efficient microbial production method for L-PA using engineered strain Saccharomyces cerevisiae BY4743. Initially, an optimized biosynthetic pathway was constructed within S. cerevisiae, converting glucose to L-PA with a yield of 0.60 g/L in a 250 mL shake flask in vivo. Subsequently, a multifaceted engineering strategy was implemented to enhance L-PA production: substrate-enzyme affinity modification, global transcription machinery engineering modification, and Kozak sequence optimization for enhanced L-PA production. Approaches above led to an impressive 8.6-fold increase in L-PA yield, reaching 5.47 g/L in shake flask cultures. Further scaling up in a 5 L fed-batch fermenter achieved a remarkable L-PA concentration of 74.54 g/L. This research offers innovative insights into the industrial-scale production of L-PA.

Selenium-Mediated Shaping of Citrus Rhizobiome for Promotion in Root Growth and Soil Phosphorus Activation.

Selenium (Se) has been widely reported to affect plant growth, nutrient cycling, and the rhizobiome. However, how Se shapes the rhizobiome and interacts with plants remains largely elusive. Pot and hydroponic experiments were employed to elucidate the regulatory mechanism of Se in the citrus rhizobiome. Compared to the control, soil Se application significantly increased the root biomass (34.7%) and markedly reduced rhizosphere HCl-P, H2O-P, NaHCO3-IP, and residual-P of citrus, which were related to the variation of citrus rhizobiome. Se primarily enriched Proteobacteria and Actinobacteria as well as the phosphorus (P) functional genes phod and pqqc. Further study revealed that Se altered the metabolite profile of root exudate, particularly enhancing the abundance of l-cyclopentylglycine, cycloleucine, l-proline, l-pipecolic acid, and inositol, which played a key role in reshaping the citrus rhizobiome. These metabolites could serve as both nutrient sources and signaling molecules, thus supporting the growth or chemotaxis of the functional microbes. These bacterial taxa have the potential to solubilize P or stimulate plant growth. These findings provide a novel mechanistic understanding of the intriguing interactions between Se, root exudate, and rhizosphere microbiomes, and demonstrate the potential for utilizing Se to regulate rhizobiome function and enhance soil P utilization in citrus cultivation.

Precursor-directed biosynthesis and biological activity of tripropeptin Cpip, a new tripropeptin C analog containing pipecolic acid.

Tripropeptin C, a non-ribosomal cyclic lipopeptide containing three proline residues, exhibits excellent efficacy in the mouse-methicillin-resistant Staphylococcus aureus septicemia model. Since tripropeptins contain L-prolyl-D-proline and, as a result, are known to form a hairpin structure in proteins, it was of interest to determine whether this substructure contributes to their antibacterial activity. In this study, prolines in tripropeptin C were replaced with pipecolic acid(s) using precursor-directed biosynthesis. Only a new tripropeptin analog, tripropeptin Cpip, which has one L-pipecolic acid in place of L-proline, was isolated. The in vitro antimicrobial activity of the new analog was approximately two to four times weaker activity against Gram-positive bacteria, including drug-resistant species, compared with that of tripropeptin C. These results suggest that the L-prolyl-D-proline substructure plays an important role in the observed potency of tripropeptins.

Transcriptomic Screening of Alternaria oxytropis Isolated from Locoweed Plants for Genes Involved in Mycotoxin Swaisonine Production.

