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.

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.

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.

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.

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.

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.

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.

Cardiac remodeling after tricuspid valve repair in Ebstein's anomaly: a magnetic resonance study.

OBJECTIVES: We aimed to evaluate immediate and midterm cardiac remodeling after surgery by cardiac magnetic resonance (CMR) in Ebstein's anomaly (EA), and also to investigate preoperative predictors of right ventricular (RV) normalization. METHODS: We retrospectively analyzed CMR parameters of the whole heart in adult patients with EA before surgery, at discharge and follow-up. RESULTS: A total of 26 patients were included and performed CMR at 7 days (interquartile range, 3-13 days) before surgery. Immediate postoperative CMR was finished at discharge (median: 8 [7-9] days; n = 18) and follow-up CMR at 187 days (interquartile range, 167-356 days; n = 17). RV and right atrial (RA) volumes promptly decreased immediately after surgery and at follow-up (all p < 0.05). RV ejection fraction decreased significantly at discharge (p < 0.05) but recovered at follow-up (p = 0.18). However, RV global longitudinal strain and RA reservoir strain were significantly impaired immediately and midterm after surgery (all p < 0.05). Indexed left ventricular (LV) end-diastolic volume, stroke volume, as well as global longitudinal strain increased from preoperative to follow-up (all p < 0.05). Patients who achieved normalization of RV volumes after surgery had smaller severity index and RV and RA volumes and higher LV ejection fraction and RA reservoir strain at baseline than patients without RV normalization (all p < 0.05). CONCLUSIONS: Reverse biventricular remodeling took place in EA after tricuspid valve surgery. Tricuspid valve reconstruction should be performed before deterioration of RV volume overload and LV function to achieve reverse RV remodeling. Key Points • After removing the volume load of tricuspid regurgitation in Ebstein's anomaly, reverse remodeling was detected by CMR in both left and right heart at midterm follow-up. • Tricuspid valve reconstruction should be performed before deterioration of RV volume overload and LV function to achieve reverse RV remodeling.

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.

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.

Engineering Gluconobacter cerinus CGMCC 1.110 for direct 2-keto-L-gulonic acid production.

Gluconobacter is a potential strain for single-step production of 2-keto-L-gulonic acid (2-KLG), which is the direct precursor of vitamin C. Three dehydrogenases, namely, sorbitol dehydrogenase (SLDH), sorbose dehydrogenase (SDH), and sorbosone dehydrogenase (SNDH), are involved in the production of 2-KLG from D-sorbitol. In the present study, the potential SNDH/SDH gene cluster in the strain Gluconobacter cerinus CGMCC 1.110 was mined by genome analysis, and its function in transforming L-sorbose to 2-KLG was verified. Proteomic analysis showed that the expression level of SNDH/SDH had a great influence on the titer of 2-KLG, and fermentation results showed that SDH was the rate-limiting enzyme. A systematic metabolic engineering process, which was theoretically suitable for increasing the titer of many products involving membrane-bound dehydrogenase from Gluconobacter, was then performed to improve the 2-KLG titer in G. cerinus CGMCC 1.110 from undetectable to 51.9 g/L in a 5-L bioreactor after fermentation optimization. The strategies used in this study may provide a reference for mining other potential applications of Gluconobacter. KEY POINTS: • The potential SNDH/SDH gene cluster in G. cerinus CGMCC 1.110 was mined. • A systematic engineering process was performed to improve the titer of 2-KLG. • The 2-KLG titer was successfully increased from undetectable to 51.9 g/L.

Patterns of Replacement Fibrosis in Hypertrophic Cardiomyopathy.

