Multi-omics analysis uncovers the transcriptional regulatory mechanism of magnesium Ions in the synthesis of active ingredients in Sophora tonkinensis.

Magnesium (Mg) plays a pivotal role as an essential component of plant chlorophyll and functions as a critical coenzyme. However, research exploring the regulatory mechanisms of magnesium ions on the synthesis of secondary metabolites is still in its early stages. Sophora tonkinensis is a widely utilized medicinal plant in China, recognized for its diverse secondary metabolites with active properties. This study investigates variations in these ingredients in tissue-cultured seedlings under varying magnesium concentrations. Simultaneously, an omics data analysis was conducted on tissue-cultured seedlings subjected to treatments with magnesium and low magnesium. These comprehensive omics analyses aimed to elucidate the mechanisms through which magnesium influences active components, growth, and development. Magnesium exerts a pervasive influence on various metabolic pathways, forming an intricate network. Research findings indicate that magnesium impacts diverse metabolic processes, including the absorption of potassium and calcium, as well as photosynthetic activity. Consequently, these influences lead to discernible changes in the levels of pharmacologically active compounds and the growth and developmental status.This study is the first to employ a multi-omics data analysis in S. tonkinensis. This methodology allows us to uncover the overarching impact of metabolic networks on the levels of various active ingredients and specific phenotypes.

Optimal treatment strategies for hepatoid adenocarcinoma of the lung: insights from a comprehensive analysis.

BACKGROUND: Hepatoid adenocarcinoma of the lung (HAL) is a distinctly uncommon subtype of lung adenocarcinoma (LAC), characterized by hepatoid features and an alarmingly low 5-year survival rate of approximately 8%. The scarcity of information on this condition has contributed to the absence of standardized treatment protocols, and the molecular underpinnings of its pathogenesis remain largely unexplored. To bridge these gaps, this study compiled data from 191 primary HAL patients to delineate treatment patterns, prognostic factors, and potential pathogenic mechanisms. METHODS: This study was divided into two cohorts: cohort 1, comprising 110 patients extracted from the Surveillance, Epidemiology, and End Results (SEER) database, and cohort 2, consisting of 70 patients identified through a comprehensive literature review via the PubMed, Web of Science, and Scopus databases, in addition to 11 patients from Tongji Hospital. The Cox proportional hazards regression model was employed to identify independent prognostic factors. Kaplan-Meier survival curves were generated to assess the impact of treatment modalities centered around surgery and chemotherapy. Moreover, this study evaluated the efficacy of first-line treatment regimens and conducted Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses on identified mutated genes. RESULTS: The demographic and clinical profile of HAL patients typically comprises older individuals who are smokers, with a predisposition for diagnosis at advanced disease stages, culminating in a high mortality rate. Key prognostic indicators identified included disease stage, chemotherapy and surgical interventions. The study suggests a treatment strategy that advocates chemotherapy for patients with stage IV HAL and surgery for those with non-stage IV disease. The combination of paclitaxel and platinum-based chemotherapy emerged as an efficacious first-line treatment, with the integration of immunotherapy and targeted therapies showing potential benefits. Genetic analysis underscored similarities between HAL and LAC, particularly highlighting aberrant kinase activity (serine, threonine, and tyrosine) and the activation of PI3K-Akt and MAPK signaling pathways as contributing factors to HAL pathogenesis. CONCLUSION: Despite its relatively rare occurrence, this study underscores the significance of treatment strategies and concludes probable prognostic factors. Due to limited reports, a deeper understanding of the molecular mechanisms driving tumorigenesis and progression in HAL is needed.

Results of a defined surgical protocol for treating pediatric neurogenic bladder incontinence in a single institution.

