A comprehensive review on the recent advances for 5-aminolevulinic acid production by the engineered bacteria.

5-Aminolevulinic acid (5-ALA) is a non-proteinogenic amino acid essential for synthesizing tetrapyrrole compounds, including heme, chlorophyll, cytochrome, and vitamin B12. As a plant growth regulator, 5-ALA is extensively used in agriculture to enhance crop yield and quality. The complexity and low yield of chemical synthesis methods have led to significant interest in the microbial synthesis of 5-ALA. Advanced strategies, including the: enhancement of precursor and cofactor supply, compartmentalization of key enzymes, product transporters engineering, by-product formation reduction, and biosensor-based dynamic regulation, have been implemented in bacteria for 5-ALA production, significantly advancing its industrialization. This article offers a comprehensive review of recent developments in 5-ALA production using engineered bacteria and presents new insights to propel the field forward.

Clonal hematopoiesis in people with advanced HIV and associated inflammatory syndromes.

People with HIV (PWH) have a higher age-adjusted mortality due to chronic immune activation and age-related comorbidities. PWH also have higher rates of clonal hematopoiesis (CH) than age-matched non-HIV cohorts; however, risk factors influencing the development and expansion of CH in PWH remain incompletely explored. We investigated the relationship between CH, immune biomarkers, and HIV-associated risk factors (CD4+ and CD8+ T cells, nadir CD4+ count, opportunistic infections [OIs], and immune reconstitution inflammatory syndrome [IRIS]) in a diverse cohort of 197 PWH with median age of 42 years, using a 56-gene panel. Seventy-nine percent had a CD4+ nadir below 200 cells/µL, 58.9% had prior OIs, and 34.5% had a history of IRIS. The prevalence of CH was high (27.4%), even in younger individuals, and CD8+ T cells and nadir CD4+ counts strongly associated with CH after controlling for age. A history of IRIS was associated with CH in a subgroup analysis of patients 35 years of age and older. Inflammatory biomarkers were higher in CH carriers compared with noncarriers, supporting a dysregulated immune state. These findings suggest PWH with low nadir CD4+ and/or inflammatory complications may be at high risk of CH regardless of age and represent a high-risk group that could benefit from risk reduction and potentially targeted immunomodulation.

CMR feature tracking-based left atrial mechanics predicts response to cardiac resynchronization therapy and adverse outcomes.

BACKGROUND: Cardiac resynchronization therapy (CRT) is an established therapy for advanced heart failure (HF) with prolonged QRS duration. However, 30% of patients have shown no benefit from the treatment. OBJECTIVE: This study aimed to examine the value of left atrial (LA) mechanics by cardiac magnetic resonance (CMR) to predict response to CRT and clinical outcomes. METHODS: A total of 163 CRT recipients with preimplantation CMR examination were retrospectively recruited. CMR feature tracking was used to evaluate LA size and function. The end points include (1) improvement of at least 5% in left ventricular ejection fraction combined with a reduction of at least 1 New York Heart Association functional class at 6-month follow-up and (2) any all-cause death or HF hospitalization during follow-up. RESULTS: Overall, 82 (50.3%) were CRT responders. CRT nonresponders had larger LA and worse LA reservoir and booster pump function than did responders (P < .001 for all). LA structural (maximum volume index < 47 mL/m2) and functional (booster pump strain > 8.5%) criteria were incremental to traditional indicators in detecting CRT response (χ2, 40.83 vs 9.98; P < .001). During follow-up (median 41 months), survival free from death or HF hospitalization increased with the number of positive LA criteria (log-rank, P < .001). After adjustment for clinical confounders, the absence of the 2 criteria remained associated with a considerably increased risk of death or HF hospitalization (adjusted hazard ratio 6.2; 95% confidence interval 2.15-17.88; P = .001). CONCLUSION: The preprocedure LA mechanics evaluated using CMR may be useful to predict response to CRT and improve risk stratification in CRT recipients.

Ultrahigh-throughput screening-assisted in vivo directed evolution for enzyme engineering.