Background Myocardial replacement fibrosis is one of the major histologic features of hypertrophic cardiomyopathy (HCM), but its characteristics have not been well delineated. Purpose To clarify the characteristics of replacement fibrosis in HCM and to evaluate the prognostic value of the regional extent of fibrosis. Materials and Methods This prospective study evaluated participants with HCM who underwent contrast-enhanced cardiac MRI from March 2011 to April 2019. For each participant, global and 16-segment extent of late gadolinium enhancement (LGE) in the left ventricle (LV) at cardiac MRI was analyzed. The primary end point was all-cause death. Results Among the 798 study participants enrolled (median age, 49 years [interquartile range {IQR}: 38-59 years]; 508 men), 588 (74%) underwent whole-exome sequencing. Thirty-five participants (4%) experienced death from any cause during a median follow-up of 2.9 years (IQR: 1.5-4.7 years). Spearman analysis showed weak correlations between the extent of LGE and wall thickness (LGE of global LV and maximal LV wall thickness, r = 0.35 [P < .001]; LGE and thickness of septum, r = 0.30 [P < .001]). In the 16-segment model, the distribution of LGE was visually inhomogeneous and higher in the basal anterior, basal septal, midanterior, and midseptal regions (P < .001). This similar distribution of LGE was observed in participants with asymmetric septal hypertrophy, those with apical HCM, participants positive for mutation and those negative for mutation, and participants with MYH7 and MYBPC3 mutations. Cox analysis indicated that both the global extent of LGE (adjusted hazard ratio = 1.68 per 10% increase in LGE; P < .001) and the regional extent of LGE (ie, basal, midventricular, and apical regions of LV when on the short-axis view; septum, anterior free wall, inferior free wall, and lateral free wall when on the long-axis view) were associated with adverse outcomes. Conclusion In hypertrophic cardiomyopathy, myocardial replacement fibrosis weakly correlated with hypertrophy, was inhomogeneous and asymmetric, and was predominantly distributed in the interventricular septal wall and anterior free wall at the basal and mid levels. Greater extent of fibrosis was associated with poor prognosis, regardless of its location in the left ventricle. © RSNA, 2021 See also the editorial by Hanneman in this issue.

Glucocorticoid-induced eosinopenia results from CXCR4-dependent bone marrow migration.

Glucocorticoids are considered first-line therapy in a variety of eosinophilic disorders. They lead to a transient, profound decrease in circulating human eosinophils within hours of administration. The phenomenon of glucocorticoid-induced eosinopenia has been the basis for the use of glucocorticoids in eosinophilic disorders, and it has intrigued clinicians for 7 decades, yet its mechanism remains unexplained. To investigate, we first studied the response of circulating eosinophils to in vivo glucocorticoid administration in 3 species and found that the response in rhesus macaques, but not in mice, closely resembled that in humans. We then developed an isolation technique to purify rhesus macaque eosinophils from peripheral blood and performed live tracking of zirconium-89-oxine-labeled eosinophils by serial positron emission tomography/computed tomography imaging, before and after administration of glucocorticoids. Glucocorticoids induced rapid bone marrow homing of eosinophils. The kinetics of glucocorticoid-induced eosinopenia and bone marrow migration were consistent with those of the induction of the glucocorticoid-responsive chemokine receptor CXCR4, and selective blockade of CXCR4 reduced or eliminated the early glucocorticoid-induced reduction in blood eosinophils. Our results indicate that glucocorticoid-induced eosinopenia results from CXCR4-dependent migration of eosinophils to the bone marrow. These findings provide insight into the mechanism of action of glucocorticoids in eosinophilic disorders, with implications for the study of glucocorticoid resistance and the development of more targeted therapies. The human study was registered at ClinicalTrials.gov as #NCT02798523.

Correlation between left ventricular fractal dimension and impaired strain assessed by cardiac MRI feature tracking in patients with left ventricular noncompaction and normal left ventricular ejection fraction.

