Novel mitophagy inducer alleviates lupus nephritis by reducing myeloid cell activation and autoantigen presentation.

Lupus nephritis (LN) is one of the most severe manifestations of systemic lupus erythematosus (SLE), but its mechanism of onset remains unclear. Since impaired mitophagy has been implicated in multiple organs in SLE, we hypothesized that mitophagy dysfunction is critical in the development of LN and that pharmacologically targeting mitophagy would ameliorate this disease. Therefore, lupus-prone MRL/MpJ-Faslpr (MRL/lpr) and NZBWF1/J mice were treated with a novel mitophagy inducer, UMI-77, during their onset of LN. This treatment effectively mitigated kidney inflammation and damage as assessed by histology and flow cytometry. Furthermore, dendritic cell (DC)-T-cell coculture assay indicated that UMI-77 treatment attenuated DC function that would drive T-cell proliferation but did not directly influence the potent T-cell proliferation in lupus mice. UMI-77 also restored mitochondrial function and attenuated proinflammatory phenotypes in lupus DCs. Adoptive transfer of DCs from MRL/lpr mice augmented serum anti-dsDNA IgG, urine protein and T-cell infiltration of the kidney in MRL/MpJ mice, which could be prevented by either treating lupus donors in vivo or lupus DCs directly with UMI-77. UMI-77 also restored mitochondrial function in myeloid cells from patients with LN in vitro as evidenced by increased ATP levels. Thus, enhancing mitophagy in SLE restrains autoimmunity and limits kidney inflammation for LN development. Hence, our findings suggest targeting mitophagy as a tangible pathway to treat LN.

Alterations of B-Cell subsets in Peripheral Blood from Adult Patients with Idiopathic Membranous Nephropathy.

OBJECTIVES: Idiopathic membranous nephropathy (MN) is an autoimmune disease characterized by specific antibodies. However, the underlying mechanisms by which lymphocytes promote the development of MN remain poorly understood. This study aims to determine the changes of B-cell subsets and their clinical significance in MN patients. METHODS: We included a cohort of 21 idiopathic MN patients with new onset or a relapse, 19 healthy controls (HCs) and 10 patients with minimal change disease (MCD). Immunohistochemistry and flow cytometry were performed to assess the B-cell infiltration in renal biopsy tissues and peripheral blood, respectively. RESULTS: Idiopathic MN patients (including new-onset and relapse groups) had lower percentages of marginal-zone B (MZB) and non-switched memory B cells, and higher percentages of plasmablasts than HCs (P < 0.01). Particularly, the new-onset group had lower percentages of switched memory B cells and MZB cells, and higher percentages of Naïve B cells than HCs (P<0.05). Interestingly, the percentage of plasmablasts was significantly correlated with urine protein to creatinine ratio, serum albumin, IgG, anti-M-type phospholipase A2 receptor antibody level and age in MN patients (P < 0.05). MN with Ehrenreich-Churg stage Ⅱ-Ⅳ had a lower median percentage of MZB and non-switched memory B cells, while a higher median percentage of plasmablasts than those in MN patients with stage Ehrenreich-Churg I (P < 0.05). CONCLUSION: Idiopathic MN patients had specific changes in B-cell subsets proportions in peripheral blood. Further studies are needed to precisely determine the roles of B-cell subsets in MN.

Efficacy of Rituximab for Minimal Change Disease and Focal Segmental Glomerulosclerosis with Frequently Relapsing or Steroid-Dependent Nephrotic Syndrome in Adults: A Chinese Multicenter Retrospective Study.

INTRODUCTION: Rituximab has been proven effective and safe in pediatric patients with frequently relapsing or steroid-dependent nephrotic syndrome (FR/SDNS). We aimed to analyze the efficacy and safety of rituximab in adult FR/SDNS patients with minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS). METHODS: A retrospective cohort study at three nephrology centers in China included adult FR/SDNS patients with biopsy-proven MCD or FSGS. Primary outcomes were relapse frequency and first relapse-free survival time. Adverse events were well recorded, and logistic regression analyses were used to investigate the risk factors of relapse. RESULTS: Eighty-one patients (age, 25.0 years; interquartile range, 20.0-40.5; 67% males; 82.7% MCD) received an average rituximab dose of 1,393.8 ± 618.7 mg/2 years during the 2-year follow-up period. The relapse frequency, calculated as the ratio of relapse times to follow-up years, significantly decreased after rituximab treatment (0.04 [0.00, 0.08] vs. 1.71 [1.00, 2.45], p < 0.001). The first relapse-free survival time was 16.7 ± 8.0 months. Fifty-seven patients (70.4%) achieved cessation of corticosteroids and immunosuppressants within 3 months after the first rituximab infusion. Adverse events were mostly mild, and no severe treatment-related adverse events were observed. Low serum albumin level before rituximab and high CD56+CD16+ natural killer cell count after rituximab were independent risk factors of relapse within 2 years after rituximab treatment. CONCLUSION: Rituximab was proven an effective and safe treatment option for adult FR/SDNS patients with MCD or FSGS in maintaining disease remission and minimizing corticosteroid exposure.