BACKGROUND: Classical directed evolution is a powerful approach for engineering biomolecules with improved or novel functions. However, it traditionally relies on labour- and time-intensive iterative cycles, due in part to the need for multiple molecular biology steps, including DNA transformation, and limited screening throughput. RESULTS: In this study, we present an ultrahigh throughput in vivo continuous directed evolution system with thermosensitive inducible tunability, which is based on error-prone DNA polymerase expression modulated by engineered thermal-responsive repressor cI857, and genomic MutS mutant with temperature-sensitive defect for fixation of mutations in Escherichia coli. We demonstrated the success of the in vivo evolution platform with ß-lactamase as a model, with an approximately 600-fold increase in the targeted mutation rate. Furthermore, the platform was combined with ultrahigh-throughput screening methods and employed to evolve α-amylase and the resveratrol biosynthetic pathway. After iterative rounds of enrichment, a mutant with a 48.3% improvement in α-amylase activity was identified via microfluidic droplet screening. In addition, when coupled with an in vivo biosensor in the resveratrol biosynthetic pathway, a variant with 1.7-fold higher resveratrol production was selected by fluorescence-activated cell sorting. CONCLUSIONS: In this study, thermal-responsive targeted mutagenesis coupled with ultrahigh-throughput screening was developed for the rapid evolution of enzymes and biosynthetic pathways.

Reduced thoracic skeletal muscle size is associated with adverse outcomes in diabetes patients with heart failure and reduced ejection fraction: quantitative analysis of sarcopenia by using cardiac MRI.

BACKGROUND: Sarcopenia is frequently found in patients with heart failure with reduced ejection fraction (HFrEF) and is associated with reduced exercise capacity, poor quality of life and adverse outcomes. Recent evidence suggests that axial thoracic skeletal muscle size could be used as a surrogate to assess sarcopenia in HFrEF. Since diabetes mellitus (DM) is one of the most common comorbidities with HFrEF, we aimed to explore the potential association of axial thoracic skeletal muscle size with left ventricular (LV) remodeling and determine its prognostic significance in this condition. METHODS: A total of 243 diabetes patients with HFrEF were included in this study. Bilateral axial thoracic skeletal muscle size was obtained using cardiac MRI. Patients were stratified by the tertiles of axial thoracic skeletal muscle index (SMI). LV structural and functional indices, as well as amino-terminal pro-B-type natriuretic peptide (NT-proBNP), were measured. The determinants of elevated NT-proBNP were assessed using linear regression analysis. The associations between thoracic SMI and clinical outcomes were assessed using a multivariable Cox proportional hazards model. RESULTS: Patients in the lowest tertile of thoracic SMI displayed a deterioration in LV systolic strain in three components, together with an increase in LV mass and a heavier burden of myocardial fibrosis (all P < 0.05). Moreover, thoracic SMI (ß = -0.25; P < 0.001), rather than body mass index (ß = -0.04; P = 0.55), was independently associated with the level of NT-proBNP. The median follow-up duration was 33.6 months (IQR, 20.4-52.8 months). Patients with adverse outcomes showed a lower thoracic SMI (40.1 [34.3, 47.9] cm2/m2 vs. 45.3 [37.3, 55.0] cm2/m2; P < 0.05) but a similar BMI (P = 0.76) compared with those without adverse outcomes. A higher thoracic SMI indicated a lower risk of adverse outcomes (hazard ratio: 0.96; 95% confidence interval: 0.92-0.99; P = 0.01). CONCLUSIONS: With respect to diabetes patients with HFrEF, thoracic SMI is a novel alternative for evaluating muscle wasting in sarcopenia that can be obtained by a readily available routine cardiac MRI protocol. A reduction in thoracic skeletal muscle size predicts poor outcomes in the context of DM with HFrEF.

Association between left atrial myopathy and sarcomere mutation in patients with hypertrophic cardiomyopathy: insights into left atrial strain by MRI feature tracking.