Locoweed is a collective name for a variety of plants, such as Oxytropis and Astragalus L. When these plants are infected by some fungi or endophytes, they will produce an alkaloid (swainsonine) that is harmful to livestock. Chronic toxicity characterized by neurological disorders occurs in livestock overfed on locoweed, and swainsonine (SW) is considered a major toxic component. The mechanism of the SW synthesis of endophytic fungi from locoweed remains unknown. In order to further discover the possible synthetic pathway of SW, in this study, a mycotoxin (SW) producer, Alternaria oxytropis isolate, UA003, isolated from Locoweed plants, and its mutant were subjected to transcriptomic analyses to ascertain the genes involved in the synthesis of this toxin. Mutant strain A. oxytropis E02 was obtained by ethyl methanesulfonate (EMS) mutagenesis treatment, and the strains were sequenced with different culture times for transcriptomic analysis and screening of differentially expressed genes. The results show a highly significant (p < 0.01) increase in SW yield in the A. oxytropis E02 strain obtained by EMS mutagenesis treatment compared to A. oxytropis UA003. A total of 637 differentially expressed genes were screened by transcriptome sequencing analysis, including 11 genes potentially associated with SW biosynthesis. These genes were screened using GO and KEGG data annotation and analysis. Among the differential genes, evm.TU.Contig4.409, evm.TU.Contig19.10, and evm.TU.Contig50.48 were associated with L-lysine biosynthesis, the L-pipecolic acid pathway, and the α-aminoadipic acid synthesis pathway. This study provides new insights to elucidate the mechanism of SW synthesis of endophytic fungi in locoweed and provides data support for further exploration of A. oxytropis genomics studies.

Characterizing the metabolic divide: distinctive metabolites differentiating CAD-T2DM from CAD patients

ObjectiveTo delineate the metabolomic differences in plasma samples between patients with coronary artery disease (CAD) and those with concomitant CAD and type 2 diabetes mellitus (T2DM), and to pinpoint distinctive metabolites indicative of T2DM risk.MethodPlasma samples from CAD and CAD-T2DM patients across three centers underwent comprehensive metabolomic and lipidomic analyses. Multivariate logistic regression was employed to discern the relationship between the identified metabolites and T2DM risk. Characteristic metabolites' metabolic impacts were further probed through hepatocyte cellular experiments. Subsequent transcriptomic analyses elucidated the potential target sites explaining the metabolic actions of these metabolites.ResultsMetabolomic analysis revealed 192 and 95 significantly altered profiles in the discovery (FDR < 0.05) and validation (P < 0.05) cohorts, respectively, that were associated with T2DM risk in univariate logistic regression. Further multivariate regression analyses identified 22 characteristic metabolites consistently associated with T2DM risk in both cohorts. Notably, pipecolinic acid and L-pipecolic acid, lysine derivatives, exhibited negative association with CAD-T2DM and influenced cellular glucose metabolism in hepatocytes. Transcriptomic insights shed light on potential metabolic action sites of these metabolites.ConclusionsThis research underscores the metabolic disparities between CAD and CAD-T2DM patients, spotlighting the protective attributes of pipecolinic acid and L-pipecolic acid. The comprehensive metabolomic and transcriptomic findings provide novel insights into the mechanism research, prophylaxis and treatment of comorbidity of CAD and T2DM.

Electrostatic interaction and regioselectivity enhancement in proline cis-4-hydroxylase for L-pipecolic acid hydroxylation

L-Proline cis-4-hydroxylase (cis-P4H), a non-heme Fe2+/α-ketoglutarate-dependent dioxygenase (KDD), finds application in proline C4 hydroxylation and selective L-pipecolic acid (L-PA) C5 hydroxylation. Nonetheless, its regioselectivity is often ambiguous, yielding nearly equal amounts of cis-5/cis-3 hydroxylated L-PA isomers, posing challenges in separation and purification. In this study, we selected Kordia jejudonensis proline hydroxylase (KjPH) for modification and investigated the electrostatic effect's molecular mechanism on its regioselectivity. Through sequence and catalytic domain alignment of KjPH (cis-5/cis-3 = 20:1), SmP4H (cis-5/cis-3 = 1:1, from Sinorhizobium meliloti), and MlP4H (cis-5/cis-3 = 1:7, from Mesorhizobium loti), we identified four non-conserved key residues (Y35, S57, F95, and C97). We confirmed that F95 in KjPH plays a pivotal role in affecting regioselectivity. The single-site variant F95Y significantly enhanced regioselectivity, increasing the cis-5/cis-3 ratio from 20:1 to 55:1. Molecular dynamics simulations unveiled that the improved regioselectivity of the F95Y variant primarily resulted from the electrostatic repulsive interaction, which increased the distance between the substrate's C3 site and the Fe2+ catalytic core.

Varietal Authenticity Assessment of QTMJ Tea Using Non-Targeted Metabolomics and Multi-Elemental Analysis with Chemometrics.