INTRODUCTION: Incontinent pediatric neurogenic bladder (NB) patients face social ostracization and potential renal deterioration. Reconstructive surgery, after maximal medical therapy, requires a difficult decision-making process. Current literature for NB surgeries is difficult to interpret given definitions of dryness, use of augmentation cystoplasty (AC) and the lack of renal preservation. This study assesses the results of a defined surgical protocol to treat incontinent NB patients, using a new composite outcome measure, which includes upper tracts status and a definition of dryness. MATERIALS AND METHODS: This is a retrospective cohort study assessing 33 consecutive incontinent NB patients (Spina bifida 31, Sacral agenesis- 2) who underwent one of 2 procedures between 2008 and 2021. AC with a Mitrofanoff procedure (MP) was performed in patients who had a high detrusor leak point pressure (DLPP) and significant bladder trabeculations (N = 21, Group 1). Children with a low DLPP and non-trabeculated bladders, underwent a modified Young-Dees-Leadbetter/Mitchell procedure with a 360° autologous rectus fascial sling (BOP) with concomitant AC and MP (N-12, Group 2). Post-operative success was defined using a composite grading of success assessing dryness, upper tract stability and medication use. RESULTS: The mean age at surgery was 11.6 years (SD = 6 years), with 21 in Group 1 and 12 in Group 2. Mean follow-up was 3.25 years, with a minimum 24-month follow-up period. Success rate was 90% in Group 1 and 66% in Group 2. No patient had upper tract deterioration following surgery. Redo-surgical intervention, was required in 38% of Group 1 and 50% of Group 2 patients. These include 3 bladder neck injections in Group 1 and 2 bladder neck closure in Group 2, with a final success rate to 95 % in Group 1 and 83 % in Group 2. DISCUSSION: Achieving dryness and preserving upper tracts is a challenge in incontinent NB patients. Dryness rates achieved in this study is comparable, given complications and redo-surgery. Primary bladder neck closure is a radical intervention, but Group 2 patients, may benefit from an upfront discussion of the pros and cons of a bladder neck closure primarily or as a secondary procedure. CONCLUSIONS: Isolated AC obtains acceptable results for a selected subset of incontinent NB patients with significant bladder trabeculation. For those requiring a BOP, the success rate is relatively lower with the higher rate of potential complications and need for redo-surgery.

Construction and evaluation of AuNPs enhanced electrochemical immunosensors with [Fe(CN)6]3-/4- and PPy probe for highly sensitive detection of human chorionic gonadotropin.

Human chorionic gonadotropin (HCG), a vital protein for pregnancy determination and a marker for trophoblastic diseases, finds application in monitoring early pregnancy and ectopic pregnancy. This study presents an innovative approach employing electrochemical immunosensors for enhanced HCG detection, utilizing Anti-HCG antibodies and gold nanoparticles (AuNPs) in the sensor platform. Two sensor configurations were optimized: BSA/Anti-HCG/c-AuNPs/MEL/e-AuNPs/SPCE with [Fe(CN)6]3-/4- as a redox probe (1) and BSA/Anti-HCG/PPy/e-AuNPs/SPCE using polypyrrole (PPy) as a redox probe (2). The first sensor offers linear correlation in the 0.10-500.00 pg∙mL-1 HCG range, with a limit of detection (LOD) of 0.06 pg∙mL-1, sensitivity of 32.25 µA∙pg-1∙mL∙cm-2, RSD <2.47 %, and a recovery rate of 101.03-104.81 %. The second sensor widens the HCG detection range (40.00 fg∙mL-1-5.00 pg∙mL-1) with a LOD of 16.53 fg∙mL-1, ensuring precision (RSD <1.04 %) and a recovery range of 94.61-106.07 % in serum samples. These electrochemical immunosensors have transformative potential in biomarker detection, offering enhanced sensitivity, selectivity, and stability for advanced healthcare diagnostics.

Optimizing enzyme properties to enhance dihydroxyacetone production via methylglyoxal biosensor development.