OBJECTIVES: Left atrial (LA) myopathy, characterized by LA enlargement and mechanical dysfunction, is associated with worse prognosis in hypertrophic cardiomyopathy (HCM) while the impact of sarcomere mutation on LA myopathy remains unclear. We aimed to assess the association between LA myopathy and sarcomere mutation and to explore the incremental utility of LA strain in mutation prediction. METHODS: A total of 105 consecutive HCM patients (mean age 47.8 ± 11.9 years, 71% male) who underwent HCM-related gene screening and cardiac MRI were retrospectively enrolled. LA volume, ejection fraction and strain indices in reservoir, conduit, and booster-pump phases were investigated respectively. RESULTS: Fifty mutation-positive patients showed higher LA maximal volume index (59.4 ± 28.2 vs 43.8 ± 18.1 mL/m2, p = 0.001), lower reservoir (21.3 ± 7.9 vs 26.2 ± 6.6%, p < 0.001), and booster-pump strain (12.1 ± 5.4 vs 17.1 ± 5.0%, p < 0.001) but similar conduit strain (9.2 ± 4.5 vs 9.1 ± 4.5%, p = 0.909) compared with mutation-negative patients. In multivariate logistic regression, LA booster-pump strain was associated with sarcomere mutation (odds ratio = 0.86, 95% confidence interval: 0.77-0.96, p = 0.010) independent of maximal wall thickness, late gadolinium enhancement, and LA volume. Furthermore, LA booster-pump strain showed incremental value for mutation prediction added to Mayo II score (AUC 0.798 vs 0.709, p = 0.024). CONCLUSIONS: In HCM, mutation-positive patients suffered worse LA enlargement and worse reservoir and booster-pump functions. LA booster-pump strain was a strong factor for sarcomere mutation prediction added to Mayo II score. CLINICAL RELEVANCE STATEMENT: The independent association between sarcomere mutation and left atrial mechanical dysfunction provide new insights into the pathogenesis of atrial myopathy and is helpful to understand the adverse prognosis regarding atrial fibrillation and stroke in mutation-positive patients. KEY POINTS: • In patients with hypertrophic cardiomyopathy, left atrial (LA) reservoir and booster-pump function, but not conduit function, were significantly impaired in mutation-positive patients compared with mutation-negative patients. • LA booster-pump strain measured by MRI-derived feature tracking is feasible to predict sarcomere mutation with high incremental value added to Mayo II score.

Incremental Diagnostic Value of Right Ventricular Strain Analysis in Arrhythmogenic Right Ventricular Cardiomyopathy.

BACKGROUND: Strain analysis is a sensitive method for the assessment of ventricular structural or functional alterations. The authors aimed to determine whether right ventricular (RV) strain parameters can discriminate patients with revised Task Force Criteria-diagnosed arrhythmogenic RV cardiomyopathy (ARVC) incremental to the existing cardiovascular magnetic resonance (CMR) criteria, thus improving the diagnostic yield of CMR in ARVC. METHODS AND RESULTS: A total of 74 patients with revised Task Force Criteria-diagnosed ARVC (37 borderline and 37 definite) and 37 controls were retrospectively enrolled for analysis. Using CMR feature tracking, RV global longitudinal (GLS), circumferential, and radial strain of all participants were evaluated. Compared with controls, the study patients demonstrated significantly impaired global biventricular strain in all 3 directions (all P<0.001). Receiver operating characteristic curve analysis indicated that RV GLS was the strongest discriminator among all RV strain parameters for the identification of patients with ARVC (area under the curve, 0.92). Using the Youden index, the authors determined RV GLS ≥-19.95% as the diagnostic criterion of ARVC. In patients diagnosed with borderline ARVC according to revised Task Force Criteria but with no or only minor CMR criteria, there were >50% presenting with impaired RV GLS. When both conventional criteria and RV GLS were considered together, this new diagnostic method demonstrated an overall diagnostic accuracy of 90%. The likelihood ratio test showed a significant incremental diagnostic value of RV GLS (P=0.02) over the existing CMR major criteria. CONCLUSIONS: The current study showed an improved diagnostic accuracy when both RV GLS and the existing CMR criteria were considered together, especially for patients with borderline diagnosis, suggesting the incremental value of strain analysis to the initial assessment of ARVC.

Incremental prognostic value of left atrial and biventricular feature tracking in dilated cardiomyopathy: a long-term study.