In this paper, a combination of non-targeted metabolomics and multi-element analysis was used to investigate the impact of five different cultivars on the sensory quality of QTMJ tea and identify candidate markers for varietal authenticity assessment. With chemometric analysis, a total of 54 differential metabolites were screened, with the abundances significantly varied in the tea cultivars. By contrast, the QTMJ tea from the Yaoshan Xiulv (XL) monovariety presents a much better sensory quality as result of the relatively more abundant anthocyanin glycosides and the lower levels of 2'-o-methyladenosine, denudatine, kynurenic acid and L-pipecolic acid. In addition, multi-elemental analysis found 14 significantly differential elements among the cultivars (VIP > 1 and p < 0.05). The differences and correlations of metabolites and elemental signatures of QTMJ tea between five cultivars were discussed using a Pearson correlation analysis. Element characteristics can be used as the best discriminant index for different cultivars of QTMJT, with a predictive accuracy of 100%.

Untargeted metabolomics analysis of plasma metabolic characteristics in patients with acne and insulin resistance.

Acne vulgaris is a chronic inflammatory disease with high incidence, diverse clinical manifestations, poor clinical efficacy, and easy recurrence. Recent studies have found that the occurrence of acne is related to metabolic factors such as insulin resistance; however, the specific mechanism of action remains unclear. This study aimed to identify significantly different metabolites and related metabolic pathways in the serum of acne vulgaris patients with or without insulin resistance. LC-MS/MS was used to analyze serum samples from patients about acne with insulin resistance (n = 51) and acne without insulin resistance (n = 69) to identify significant metabolites and metabolic pathways. In this study, 18 significant differential metabolites were screened for the first time. In the positive-ion mode, the upregulated substances were creatine, sarcosine, D-proline, uracil, Phe-Phe, L-pipecolic acid, and DL-phenylalanine; the downregulated substances were tridecanoic acid (tridecylic acid), L-lysine, cyclohexylamine, sphingomyelin (d18:1/18:0), gamma-L-Glu-epsilon-L-Lys, and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine. In the negative-ion mode, the upregulated substance was cholesterol sulfate, and the downregulated substances were D(-)-beta-hydroxybutyric acid, myristic acid, D-galacturonic acid, and dihydrothymine. Cholesterol sulfate showed the most significant expression among all differential metabolites (VIP = 7.3411). Based on the KEGG database, necroptosis and ABC transporters were the most significantly enriched metabolic pathways in this experiment. The differential metabolites and pathways identified in this study may provide new possibilities for the clinical diagnosis and development of targeted drugs for acne patients with insulin resistance.

Identification of salivary metabolic biomarker signatures for oral tongue squamous cell carcinoma

ObjectiveTo identify the salivary metabolites associated with squamous cell carcinoma of the tongue to develop easy and non-invasive potential biomarkers for disease diagnosis. DesignInitially, the study utilized untargeted metabolomics to analyze 20 samples of tongue squamous cell carcinoma and 10 control samples. The objective was to determine the salivary metabolites that exhibited differential expression in tongue squamous cell carcinoma. Then the selected metabolites were validated using targeted metabolomics in saliva samples of 100 patients diagnosed with squamous cell carcinoma of the tongue, as well as 30 healthy control individuals. ResultsFrom the analysis of untargeted metabolomics, 10 metabolites were selected as potential biomarkers. In the subsequent targeted metabolomics study on these selected metabolites, it was observed that N-Acetyl-D-glucosamine, L-Pipecolic acid, L-Carnitine, Phosphorylcholine, and Deoxyguanosine exhibited significant differences. The receiver operating characteristic curve analysis indicates a combination of three important metabolites such as N-Acetyl-D-glucosamine, L-Pipecolic acid and L-Carnitine provided the best prediction with an area under the curve of 0.901. ConclusionsThe present result reveals that the N-Acetyl-D-glucosamine, L-Pipecolic acid and L-Carnitine are the signature diagnostic biomarkers for oral tongue squamous cell carcinoma. These findings can be used to develop a rapid and non-invasive method for disease monitoring and prognosis in oral tongue cancer.