BACKGROUND: Dihydroxyacetone (DHA) stands as a crucial chemical material extensively utilized in the cosmetics industry. DHA production through the dephosphorylation of dihydroxyacetone phosphate, an intermediate product of the glycolysis pathway in Escherichia coli, presents a prospective alternative for industrial production. However, insights into the pivotal enzyme, dihydroxyacetone phosphate dephosphorylase (HdpA), remain limited for informed engineering. Consequently, the development of an efficient tool for high-throughput screening of HdpA hypermutants becomes imperative. RESULTS: This study introduces a methylglyoxal biosensor, based on the formaldehyde-responding regulator FrmR, for the selection of HdpA. Initial modifications involved the insertion of the FrmR binding site upstream of the -35 region and into the spacer region between the -10 and -35 regions of the constitutive promoter J23110. Although the hybrid promoter retained constitutive expression, expression of FrmR led to complete repression. The addition of 350 µM methylglyoxal promptly alleviated FrmR inhibition, enhancing promoter activity by more than 40-fold. The methylglyoxal biosensor system exhibited a gradual increase in fluorescence intensity with methylglyoxal concentrations ranging from 10 to 500 µM. Notably, the biosensor system responded to methylglyoxal spontaneously converted from added DHA, facilitating the separation of DHA producing and non-producing strains through flow cytometry sorting. Subsequently, the methylglyoxal biosensor was successfully applied to screen a library of HdpA mutants, identifying two strains harboring specific mutants 267G > T and D110G/G151C that showed improved DHA production by 68% and 114%, respectively. Expressing of these two HdpA mutants directly in a DHA-producing strain also increased DHA production from 1.45 to 1.92 and 2.29 g/L, respectively, demonstrating the enhanced enzyme properties of the HdpA mutants. CONCLUSIONS: The methylglyoxal biosensor offers a novel strategy for constructing genetically encoded biosensors and serves as a robust platform for indirectly determining DHA levels by responding to methylglyoxal. This property enables efficiently screening of HdpA hypermutants to enhance DHA production.

Fever of unknown origin associated with immune checkpoint inhibitors.

Since the approval for the treatment of melanoma in 2014, immune checkpoint inhibitors (ICIs) have revolutionized the therapy pattern across various malignancies. Coinciding with their frequent usage, their adverse effects, including fever, cannot be neglected. In the context of cancer diseases and cancer treatments, fever of unknown origin (FUO), which has long posed a challenge for clinicians in terms of diagnosis and management, brings forth new connotation and significance. In this paper review, we present the concept of ICIs-associated FUO, consider activated immune system and elevated cytokines as common mechanisms by which ICIs induce fever and various immune-related adverse events (irAEs), summarize and compare the primary etiologies of ICI-associated FUO, and compare it with conventional types of FUO.

Genetic polymorphisms associated with urinary tract infection in children and adults: a systematic review and meta-analysis.

INTRODUCTION: The lifetime risk of urinary tract infection is known from first-degree relative studies to be highly heritable. Associations have also been observed across the life course from pediatric urinary tract infection to recurrent urinary tract infection in adulthood, suggesting lifelong susceptibility factors. Candidate gene studies and genome-wide association studies have tested for genetic associations of urinary tract infection; however, no contemporary systematic synthesis of studies is available. OBJECTIVE: We conducted a systematic review to identify all genetic polymorphisms tested for an association with urinary tract infection in children and adults; and to assess their strength, consistency, and risk of bias among reported associations. DATA SOURCES AND STUDY ELIGIBILITY CRITERIA: PubMed, HuGE Navigator and Embase were searched from January 1, 2005 to November 16, 2023, using a combination of genetic and phenotype key words. STUDY APPRAISAL AND SYNTHESIS METHODS: Fixed and random effects meta-analyses were conducted using codominant models of inheritance in metan. The interim Venice criteria were used to assess their credibility of pooled associations. RESULTS: After removing 451 duplicates, 1821 studies reports were screened, with 106 selected for full-text review, 22 were included in the meta-analysis (7 adult studies and 15 pediatric studies). Our meta-analyses demonstrated significant pooled associations for pediatric urinary tract infection with variation in CXCR1, IL8, TGF, TLR4 and VDR; all of which have plausible roles in the pathogenesis of urinary tract infection. Our meta-analyses also demonstrated a significant pooled association for adult urinary tract infection with variation in CXCR1. All significant pooled associations were graded according to their epidemiological credibility, sample sizes, heterogeneity between studies, and risk of bias. CONCLUSION: This systematic review provides a current synthesis of the known genetic architecture of urinary tract infection in childhood and adulthood; and should provide important information for researchers analysing future genetic association studies. Although, overall, the credibility of pooled associations was weak, the consistency of findings for rs2234671 single nucleotide polymorphisms of CXCR1 in both populations suggest a key role in the urinary tract infection pathogenesis.