OBJECTIVES: To investigate the correlation between the extent of excessive trabeculation assessed by fractal dimension (FD) and myocardial contractility assessed by cardiac MRI feature tracking in patients with left ventricular noncompaction (LVNC) and normal left ventricular ejection fraction (LVEF). METHODS: Forty-one LVNC patients with normal LVEF (≥ 50%) and 41 healthy controls were retrospectively included. All patients fulfilled three available diagnostic criteria on MRI. Cardiac MRI feature tracking was performed on cine images to determine left ventricular (LV) peak strains in three directions: global radial strain (GRS), global circumferential strain (GCS), and global longitudinal strain (GLS). The complexity of excessive trabeculation was quantified by fractal analysis on short-axis cine stacks. RESULTS: Compared with controls, patients with LVNC had impaired GRS, GCS, and GLS (all p < 0.05). The global, maximal, and regional FD values of the LVNC population were all significantly higher than those of the controls (all p < 0.05). Global FD was positively correlated with the end-diastolic volume index, end-systolic volume index, and stroke volume index (r = 0.483, 0.505, and 0.335, respectively, all p < 0.05), but negatively correlated with GRS and GCS (r = - 0.458 and 0.508, respectively, both p < 0.001). Moreover, apical FD was also weakly associated with LVEF and GLS (r = - 0.249 and 0.252, respectively, both p < 0.05). CONCLUSION: In patients with LVNC, LV systolic dysfunction was detected early by cardiac MRI feature tracking despite the presence of normal LVEF and was associated with excessive trabecular complexity assessed by FD. KEY POINTS: • Left ventricular global strain was already impaired in patients with extremely prominent excessive trabeculation but normal left ventricular ejection fraction. • An increased fractal dimension was associated with impaired deformation in left ventricular noncompaction.

Engineering Saccharomyces cerevisiae for the production of dihydroquercetin from naringenin.

BACKGROUND: Dihydroquercetin (DHQ), a powerful bioflavonoid, has a number of health-promoting qualities and shows potential as a treatment for a number of disorders. Dihydroquercetin biosynthesis is a promising solution to meet the rising demand for dihydroquercetin. However, due to the significant accumulation of eriodietyol (ERI), naringenin (NAR), dihydrokaempferol (DHK), and other metabolites, the yield of DHQ biosynthesis is low. As a result, this is the hindrance to the biosynthesis of DHQ. RESULTS: In this study, we proposed several strategies to enhance the product formation and reduce the metabolites in accumulation. The flavonoid 3'-hydroxylase (F3'H) and cytochrome P450 reductase from different species were co-expressed in S. cerevisiae, and the best strain expressing the P450-reductase enzyme complex (SmF3'H/ScCPR) yielded 435.7 ± 7.6 mg/L of ERI from NAR in the deepwell microplate. The product conversion rate was improved further by mutating the predicted potential ubiquitination sites to improve SmF3'H stability, resulting in a 12.8% increase in titre using the mutant SmF3'H (K290R). Besides, different F3Hs from various sources and promoters were tested for the improved DHQ production, with the best strain producing 381.2 ± 10.7 mg/L of DHQ from 1 g/L of NAR, suggesting the temporal regulation the expression of F3H is important for maximization the function of F3'H and F3H. CONCLUSION: This study offers effective strategies for improving DHQ production from NAR and could be used as a reference for related research.

Simultaneous transformation of five vectors in Gluconobacter oxydans.

Gluconobacter oxydans is an obligate Gram-negative bacterium that belongs to the family Acetobacteraceae. It is one of the most frequently used microorganisms in industrial biotechnology to produce chemicals related to incomplete oxidation. However, the fine-tuning of G. oxydans is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. Thus, a series of shuttle vectors for G. oxydans inspired by a series of wild-type plasmids in different G. oxydans strains were constructed. Fifteen shuttle vectors were employed to express mCherry in G. oxydans WSH-003 using the replication origin of these wild-type plasmids. Among them, the intensity of fluorescent proteins expressed by p15-K-mCherry was about 10 times that of fluorescent proteins expressed by p5-K-mCherry. Quantitative real-time polymerase chain reaction showed that the relative copy number of p15-K-mCherry reached 19 and had high stability. In contrast, some of the plasmids had a relative copy number of less than 10. The co-expression of multiple shuttle vectors revealed five shuttle vectors that could be transformed into G. oxydans WSH-003 and could express five different fluorescent proteins. The shuttle vectors will facilitate genetic operations for Gluconobacter strains to produce useful compounds more efficiently.