Superhydrophobic Nanoparticles: An Efficiently Selective Adsorbent for Surfactant-Like Contaminants from Complex Wastewater Matrices.

Surfactant-like contaminants (SLCs) with distinctive amphiphilic structures have become a global concern in wastewater due to their toxicity and persistency. Despite extensive efforts, achieving efficient and selective SLCs removal remains challenging because of their wide range of molecular weights and complex functional group compositions. Superhydrophobic nanoparticles can potentially tackle this challenge by targeting the long oleophilic chains of SLCs. However, conventional contact angle measurements hinder hydrophobicity characterization and corresponding selectivity research because of the powder morphology of nanoparticles. Herein, the authors offered information regarding the distribution of water molecular probes in surfaces and proposed a quantitative characterization approach based on low-field nuclear magnetic resonance. Through synthesizing superhydrophobic and hydrophilic polydopamine nanospheres with similar morphologies, the selective adsorption potential of superhydrophobic nanoparticles for SLCs is systematically demonstrated. As revealed by the interaction mechanisms, the superhydrophobic surface of nanospheres increased its affinity and selectivity for SLCs adsorption by enhancing hydrophobic interactions. Superhydrophobic modification achieved ten times the adsorption capacity of sodium dodecyl benzene sulfonate, an exemplified surfactant, compared with pristine nanoparticles. By regulated self-polymerization, the superhydrophobic nanospheres are coated onto the surface of a 3D sponge and enable efficient selective SLCs adsorption from highly polluted leachate matrices with long-term stability and reusability.

Efficient De Novo Biosynthesis of Curcumin in Escherichia coli by Optimizing Pathway Modules and Increasing the Malonyl-CoA Supply.

Curcumin is a natural phenylpropanoid compound with various biological activities and is widely used in food and pharmaceuticals. A de novo curcumin biosynthetic pathway was constructed in Escherichia coli BL21(DE3). Optimization of the curcumin biosynthesis module achieved a curcumin titer of 26.8 ± 0.6 mg/L. Regulating the metabolic fluxes of the ß-oxidation pathway and fatty acid elongation cycle and blocking the endogenous malonyl-CoA consumption pathway increased the titer to 113.6 ± 7.1 mg/L. Knockout of endogenous curcumin reductase (curA) and intermediate product detoxification by heterologous expression of the solvent-resistant pump (srpB) increased the titer to 137.5 ± 3.0 mg/L. A 5 L pilot-scale fermentation, using a three-stage pH alternation strategy, increased the titer to 696.2 ± 20.9 mg/L, 178.5-fold higher than the highest curcumin titer from de novo biosynthesis previously reported, thereby laying the foundation for efficient biosynthesis of curcumin and its derivatives.

α-Lipoic acid chemistry: the past 70 years.

α-Lipoic acid (ALA) is a naturally occurring sulfur-containing fatty acid with high antioxidant activity. It is also used to treat diabetes, nerve pain, weight loss, heart disease, and primary mitochondrial disorders. Moreover, numerous therapeutic agents have been studied for managing other clinical conditions, including for anticancer, anti-HIV, anti-inflammatory, and anti-AD treatments. The medicinal importance of ALA, especially its biologically active form (R-ALA), has attracted considerable attention from synthetic chemists in industrial and academic fields. In this review, we discuss synthetic approaches to ALA and R-ALA over the past 70 years (1952 to the present), which will help medicinal chemists further develop novel routes for their synthesis.

Clinical characteristics and prognostic analysis of idiopathic inflammatory myopathy with positive anti-aminoacyl-tRNA synthetase antibodies: A single center experience.