Constructing a nomogram based on the distribution of thyroid nodules and suspicious lateral cervical lymph nodes in fine-needle aspiration biopsies to predict metastasis in papillary thyroid carcinoma.

Purpose: Elevated concentrations of thyroglobulin eluent is a risk factor for lateral cervical lymph node metastasis (LLNM) in patients with papillary thyroid cancer (PTC). We aimed to develop a practical nomogram based on the distribution of thyroid nodules and the presence of suspicious lateral cervical lymph nodes in fine-needle aspiration biopsies (LN-FNABs), including the cytopathology and the suspicious lateral cervical lymph node (LLN) thyroglobulin eluent (Tg), to predict the possibility of LLNM preoperatively in patients with PTC. Methods: The clinical data of PTC patients who were admitted to the Third Affiliated Hospital of Soochow University from January 2022 to May 2023 to undergo fine-needle aspiration biopsy (FNAB) were included in this study. A total of 208 patients in 2022 served as the training set (70%), and 89 patients in 2023 served as the validation set (30%). The clinical characteristics and LN-FNAB results were collected to determine the risk factors of LLNM. A preoperative nomogram was developed for predicting LLNM based on the results of the univariate and multivariate analyses. Internal calibration, external calibration, and decision curve analysis (DCA) were performed for these models. Results: The multivariate logistic regression analysis showed that the maximum thyroid nodule diameter (Odds Ratio (OR) 2.323, 95% CI 1.383 to 3.904; p = 0.001), Tg level (OR 1.007, 95% CI 1.005 to 1.009; p = 0.000), Tg divided by serum thyroglobulin, (Tg/sTg) [odds ratio (OR) 1.005, 95% CI 1.001 to 1.008; p = 0.009], and cytopathology (OR 9.738, 95% CI 3.678 to 25.783; p = 0.000) (all p < 0.05) had a significant impact on the LLNM of patients with suspicious LLNs. The nomogram showed a better predictive value in both the training cohort [area under the curve, (AUC) 0.937, 95% CI 0.895 to 0.966] and the validation cohort (AUC 0.957, 95% CI 0.892 to 0.989). The nomogram also showed excellent internal and external calibration in predicting LLNM. According to the DCA, the diagnostic performance of this model was dependent on the following variables: maximum thyroid nodule diameter, Tg level, Tg/sTg, and cytopathology. Conclusion: Based on the aforementioned risk factors, we believe that it is necessary to establish a personalized LLNM model for patients with PTC. Using this practical nomogram, which combines clinical and Tg risk factors, surgeons could accurately predict the possibility of LLNM preoperatively. The nomogram will also help surgeons to establish personalized treatment plans before surgery.

Time course efficiency of MICE and HIIE on inhibitory control and HRV in adolescents with obesity and different cardiorespiratory fitness.