BACKGROUND: Despite the use of cardiovascular magnetic resonance (CMR) feature tracking (FT) imaging to detect myocardial deformation, the optimal strain index in dilated cardiomyopathy (DCM) is unclear. This study aimed to determine whether atrial and biventricular strains can provide the greatest or joint incremental prognostic value in patients with DCM over a long follow-up period. METHODS: Four hundred-twelve DCM patients were included retrospectively. Comprehensive clinical evaluation and imaging investigations were obtained, including measurements of CMR-FT derived left atrial (LA) reservoir, conduit, booster strain (εs, εe, εa); left ventricular (LV) and right ventricular (RV) global longitudinal, radial, circumferential strain (GLS, GRS, GCS). All patients were followed up for major adverse cardiac events (MACE) including all-cause mortality, heart transplantation, and implantable cardioverter defibrillator discharge. The predictors of MACE were examined with univariable and multivariable Cox regression analysis. Subsequently, nested Cox regression models were built to evaluate the incremental prognostic value of strain parameters. The incremental predictive power of strain parameters was assessed by Omnibus tests, and the model performance and discrimination were evaluated by Harrell C-index and integrated discrimination improvement (IDI) analysis. Patient survival was illustrated by Kaplan-Meier curves and differences were evaluated by log-rank test. RESULTS: During a median follow-up of 5.0 years, MACE were identified in 149 (36%) patients. LAεe, LVGLS, and RVGLS were the most predictive strain parameters for MACE (AUC: 0.854, 0.733, 0.733, respectively). Cox regression models showed that the predictive value of LAεe was independent from and incremental to LVGLS, RVGLS, and baseline variables (HR 0.74, 95% CI 0.68-0.81, P < 0.001). In reclassification analysis, the addition of LAεe provided the best discrimination of the model (χ2 223.34, P < 0.001; C-index 0.833; IDI 0.090, P < 0.001) compared with LVGLS and RVGLS models. Moreover, LAεe with a cutoff of 5.3% further discriminated the survival probability in subgroups of patients with positive LGE or reduced LVEF (all log-rank P < 0.001). CONCLUSION: LAεe provided the best prognostic value over biventricular strains and added incremental value to conventional clinical predictors for patients with DCM.

Assessment of late gadolinium enhancement in hypertrophic cardiomyopathy improves risk stratification based on current guidelines.

BACKGROUND AND AIMS: Identifying patients with hypertrophic cardiomyopathy (HCM) who are candidates for implantable cardioverter defibrillator (ICD) implantation in primary prevention for sudden cardiac death (SCD) is crucial. The aim of this study was to externally validate the 2022 European Society of Cardiology (ESC) model and other guideline-based ICD class of recommendation (ICD-COR) models and explore the utility of late gadolinium enhancement (LGE) in further risk stratification. METHODS: Seven hundred and seventy-four consecutive patients who underwent cardiac magnetic resonance imaging were retrospectively enrolled. RESULTS: Forty-six (5.9%) patients reached the SCD-related endpoint during 7.4 ± 2.5 years of follow-up. Patients suffering from SCD had higher ESC Risk-SCD score (4.3 ± 2.4% vs. 2.8 ± 2.1%, P < .001) and LGE extent (13.7 ± 9.4% vs. 4.9 ± 6.6%, P < .001). Compared with the 2014 ESC model, the 2022 ESC model showed increased area under the curve (.76 vs. .63), sensitivity (76.1% vs. 43.5%), positive predictive value (16.8% vs. 13.6%), and negative predictive value (98.1% vs. 95.9%). The C-statistics for SCD prediction of 2011 American College of Cardiology (ACC)/American Heart Association (AHA), 2014 ESC, 2020 AHA/ACC, and 2022 ESC models were .68, .64, .76 and .78, respectively. Furthermore, in patients without extensive LGE, LGE ≥5% was responsible for seven-fold SCD risk after multivariable adjustment. Whether in ICD-COR II or ICD-COR III, patients with LGE ≥5% and <15% showed significantly worse prognosis than those with LGE <5% (all P < .001). CONCLUSIONS: The 2022 ESC model performed better than the 2014 ESC model with especially improved sensitivity. LGE enabled further risk stratification based on current guidelines.