Protective effect of L-pipecolic acid on constipation in C57BL/6 mice based on gut microbiome and serum metabolomic

BackgroundFunctional constipation (FC) in children affects their growth, development and quality of life. L-pipecolic acid (L-PA) was decreased in FC children based on gut microbiome and serum metabolomic. In this study, loperamide-induced constipation in mice was used to evaluate the effects of L-PA on constipated mice.Method26 FC and 28 healthy children were recruited. Stool samples and serum samples were subjected to 16S rDNA sequencing and ultra-performance liquid chromatography/quadrupole time of flight (UPLC-Q/TOF-MS) approach, respectively. A loperamide-induced mouse constipation model was developed, and all mice were randomly divided into control (Con), loperamide (Lop) and L-PA (Lop + L-PA) treatment groups (6 mice per group). The mice in the Lop + L-PA group were given L-PA (250 mg/kg, once a day) and loperamide; the Lop group was given loperamide for 1 week, and the Con group was given saline. The fecal parameters and intestinal motility of mice in each group were detected. serum 5-HT levels and colon 5-HT expression were detected by ELISA and immunohistochemistry, respectively; qRT-PCR was used to detect the expression of AQP3 and 5-HT4R mRNA in each group.Results45 differential metabolites and 18 significantly different microbiota were found in FC children. The α and β diversity of gut microbiota in FC children was significantly reduced. Importantly, serum L-PA was significantly reduced in FC children. The KEGG pathway enrichment were mainly enriched in fatty acid biosynthesis, lysine degradation, and choline metabolism. L-PA was negatively associated with Ochrobactrum, and N6, N6, N6-trimethyl-l-lysine was positively associated with Phascolarcrobacterium. In addition, L-PA improved the fecal water content, intestinal transit rate, and increased the serum 5-HT levels in constipated mice. Moreover, L-PA increased the expression of 5-HT4R, reduced AQP3, and regulated constipation-associated genes.ConclusionsGut microbiota and serum metabolites were significantly altered in children with FC. The abundance of Phascolarctobacterium and Ochrobactrum and serum L-PA content were decreased in FC children. L-PA was found to alleviate the fecal water content, increase intestinal transit rate and the first black stool defecation time. L-PA improved constipation by increasing 5-HT and 5-HT4R expression while down-regulating AQP3 expression.

Plasma and milk metabolomics in lactating sheep divergent for feed efficiency

Enhancing the ability of animals to convert feed into meat or milk by optimizing feed efficiency (FE) has become a priority in livestock research. Although untargeted metabolomics is increasingly used in this field and may improve our understanding of FE, no information in this regard is available in dairy ewes. This study was conducted to (1) discriminate sheep divergent for FE and (2) provide insights into the physiological mechanisms contributing to FE through high-throughput metabolomics. The ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q/TOF-MS) technique was applied to easily accessible animal fluids (plasma and milk) to assess whether their metabolome differs between high- and low-feed efficient lactating ewes (H-FE and L-FE groups, respectively; 8 animals/group). Blood and milk samples were collected on the last day of the 3-wk period used for FE estimation. A total of 793 features were detected in plasma and 334 in milk, with 100 and 38 of them, respectively, showing differences between H-FE and L-FE. The partial least-squares discriminant analysis separated both groups of animals regardless of the type of sample. Plasma allowed the detection of a greater number of differential features; however, results also supported the usefulness of milk, more easily accessible, to discriminate dairy sheep divergent for FE. Regarding pathway analysis, nitrogen metabolism (either anabolism or catabolism) seemed to play a central role in FE, with plasma and milk consistently indicating a great impact of AA metabolism. A potential influence of pathways related to energy/lipid metabolism on FE was also observed. The variable importance in the projection plot revealed 15 differential features in each matrix that contributed the most for the separation in H-FE and L-FE, such as l-proline and phosphatidylcholine 20:4e in plasma or l-pipecolic acid and phosphatidylethanolamine (18:2) in milk. Overall, untargeted metabolomics provided valuable information into metabolic pathways that may underlie FE in dairy ewes, with a special relevance of AA metabolism in determining this complex phenotype in the ovine. Further research is warranted to validate these findings.