Background: Adolescent obesity is associated with impaired inhibitory control. Acute exercise can improve executive function. However, due to the influence of exercise intensity, cognitive test timing, and cardiorespiratory fitness (CF) level, the most effective exercise program remains controversial. Methods: The current study investigated the time-course effects of moderate-intensity continuous exercise (MICE) and high-intensity interval exercise (HIIE) on inhibitory control (Stroop) and task-related heart rate variability (HRV) in adolescents with different CF. A mixed experimental design of 2 CF levels (high CF, HCF; low CF, LCF) × 3 exercise methods (MICE, HIIE, CON) × 3 test timing (pre, post-0, post-20) was adopted. Heart rate variability (HRV) and Stroop task tests were conducted before exercise (pre), immediately after exercise (post-0), and 20 min after exercise (post-20). Results: Individuals with HCF exhibited a positive decrease in Stroop response time immediately and 20 min after MICE and HIIE, compared to pretest response times (RT). Conversely, individuals with LCF showed a slight increase in Stroop task (RT) only immediately after HIIE. All individuals had a slight increase in ACC after MICE and HIIE compared to before exercise. In addition, compared with the control group, the time-domain index (the square root of the mean squared differences of successive NN intervals, RMSSD) of HRV was significantly decreased, the frequency-domain index (the absolute power of the Low-Frequency band/the absolute power of the High-Frequency band ratio, LF/HF) was significantly increased after MICE and HIIE, and the effect of HIIE on RMSSD and LF/HF was significantly greater than that of MICE. Conclusion: The current study found that the acute effects of MICE and HIIE on inhibitory control in obese adolescents were influenced by the interaction of cognitive test timing and cardiorespiratory fitness. Individuals with high cardiorespiratory fitness performed better on the Stroop task than individuals with low cardiorespiratory fitness. The inhibitory control of HIIE in high-cardiorespiratory obese adolescents produced positive effects similar to those in MICE but more lasting, suggesting that HIIE is more beneficial for high-cardiorespiratory obese adolescents. MICE promoted inhibitory control in obese adolescents with low cardiorespiratory fitness, but HIIE impaired inhibitory control in obese adolescents with low cardiorespiratory fitness immediately after exercise, suggesting that low cardiopulmonary fitness obese adolescents may be suitable for MICE rather than HIIE exercise intervention. The shift from balanced HRV to sympathetic dominance after acute exercise reflects increased arousal levels and may be one of the underlying mechanisms by which acute exercise brings benefits to executive function.

Electrochemical sensor for ultrasensitive sensing of biotin based on heme conjugated with gold nanoparticles and its electrooxidation mechanism.

We report the fabrication of a facile sensor using heme conjugated with gold nanoparticles (AuNPs) in situ on a glass carbon electrode (GCE) for the ultrasensitive determination of biotin without antibody or streptavidin. The use of heme and AuNPs as dual amplifiers allows a very broad detection range from 0.0050 to 50.0000 µmol·L-1 and a very low detection limit of 0.0016 µmol·L-1. The mechanistic aspects were elucidated using electrochemical analyses and frontier orbital calculations showing that the electrooxidation of biotin involves a one-electron and a one-proton transfer, generating biotin sulfoxide. The heme/AuNPs/GCE sensor exhibited excellent selectivity, reproducibility and stability, indicating high robustness. The recovery was between 97.20 and 105.70% with RSD less than 8.71%, suggesting good practicability. Our studies demonstrate that this approach can be used to detect and quantify biotin in a range of foods, including milk, infant formula, flour, orange juice, mango juice, egg white and egg yolk. Furthermore, all measurements do not require any intricate preparation or pre-treatment of the foods, thus representing a great potential for point-of-care testing.

Anterior inferior cerebellar artery occlusion accompanied by hemorheology-documented increased blood viscosity: a case report.