Core-Shell Droplet-Based Microfluidic Screening System for Filamentous Fungi.

Filamentous fungi are competitive hosts for the production of drugs, proteins, and chemicals. However, their utility is limited by screening methods and low throughput. In this work, a universal high-throughput system for optimizing protein production in filamentous fungi was described. Droplet microfluidics was used to encapsulate large mutant strain pools in biocompatible core-shell microdroplets designed to avoid mycelial punctures and thus sustain prolonged culture. The self-assembled split GFP was then used to characterize the secretory capacity of the strains and isolate strains with superior production titers according to the fluorescence signals. The platform was applied to optimize the α-amylase secretion of Aspergillus niger, resulting in the isolation of a strain with 2.02-fold higher secretion capacity. The system allows the analysis of >105 single cells per h and will facilitate ultrahigh-throughput screening experiments of filamentous fungi. This method could help identify improved hosts for the large-scale production of biotechnology-relevant proteins. This is a broadly applicable system that can be equally used in other hosts.

T1 Mapping for Identifying the Substrate in Patients With Apparently Idiopathic Premature Ventricular Complexes.

BACKGROUND: Myocardial fibrosis is implicated as a potential substrate responsible for arrhythmias. OBJECTIVES: This study aimed to investigate myocardial fibrosis assessed by T1 mapping in patients with apparently idiopathic premature ventricular complexes (PVCs), and to determine the relationship between this tissue biomarker and PVC features. METHODS: Patients with frequent PVC (>1,000/24 h) who underwent cardiac magnetic resonance imaging (MRI) between 2020 and 2021 were retrospectively evaluated. Patients were included if they had no indicator of known heart diseases on MRI. Sex- and age-matched healthy subjects underwent noncontrast MRI with native T1 mapping. High PVC burden was defined as the percentage of PVC >20%/24 h. RESULTS: A total of 70 patients and 70 healthy controls were included. Global T1 value was significantly higher in patients than in controls (P < 0.001). Extracellular volume was 26.03% ± 2.16% in the patients. Moreover, global T1 value showed a stepwise increase in PVC tertiles (P = 0.03) but not for extracellular volume (P = 0.85). Patients with a non-left bundle branch block (LBBB) inferior axis morphology showed higher global native T1 values than LBBB inferior axis pattern (P = 0.005). In addition, global T1 values correlated significantly with PVC burden (r = 0.28, P = 0.02). In the multivariate analysis, global T1 value independently correlated with high PVC burden (odds ratio: 1.22 per 10-ms increase, P = 0.02). CONCLUSIONS: Increased global T1, a marker of interstitial fibrosis, was detected in patients with apparently idiopathic PVC and was significantly associated with non-LBBB inferior axis morphology and high PVC burden.

Prediction of adverse outcomes in nonischemic dilated cardiomyopathy: A CMR-based nomogram.

OBJECTIVES: To develop and validate a novel nomogram score to predict outcomes in patients with nonischemic dilated cardiomyopathy (NIDCM) over a long follow-up period. METHODS: A total of 335 consecutive NIDCM patients who underwent cardiac magnetic resonance (CMR) imaging were retrospectively enrolled. Comprehensive clinical evaluation and imaging investigation were obtained, including measurements of late gadolinium enhancement (LGE) and feature tracking (FT) images. All patients were followed up for a composite endpoint of major adverse cardiac events (MACE) including all-cause mortality and heart transplantation. These patients were randomly divided into development and validation cohorts (7:3). RESULTS: MACE occurred in 87 (37.2%) out of 234 patients in the development cohort, and in 31 (30.7%) out of 101 patients in the validation cohort. Five variables including NYHA class III-IV, NT-proBNP, beta-blocker medication, LGE presence, and LV global longitudinal strain (GLS) were found to be significantly associated with MACE and were used for constructing the nomogram. The nomogram achieved good discrimination with C-indexes in development and validation cohorts respectively. The calibration curve for 1-, 3-, and 5-year survival probability also showed high coherence between the predicted and actual probability of MACE. Decision curve analysis identified the model was significantly better net benefit in predicting MACE. CONCLUSION: A novel nomogram score of a predictive model that incorporates clinical factors and imaging features was constructed, which could be conveniently used to facilitate risk evaluation in patients with NIDCM.