Systems metabolic engineering upgrades Corynebacterium glutamicum for selective high-level production of the chiral drug precursor and cell-protective extremolyte L-pipecolic acid

The nonproteinogenic cyclic metabolite l-pipecolic acid is a chiral precursor for the synthesis of various commercial drugs and functions as a cell-protective extremolyte and mediator of defense in plants, enabling high-value applications in the pharmaceutical, medical, cosmetic, and agrochemical markets. To date, the production of the compound is unfavorably fossil-based. Here, we upgraded the strain Corynebacterium glutamicum for l-pipecolic acid production using systems metabolic engineering. Heterologous expression of the l-lysine 6-dehydrogenase pathway, apparently the best route to be used in the microbe, yielded a family of strains that enabled successful de novo synthesis from glucose but approached a limit of performance at a yield of 180 mmol mol−1. Detailed analysis of the producers at the transcriptome, proteome, and metabolome levels revealed that the requirements of the introduced route were largely incompatible with the cellular environment, which could not be overcome after several further rounds of metabolic engineering. Based on the gained knowledge, we based the strain design on l-lysine 6-aminotransferase instead, which enabled a substantially higher in vivo flux toward l-pipecolic acid. The tailormade producer C. glutamicum PIA-7 formed l-pipecolic acid up to a yield of 562 mmol mol−1, representing 75% of the theoretical maximum. Ultimately, the advanced mutant PIA-10B achieved a titer of 93 g L−1 in a fed-batch process on glucose, outperforming all previous efforts to synthesize this valuable molecule de novo and even approaching the level of biotransformation from l-lysine. Notably, the use of C. glutamicum allows the safe production of GRAS-designated l-pipecolic acid, providing extra benefit toward addressing the high-value pharmaceutical, medical, and cosmetic markets. In summary, our development sets a milestone toward the commercialization of biobased l-pipecolic acid.

Recent Advances in the Hydroxylation of Amino Acids and Its Derivatives

Hydroxy amino acids (HAAs) are of unique value in the chemical and pharmaceutical industry with antiviral, antifungal, antibacterial, and anticancer properties. At present, the hydroxylated amino acids most studied are tryptophan, lysine, aspartic acid, leucine, proline, etc., and some of their derivatives. The hydroxylation of amino acids is inextricably linked to the catalysis of various biological enzymes, such as tryptophan hydroxylase, L-pipecolic acid trans-4-hydroxylase, lysine hydroxylase, etc. Hydroxylase conspicuously increases the variety of amino acid derivatives. For the manufacture of HAAs, the high regioselectivity biocatalytic synthesis approach is favored over chemical synthesis. Nowadays, the widely used method is to transcribe the hydroxylation pathway of various amino acids, including various catalytic enzymes, into Corynebacterium glutamicum or Escherichia coli for heterologous expression and then produce hydroxyamino acids. In this paper, we systematically reviewed the biosynthetic hydroxylation of aliphatic, heterocyclic, and aromatic amino acids and introduced the basic research and application of HAAs.

Ultra-High Performance Liquid Chromatography-Mass Spectrometry-based Serum Metabolomics for Early Diagnosis of Refractory Tumor-induced Osteomalacia: A Case-control Study.

Nearly 20% patients with tumor-induced osteomalacia (TIO) experienced recurrence or nonrecovery after surgery. Serum fibroblast growth factor 23 and phosphate concentrations are not sufficient for prognosis in such cases. Despite its importance for understanding of prognosis and underlying pathogenesis, the alteration of systemic metabolism in refractory TIO remains unclear. We aimed to find the metabolomic characteristics of refractory TIO and establish a novel predictive model for early discriminating refractory TIO based on their serum metabolomics. Cross-section study for comparison of metabolomic profile between TIO and normal control and longitudinal study for identifying prognostic model. Based on liquid chromatography-tandem mass spectrometry, we analyzed the global metabolomes of preoperative sera from 86 samples (32 TIO recovery patients, 11 nonremission patients, and 43 matched controls). Statistical analyses, pathway enrichment, and receiver operating characteristic analysis were performed to identified and evaluate potential markers. Sparse partial least squares discriminant analysis indicated a clear separation of metabolomic profiles between healthy controls (HC) and TIO patients. The serum metabolites altered in different prognostic groups. L-pipecolic acid, 2-dodecylbenzenesulfonic acid, and 2-deoxygalactopyranose were the top 3 metabolites that were significantly perturbed. A combination of L-pipecolic acid and 2-dodecylbenzenesulfonic acid demonstrated a high-performance panel for TIO prognosis evaluated by random forest algorithm (area under the curve = 0.921, 95% CI, 0.787-0.995). We investigate the global metabolomes of refractory TIO and identify potential prognostic biomarkers preliminarily. A high sensitivity and specificity panel were identified as promising discriminating predictors, which need to be verified in more patients. This work may demonstrate novel insights into TIO prognosis and pathogenesis.