Anterior inferior cerebellar artery (AICA) occlusion is a subtype of posterior circulation stroke. Confirmation of its angiomorphology and etiology is challenging because of the complex mechanisms underlying small-artery thrombogenesis. In addition to conventional factors, physicians frequently overlook hemorheological changes. In this case report, we describe right AICA occlusion in a 50-year-old man. He presented with an unsteady walk, tinnitus, dizziness, and left-sided peripheral facial palsy observed over 36 hours, accompanied by increased blood viscosity on hemorheological evaluation. Magnetic resonance imaging revealed acute infarction in the left cerebellar hemisphere and middle cerebellar peduncles. Magnetic resonance angiography (MRA) and computed tomographic angiography (CTA) failed to detect AICA occlusion, which was later confirmed using digital subtraction angiography. Repeat routine blood examinations showed elevated erythrocyte and leukocyte counts and serum hemoglobin concentrations that persisted over many days. Hemorheological evaluation revealed increased whole blood viscosity at a low shear rate. AICA occlusion should thus be diagnosed based on its initial characteristic manifestations; notably, MRA and CTA may fail to detect arterial occlusion. The importance of hemorheological change as a factor of stroke is frequently neglected. We therefore report this case hoping to emphasize its relevance, especially in small-artery occlusion.

LNX2 involves in the role of ghrelin to promote the neuronal differentiation of adipose tissue-derived mesenchymal stem cells.

Adipose tissue-derived mesenchymal stem cells (ADSCs) have promising effects on nerve repair due to the differentiation ability to neural cells. Ghrelin has been shown to promote the neural differentiation of ADSCs. This work was designed to explore its underlying mechanism. Herein, we found high expression of LNX2 in ADSCs after neuronal differentiation. Knockdown of LNX2 might block neuronal differentiation of ADSCs, as evidenced by the decreased number of neural-like cells and dendrites per cell, and the reduced expressions of neural markers (including ß-Tubulin III, Nestin, and MAP2). We also demonstrated that LNX2 silencing suppressed the nuclear translocation of ß-catenin in differentiated ADSCs. Luciferase reporter assay indicated that LNX2 inhibited wnt/ß-catenin pathway by reducing its transcriptional activity. In addition, results showed that LNX2 expression was increased by ghrelin, and its inhibition diminished the effects of ghrelin on neuronal differentiation. Altogether, the results suggest that LNX2 is involved in the role of ghrelin to facilitate neuronal differentiation of ADSCs.

A facile indole probe for ultrasensitive immunosensor fabrication toward C-reactive protein sensing.

C-reactive protein (CRP) is a protein biomarker for acute phase response. Herein, we fabricate a highly sensitive electrochemical immunosensor for CRP on a screen-printed carbon electrode (SPCE) with indole as a novel electrochemical probe and Au nanoparticles for signal amplification. Amongst, indole appeared as transparent nanofilms on the electrode surface, and underwent a one-electron and one-proton transfer to form oxindole during the oxidation process. Upon optimization of experimental conditions, a logarithmic correlation between CRP concentration (0.0001-100 µg∙mL-1) and response current was revealed with a detection limit of 0.03 ng∙mL-1 and a sensitivity of 5.7055 µA∙µg-1∙mL∙cm-2. The sensor exhibited exceptionally distinction selectivity, reproducibility and stability of the electrochemical immunosensor studied. The recovery rate of CRP in human serum samples determined by the standard addition method, ranged between 98.2-102.2%. Overall, the developed immunosensor is promising, and has the potential for CRP detection in real human serum samples.

Boosting the epoxidation of squalene to produce triterpenoids in Saccharomyces cerevisiae.

BACKGROUND: Polycyclic triterpenoids (PTs) are common in plants, and have attracted considerable interest due to their remarkable biological activities. Currently, engineering the ergosterol synthesis pathway in Saccharomyces cerevisiae is a safe and cost-competitive way to produce triterpenoids. However, the strict regulation of ERG1 involved in the epoxidation of squalene limits the triterpenoid production. RESULTS: In this study, we found that the decrease in ERG7 protein level could dramatically boost the epoxidation of squalene by improving the protein stability of ERG1. We next explored the potential factors that affected the degradation process of ERG1 and confirmed that ERG7 was involved in the degradation process of ERG1. Subsequently, expression of four different triterpene cyclases utilizing either 2,3-oxidosqualene or 2,3:22,23-dioxidosqualene as the substrate in ERG7-degraded strains showed that the degradation of ERG7 to prompt the epoxidation of squalene could significantly increase triterpenoid production. To better display the potential of the strategy, we increased the supply of 2,3-oxidosqualene, optimized flux distribution between ergosterol synthesis pathway and ß-amyrin synthesis pathway, and modified the GAL-regulation system to separate the growth stage from the production stage. The best-performing strain ultimately produced 4216.6 ± 68.4 mg/L of ß-amyrin in a two-stage fed-fermentation (a 47-fold improvement over the initial strain). CONCLUSIONS: This study showed that deregulation of the native restriction in ergosterol pathway was an effective strategy to increase triterpenoid production in yeast, which provided a new insight into triterpenoids biosynthesis.