Subendocardial Involvement as an Underrecognized LGE Subtype Related to Adverse Outcomes in Hypertrophic Cardiomyopathy.

BACKGROUND: Late gadolinium enhancement (LGE) has been established as an independent predictor for adverse outcomes in hypertrophic cardiomyopathy (HCM). However, the prevalence and clinical significance of some LGE subtypes have not been well demonstrated. OBJECTIVES: In this study, the authors sought to investigate the prognostic value of subendocardium-involved LGE pattern and location of right ventricle insertion points (RVIPs) with LGE in HCM patients. METHODS: In this single-center retrospective study, 497 consecutive HCM patients with LGE confirmed by cardiac magnetic resonance (CMR) were included. Subendocardium-involved LGE was defined as LGE involving subendocardium not corresponding to a coronary vascular distribution. Subjects with ischemic heart disease that would contribute to subendocardial LGE were excluded. Endpoints included a composite of heart failure-related events, arrhythmic events, and stroke. RESULTS: Of the 497 patients, subendocardium-involved LGE and RVIP LGE were observed in 184 (37.0%) and 414 (83.3%), respectively. Extensive LGE (≥15% of left ventricular mass) was detected in 135 patients. During a median follow-up of 57.9 months, 66 patients (13.3%) experienced composite endpoints. Patients with extensive LGE had a significantly higher annual incidence of adverse events (5.1% vs 1.9% per year; P < 0.001). However, spline analysis showed that the association between LGE extent and HRs for adverse outcomes tended to be nonlinear. The risk of composite endpoint increased with percentage increase in LGE extent in patients with extensive LGE, whereas a similar trend was not observed in patients with nonextensive LGE (<15%). In patients with extensive LGE, LGE extent significantly correlated with composite endpoints (HR: 1.05; P = 0.03) after adjusting for left ventricular ejection fraction <50%, atrial fibrillation, and nonsustained ventricular tachycardia, whereas in patients with nonextensive LGE, subendocardium-involved LGE rather than LGE extent was independently associated with adverse outcomes (HR: 2.12; P = 0.03). RVIP LGE was not significantly associated with poor outcomes. CONCLUSIONS: In HCM patients with nonextensive LGE, the presence of subendocardium-involved LGE rather than LGE extent is associated with unfavorable outcomes. Given that the prognostic value of extensive LGE has been broadly recognized, subendocardial involvement as an underrecognized LGE pattern shows the potential to improve risk stratification in HCM patients with nonextensive LGE.

Chromatin regulator Ahc1p co-regulates nitrogen metabolism via interactions with multiple transcription factors in Saccharomyces cerevisiae.

Chromatin regulation is an important gene expression/regulation system, but little is known about how it affects nitrogen metabolism in Saccharomyces cerevisiae. A previous study demonstrated the regulatory role of the chromatin regulator Ahc1p on multiple key genes of nitrogen metabolism in S. cerevisiae, but the regulatory mechanism remains unknown. In this study, multiple key nitrogen metabolism genes directly regulated by Ahc1p were identified, and the transcription factors interacting with Ahc1p were analyzed. It was ultimately found that Ahc1p may regulate some key nitrogen metabolism genes in two ways. First, Ahc1p acts as a co-factor and is recruited with transcription factors such as Rtg3p or Gcr1p to facilitate transcription complex binding to target gene core promoters and promote transcription initiation. Second, Ahc1p binds at enhancers to promote the transcription of target genes in concert with transcription factors. This study furthers the understanding of the regulatory network of nitrogen metabolism in S. cerevisiae from an epigenetic perspective.

Reverse remodeling of left atrium assessed by cardiovascular magnetic resonance feature tracking in hypertrophic obstructive cardiomyopathy after septal myectomy.