Integrative metagenomic and metabolomic analyses reveal the role of gut microbiota in antibody-mediated renal allograft rejection

BackgroundAntibody-mediated rejection (AMR) remains one of the major barriers for graft survival after kidney transplantation. Our previous study suggested a gut microbiota dysbiosis in kidney transplantation recipients with AMR. However, alternations in gut microbial function and structure at species level have not been identified. In the present study, we investigated the metagenomic and metabolic patterns of gut microbiota in AMR patients to provide a comprehensive and in-depth understanding of gut microbiota dysbiosis in AMR.MethodsWe enrolled 60 kidney transplantation recipients, 28 showed AMR and 32 were non-AMR controls with stable post-transplant renal functions. Shotgun sequencing and untargeted LC/MS metabolomic profiling of fecal samples were performed in kidney transplantation recipients with AMR and controls.ResultsTotally, we identified 311 down-regulated and 27 up-regulated gut microbial species associated with AMR after kidney transplantation, resulting in the altered expression levels of 437 genes enriched in 22 pathways, of which 13 were related to metabolism. Moreover, 32 differential fecal metabolites were found in recipients with AMR. Among them, alterations in 3b-hydroxy-5-cholenoic acid, l-pipecolic acid, taurocholate, and 6k-PGF1alpha-d4 directly correlated with changes in gut microbial species and functions. Specific differential fecal species and metabolites were strongly associated with clinical indexes (Cr, BUN, etc.), and could distinguish the recipients with AMR from controls as potential biomarkers.ConclusionsAltogether, our findings provided a comprehensive and in-depth understanding of the correlation between AMR and gut microbiota, which is important for the etiological and diagnostic study of AMR after kidney transplantation.

Potential Plasma Metabolic Biomarkers of Tourette Syndrome Discovery Based on Integrated Nontargeted and Targeted Metabolomics Screening Plasma Metabolic Biomarkers of TS.

Tourette syndrome (TS) is a chronic neuropsychiatric disorder characterized by abnormal movements, phonations, and tics, but an accurate TS diagnosis remains challenging and indeed depends on its description of clinical symptoms. Our study was conducted to discover and verify some metabolite biomarkers based on nontargeted and targeted metabolomics. We conducted untargeted ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) for preliminary screening of potential biomarkers on 30 TS patients and 10 healthy controls and then performed validation experiments based on targeted ultrahigh-performance liquid chromatography triple quadrupole-MS (UHPLC/MS/MS) on 35 TS patients and 14 healthy controls. 1775 differentially expressed metabolites were identified by partial least squares discriminant analysis (PLS-DA), fold-change analysis, T-test, and hierarchical clustering analysis (adjusted p value <0.05 and |logFC| > 1). TS plasma samples were found to be differentiated from healthy samples in our approach. Furthermore, aspartate and asparagine metabolism pathways were considered to be a significant enrichment pathway in TS progression based on metabolite pathway enrichment analysis. For the 8 metabolites involved in this pathway that we detected, we then performed validation experiments based on targeted UHPLC/MS/MS. The t-test, Mann-Whitney U test, and receiver operating characteristic (ROC) curve analysis were used to determine potential biomarkers. Ultimately, L-arginine and L-pipecolic acid were validated as significantly differentiated metabolites (p < 0.05), with an AUC of 70.0% and 80.3%, respectively. L-pipecolic acid was defined as a potential biomarker for TS diagnosis by the combined application of nontargeted and targeted metabolomic analysis.