Exploring Perceptions of Competency-Based Medical Education in Undergraduate Medical Students and Faculty: A Program Evaluation.

Background: There is limited work exploring competency-based medical education (CBME) in undergraduate medical education. We aimed to assess medical students' and faculty's perception of CBME in the undergraduate medicine setting after its implementation at our institution through a Content, Input, Process, Product (CIPP) program evaluation model. Methods: We explored the rationale for the transition to a CBME curriculum (Content), the changes to the curriculum and the teams involved in the transition (Input), medical students' and faculty's perception of the current CBME curriculum (Process), and benefits and challenges of implementing undergraduate CBME (Product). A cross-sectional online survey was delivered over 8-weeks in October 2021 to medical students and faculty as part of the Process and Product evaluation. Results: Medical students displayed greater optimism towards CBME, compared to faculty, in terms of its role in medical education (p<0.05). Faculty were less certain about how CBME was currently implemented (p<0.05), as well as how feedback to students should be delivered (p<0.05). Students and faculty agreed on perceived benefits to CBME implementation. Faculty time commitment to teaching and logistical concerns were reported as perceived challenges. Conclusion: Education leaders must prioritize faculty engagement and continued professional development of faculty to facilitate the transition. This program evaluation identified strategies to aid the transition to CBME in the undergraduate setting.

Metabolic engineering of the acid-tolerant yeast Pichia kudriavzevii for efficient L-malic acid production at low pH.

Currently, the biological production of L-malic acid (L-MA) is mainly based on the fermentation of filamentous fungi at near-neutral pH, but this process requires large amounts of neutralizing agents, resulting in the generation of waste salts when free acid is obtained in the downstream process, and the environmental hazards associated with the waste salts limit the practical application of this process. To produce L-MA in a more environmentally friendly way, we metabolically engineered the acid-tolerant yeast Pichia kudriavzevii and achieved efficient production of L-MA through low pH fermentation. First, an initial L-MA-producing strain that relies on the reductive tricarboxylic acid (rTCA) pathway was constructed. Subsequently, the L-MA titer and yield were further increased by fine-tuning the flux between the pyruvate and oxaloacetate nodes. In addition, we found that the insufficient supply of NADH for cytoplasmic malate dehydrogenase (MDH) hindered the L-MA production at low pH, which was resolved by overexpressing the soluble pyridine nucleotide transhydrogenase SthA from E. coli. Transcriptomic and metabolomic data showed that overexpression of EcSthA contributed to the activation of the pentose phosphate pathway and provided additional reducing power for MDH by converting NADPH to NADH. Furthermore, overexpression of EcSthA was found to help reduce the accumulation of the by-product pyruvate but had no effect on the accumulation of succinate. In microaerobic batch fermentation in a 5-L fermenter, the best strain, MA009-10-URA3 produced 199.4 g/L L-MA with a yield of 0.94 g/g glucose (1.27 mol/mol), with a productivity of 1.86 g/L/h. The final pH of the fermentation broth was approximately 3.10, meaning that the amount of neutralizer used was reduced by more than 50% compared to the common fermentation processes using filamentous fungi. To our knowledge, this is the first report of the efficient bioproduction of L-MA at low pH and represents the highest yield of L-MA in yeasts reported to date.