BACKGROUND: Assessing the structure and function of left atrium (LA) is crucial in hypertrophic obstructive cardiomyopathy (HOCM) because LA remodeling correlates with atrial fibrillation. However, few studies have investigated the potential effect of myomectomy on LA phasic remodeling in HOCM after myectomy using cardiovascular magnetic resonance (CMR) feature tracking (FT). This study aims to evaluate the LA structural and functional remodeling with HOCM after myectomy by CMR-FT and to further investigate the determinants of LA reverse remodeling. METHODS: In this single-center study, we retrospectively studied 88 patients with HOCM who received CMR before and after myectomy between January 2011 and June 2021. Preoperative and postoperative LA parameters derived from CMR-FT were compared, including LA reservoir function (total ejection fraction [EF], total strain [εs], peak positive strain rate [SRs]), conduit function (passive EF, passive strain [εe], peak early negative strain rate [SRe]) and booster function (booster EF, active strain [εa], late peak negative strain rate [SRa]). Eighty-six healthy participants were collected for comparison. Univariate and multivariate linear regression identified variables associated with the rate of change of εa. RESULTS: Compared with preoperative parameters, LA reservoir function (total EF, εs, SRs), booster function (booster EF, εa, SRa), and SRe were significantly improved after myectomy (all P < 0.05), while no significant differences were observed in passive EF and εe. Postoperative patients with HOCM still had larger LA and worse LA function than healthy controls (all P < 0.05). After analyzing the rates of change in LA parameters, LA boost function, especially εa, showed the most dramatic improvement beyond the improvements in reservoir function, conduit function, and volume. In multivariable regression analysis, minimum LA volume index (adjusted ß = - 0.39, P < 0.001) and Δleft ventricular outflow tract (LVOT) pressure gradient (adjusted ß = - 0.29, P = 0.003) were significantly related to the rate of change of εa. CONCLUSIONS: Patients with HOCM after septal myectomy showed LA reverse remodeling with a reduction in LA size and restoration in LA reservoir and booster function but unchanged LA conduit function. Among volumetric and functional changes, booster function had the greatest improvement postoperatively. Besides, preoperative LAVmin index and ΔLVOT might be potential factors associated with the degree of improvement in εa.

Engineering Saccharomyces cerevisiae for enhanced (-)-α-bisabolol production.

(-)-α-Bisabolol is naturally occurring in many plants and has great potential in health products and pharmaceuticals. However, the current extraction method from natural plants is unsustainable and cannot fulfil the increasing requirement. This study aimed to develop a sustainable strategy to enhance the biosynthesis of (-)-α-bisabolol by metabolic engineering. By introducing the heterologous gene MrBBS and weakening the competitive pathway gene ERG9, a de novo (-)-α-bisabolol biosynthesis strain was constructed that could produce 221.96 mg/L (-)-α-bisabolol. Two key genes for (-)-α-bisabolol biosynthesis, ERG20 and MrBBS, were fused by a flexible linker (GGGS)3 under the GAL7 promoter control, and the titer was increased by 2.9-fold. Optimization of the mevalonic acid pathway and multi-copy integration further increased (-)-α-bisabolol production. To promote product efflux, overexpression of PDR15 led to an increase in extracellular production. Combined with the optimal strategy, (-)-α-bisabolol production in a 5 L bioreactor reached 7.02 g/L, which is the highest titer reported in yeast to date. This work provides a reference for the efficient production of (-)-α-bisabolol in yeast.

Improving the cryoprotective effect of antifreeze proteins from Daucus carota on plant-based meat by eliminating N-glycosylation.

As a novel animal meat alternative, plant-based meat (PBM) frequently suffers from quality problems as a result of freeze-thaw cycles in commercial transportation and household storage. There is a need to reduce the deterioration of PBM attributes, such as water holding capacity, as a result of these freeze-thaw cycles. In this study, Daucus carota antifreeze protein (DcAFP) and its deglycosylated mutant DcAFP-N294G were heterologously expressed in Komagataella phaffii X33. The effects of pretreatment with recombinant AFPs (rAFPs) on the microstructure, rheological properties, water mobility, and water distribution of PBM were assessed. The rDcAFP-N294G-treated PBM samples had superior viscoelasticity and water distribution features compared to the rDcAFP-treated group because the complex N-linked oligosaccharides did not interfere with the binding of rAFPs to ice molecules. In addition, rAFP pretreatment resulted in a smoother and flatter surface of the high-moisture protein extrudate matrix compared to the commercial cryoprotectant trehalose. Deglycosylated DcAFP has potential applications as a new effective cryoprotectant in meat alternatives.