A secretion-based dual fluorescence assay for high-throughput screening of alcohol dehydrogenases.

Alcohol dehydrogenases (ADHs) play key roles in the production of various chemical precursors that are essential in pharmaceutical and fine chemical industries. To achieve a practical application of ADHs in industrial processes, tailoring enzyme properties through rational design or directed evolution is often required. Here, we developed a secretion-based dual fluorescence assay (SDFA) for high-throughput screening of ADHs. In SDFA, an ADH of interest is fused to a mutated superfolder green fluorescent protein (MsfGFP), which could result in the secretion of the fusion protein to culture broth. After a simple centrifugation step to remove the cells, the supernatant can be directly used to measure the activity of ADH based on a red fluorescence signal, whose increase is coupled to the formation of NADH (a redox cofactor of ADHs) in the reaction. SDFA allows easy quantification of ADH concentration based on the green fluorescence signal of MsfGFP. This feature is useful in determining specific activity and may improve screening accuracy. Out of five ADHs we have tested with SDFA, four ADHs can be secreted and characterized. We successfully screened a combinatorial library of an ADH from Pichia finlandica and identified a variant with a 197-fold higher kcat /km value toward (S)-2-octanol compared to its wild type.

Balancing cellular redox metabolism in microbial electrosynthesis and electro fermentation - A chance for metabolic engineering.

More and more microbes are discovered that are capable of extracellular electron transfer, a process in which they use external electrodes as electron donors or acceptors for metabolic reactions. This feature can be used to overcome cellular redox limitations and thus optimizing microbial production. The technologies, termed microbial electrosynthesis and electro-fermentation, have the potential to open novel bio-electro production platforms from sustainable energy and carbon sources. However, the performance of reported systems is currently limited by low electron transport rates between microbes and electrodes and our limited ability for targeted engineering of these systems due to remaining knowledge gaps about the underlying fundamental processes. Metabolic engineering offers many opportunities to optimize these processes, for instance by genetic engineering of pathways for electron transfer on the one hand and target product synthesis on the other hand. With this review, we summarize the status quo of knowledge and engineering attempts around chemical production in bio-electrochemical systems from a microbe perspective. Challenges associated with the introduction or enhancement of extracellular electron transfer capabilities into production hosts versus the engineering of target compound synthesis pathways in natural exoelectrogens are discussed. Recent advances of the research community in both directions are examined critically. Further, systems biology approaches, for instance using metabolic modelling, are examined for their potential to provide insight into fundamental processes and to identify targets for metabolic engineering.

Improved performance of Pseudomonas putida in a bioelectrochemical system through overexpression of periplasmic glucose dehydrogenase.

It was recently demonstrated that a bioelectrochemical system (BES) with a redox mediator allowed Pseudomonas putida to perform anoxic metabolism, converting sugar to sugar acids with high yield. However, the low productivity currently limits the application of this technology. To improve productivity, the strain was optimized through improved expression of glucose dehydrogenase (GCD) and gluconate dehydrogenase (GAD). In addition, quantitative real-time RT-PCR analysis revealed the intrinsic self-regulation of GCD and GAD. Utilizing this self-regulation system, the single overexpression strain (GCD) gave an outstanding performance in the electron transfer rate and 2-ketogluconic acid (2KGA) productivity. The peak anodic current density, specific glucose uptake rate and 2KGA producing rate were 0.12 mA/cm2 , 0.27 ± 0.02 mmol/gCDW /hr and 0.25 ± 0.02 mmol/gCDW /hr, which were 327%, 477%, and 644% of the values of wild-type P. putida KT2440, respectively. This work demonstrates that expression of periplasmic dehydrogenases involved in electron transfer can significantly improve productivity in the BES.