An artificial pathway for trans-4-hydroxy-L-pipecolic acid production from L-lysine in Escherichia coli

Trans-4-hydroxy-L-pipecolic acid (trans-4-HyPip) is a hydroxylated product of L-pipecolic acid, which is widely used in the pharmaceutical and chemical industries. Here, a trans-4-HyPip biosynthesis module was designed and constructed in Escherichia coli by overexpressing lysine α-oxidase, Δ1-piperideine-2-carboxylase reductase, glucose dehydrogenase, lysine permease, catalase and L-pipecolic acid trans-4-hydroxylase for expanding the lysine catabolism pathway. A total of 4.89 g/L of trans-4-HyPip was generated in shake flasks from 8 g/L of L-pipecolic acid. By this approach, 14.86 g/L of trans-4-HyPip was produced from lysine after 48 h in a 5 L bioreactor. As far as we know, this is the first multi-enzyme cascade catalytic system for the production of trans-4-HyPip using E. coli from L-lysine. Therefore, it can be considered as a potential candidate for the industrial production of trans-4-HyPip in microorganisms.

Rational Design of the Soluble Variant of l-Pipecolic Acid Hydroxylase using the α-Helix Rule and the Hydropathy Contradiction Rule.

The production of recombinant proteins in Escherichia coli is an important application of biotechnology. 2-Oxoglutarate-dependent l-pipecolic acid hydroxylase derived from Xenorhabdus doucetiae (XdPH) is an excellent biocatalyst that catalyzes the hydroxylation of l-pipecolic acid to produce cis-5-hydroxy-l-pipecolic acid. However, the enzyme tends to form aggregates in the E. coli expression system. Our group established two rules, namely, the “α-helix rule” and the “hydropathy contradiction rule,” to select residues to be altered for improving the heterologous recombinant production of proteins, by analyzing their primary structure. We rationally designed XdPH variants that are expressed in highly soluble and active forms in the E. coli expression system using these hotspot prediction methods, and the L142R variant showed a remarkably high soluble expression level compared to the wild-type XdPH. Further mutations were introduced into the L142R gene by site-directed mutagenesis. Moreover, the I28P/L142R and C76Y/L142R double variants displayed improved soluble expression levels compared to the single variants. These variants were also more thermostable than the wild-type XdPH. To analyze the effect of the alteration on one of the hotspots, L142 was replaced with various hydrophilic and positively charged residues. The remarkable increase in soluble protein expression caused by the alterations suggests that the decrease in the hydrophobicity of the protein surface and the enhancement of the interaction between nearby residues are important factors determining the solubility of the protein. Overall, this study demonstrated the effectiveness of our protocol in identifying aggregation hotspots for recombinant protein production and in basic biochemical research.

Metabolomic Characterization of Acute Ischemic Stroke Facilitates Metabolomic Biomarker Discovery.

Acute ischemic stroke (AIS) is characterized by a sudden blockage of one of the main arteries supplying blood to the brain, leading to insufficient oxygen and nutrients for brain cells to function properly. Unfortunately, metabolic alterations in the biofluids with AIS are still not well understood. In this study, we performed high-throughput target metabolic analysis on 44 serum samples, including 22 from AIS patients and 22 from healthy controls. Multiple-reaction monitoring analysis of 180 common metabolites revealed a total of 29 metabolites that changed significantly (VIP > 1, p < 0.05). Multivariate statistical analysis unraveled a striking separation between AIS patients and healthy controls. Comparing the AIS group with the control group, the contents of argininosuccinic acid, beta-D-glucosamine, glycerophosphocholine, L-abrine, and L-pipecolic acid were remarkably downregulated in AIS patients. Twenty-nine out of 112 detected metabolites enriched in disturbed metabolic pathways, including aminoacyl-tRNA biosynthesis, glycerophospholipid metabolism, lysine degradation, phenylalanine, tyrosine, and tryptophan biosynthesis metabolic pathways. Collectively, these results will provide a sensitive, feasible diagnostic prospect for AIS patients.