De novo biosynthesis of carminic acid in Saccharomyces cerevisiae.

Carminic acid is a natural red dye extracted from the insect Dactylopius coccus. Due to its ideal dying effect and high safety, it is widely used in food and cosmetics industries. Previous study showed that introduction of polyketide synthase (OKS) from Aloe arborescens, cyclase (ZhuI) and aromatase (ZhuJ) from Streptomyces sp. R1128, and C-glucosyltransferase (UGT2) from D. coccus into Aspergillus nidulans could achieve trace amounts of de novo production. These four genes were introduced into Saccharomyces cerevisiae, but carminic acid was not detected. Analysis of the genome of A. nidulans revealed that 4'-phosphopantetheinyl transferase (NpgA) and monooxygenase (AptC) are essential for de novo biosynthesis of carminic acid in S. cerevisiae. Additionally, endogenous hydroxylase (Cat5) from S. cerevisiae was found to be responsible for hydroxylation of flavokermesic acid to kermesic acid. Therefore, all enzymes and their functions in the biosynthesis of carminic acid were explored and reconstructed in S. cerevisiae. Through systematic pathway engineering, including regulating enzyme expression, enhancing precursor supply, and modifying the ß-oxidation pathway, the carminic acid titer in a 5 L bioreactor reached 7580.9 µg/L, the highest yet reported for a microorganism. Heterologous reconstruction of the carminic acid biosynthetic pathway in S. cerevisiae has great potential for de novo biosynthesis of anthraquinone dye.

Coordinating caffeic acid and salvianic acid A pathways for efficient production of rosmarinic acid in Escherichia coli.

Rosmarinic acid is a natural hydroxycinnamic acid ester used widely in the food and pharmaceutical industries. Although many attempts have been made to screen rate-limiting enzymes and optimize modules through co-culture fermentation, the titer of rosmarinic acid remains at the microgram level by microorganisms. A de novo biosynthetic pathway for rosmarinic acid was constructed based on caffeic acid synthesis modules in Escherichia coli. Knockout of competing pathways increased the titer of rosmarinic acid and reduced the synthesis of rosmarinic acid analogues. An L-amino acid deaminase was introduced to balance metabolic flux between the synthesis of caffeic acid and salvianic acid A. The ratio of FADH2/FAD was maintained via the coordination of deaminase and HpaBC, which is responsible for caffeic acid synthesis. Knockout of menI, encoding an endogenous thioesterase, increased the stability of caffeoyl-CoA. The final strain produced 5780.6 mg/L rosmarinic acid in fed-batch fermentation, the highest yet reported for microbial production. The strategies applied in this study lay a foundation for the synthesis of other caffeic acid and rosmarinic acid derivatives.

Combined engineering of l-sorbose dehydrogenase and fermentation optimization to increase 2-keto-l-gulonic acid production in Escherichia coli.

One-step fermentation to produce 2-keto-l-gulonic acid (2-KLG), the precursor of vitamin C, is a long-term goal. Improvement of the enzyme's activity through engineering could benefit 2-KLG production. This study aimed to conduct a semi-rational design of l-sorbose dehydrogenase (SDH) through structure-directed, to screen mutants that could enhance the 2-KLG titer. First, the predicted structure of SDH was obtained using AlphaFold2. The key mutation sites in the substrate pocket were identified by Ala scanning. Subsequently, the mutant V336I/V368A was obtained by iterative saturation mutagenesis, which increased the yield of 2-KLG 1.9-fold. Finally, 5.03 g/L of 2-KLG was obtained by a two-stage temperature control fermentation method, and the conversion rate was 50%. Furthermore, experiments showed that knockdown of the l-sorbose-associated phosphotransferase system delays 2-KLG production. The results show that the production of 2-KLG was effectively increased through a combination of SDH engineering and fermentation optimization.