OBJECTIVES: To identify the differences of clinical characteristics, laboratory findings, and the long-term outcomes in patients with anti-synthetase syndrome (ASS) of different anti-aminoacyl-transfer RNA synthetase antibodies. METHODS: We retrospectively enrolled 119 patients with ASS, and the clinical characteristics and laboratory findings were collected. Additionally, multivariate COX regression analysis was performed to estimate the risk factors of prognosis in patients with ASS. RESULTS: The frequency of interstitial lung disease (ILD) reached 93.3% in our cohort, of 28 (23.5%) was classified as rapidly progressive (RP)-ILD. The highest incidence of RP-ILD was 36.4% in the PL12 group of ASS patients. The ILD group was characterized by an older age, a lower prevalence of V sign, and a higher prevalence of pulmonary symptoms when contrasted with the non-ILD group. There were statistical differences of clinical significance in arthritis, myositis, mechanic's hands, triad, shawl sign, V sign, and Raynaud's phenomenon among the four subgroups (all p < .05). Additionally, the prevalence rates of arthritis, myositis, mechanic's hands, triad, and V sign in the anti-Jo1 antibody-positive group were significantly higher than anti-Jo1 antibody-negative patients with ASS (all p < .05). Multivariate Cox regression analysis showed mechanic's hands (odds ratio [OR] = 6.47, p < .001), anti-nuclear antibodies (ANA) (OR = 2.13, p = .026), ILD (OR = 10.50, p < .001), and V sign (OR = 0.30, p = .007) were independent factors affecting the prognosis of patients with ASS. The incidences of RP-ILD, arthritis, myositis, triad, mechanic's hands, and shawl sign were more frequent in the anti-Ro52 antibody-positive group than the anti-Ro52 antibody-negative patients with ASS (all p < .05). CONCLUSIONS: Patients with ASS accompanied with ILD are highly prevalent. Mechanic's hands, ANA, and ILD may be a potential biomarker for predicting a poor prognosis in patients with ASS. Additionally, the detection of the anti-Ro52 antibody provides valuable insights for managing and predicting disease progression and long-term outcomes.

Exendin-4, a GLP-1 receptor agonist, suppresses diabetic retinopathy in vivo and in vitro.

Diabetic retinopathy (DR) is a complication of diabetes and a leading cause of blindness in adults. Studies have shown that glucagon-like peptide-1 (GLP-1) exerts a protective effect on patients with DR. Here, we investigated the protective effects of Exendin-4, a GLP-1 analogue, on DR. We established a high-glucose-induced HREC cell model and an STZ-induced rat DR Model to study the effect of Exendin-4 in DR in vitro and in vivo. The qRT-PCR, CCK-8, TUNEL, western blotting, tube formation assays, and ELISA were performed. In addition, we overexpressed TGFB2 to observe whether the protective effect of Exendin-4 was reversed. Our results showed that Exendin-4 inhibited the progression of DR. Furthermore, the protective effect of Exendin-4 was suppressed in cells overexpressing TGFB2. Our findings suggest that Exendin-4 may be involved in the regulation of TGFB2 expression levels to inhibit DR. These results indicate that Exendin-4 could be an effective therapy for DR.

Promoting FADH2 Regeneration of Hydroxylation for High-Level Production of Hydroxytyrosol from Glycerol in Escherichia coli.

Hydroxytyrosol is a natural polyphenolic compound widely used in the food and drug industries. The current commercial production of hydroxytyrosol relies mainly on plant extracts, which involve long extraction cycles and various raw materials. Microbial fermentation has potential value as an environmentally friendly and low-cost method. Here, a de novo biosynthetic pathway of hydroxytyrosol has been designed and constructed in an Escherichia coli strain with released tyrosine feedback inhibition. By introduction of hpaBC from E. coli and ARO10 and ADH6 from Saccharomyces cerevisiae, the de novo biosynthesis of hydroxytyrosol was achieved. An important finding in cofactor engineering is that the introduction of L-amino acid deaminase (LAAD) promotes not only cofactor regeneration but also metabolic flow redistribution. To further enhance the hydroxylation process, different 4-hydroxyphenylacetate 3-monooxygenase (hpaB) mutants and HpaBC proteins from different sources were screened. Finally, after optimization of the carbon source, pH, and seed medium, the optimum engineered strain produced 9.87 g/L hydroxytyrosol in a 5 L bioreactor. This represents the highest titer reported to date for de novo biosynthesis of hydroxytyrosol in microorganisms.

Efficient Production of Chlorogenic Acid in Escherichia coli Via Modular Pathway and Cofactor Engineering.

Chlorogenic acid is a natural phenolic compound widely used in the food and daily chemical industries. Compared to plant extraction, microbial cell factories provide a green and sustainable production method for the production of chlorogenic acid. However, complex metabolic flux distribution and potential byproducts limited the biosynthesis of chlorogenic acid in microorganisms. A de novo biosynthesis pathway for chlorogenic acid was constructed in Escherichia coli via modular engineering. Increasing the shikimate pathway flux greatly promoted chlorogenic acid production, and the influence of pyruvate metabolism on chlorogenic acid synthesis was also explored. The supply of cofactors for the key enzymes quinate/shikimate 5-dehydrogenase (YdiB) and 4-hydroxyphenylacetate 3-monooxygenase (HpaBC) was enhanced by a cofactor regeneration system. Furthermore, mutants of YdiB were verified for chlorogenic acid production in vivo. Chlorogenic acid browning occurred when the buffer pH of the buffer exceeded 6.0, but two-stage pH control achieved a chlorogenic acid titer of 2789.2 mg/L in a 5 L fermenter, the highest reported to date. This study provided a strategy for the efficient production of chlorogenic acid from simple carbon sources.

Vascular endothelial growth factor a modified mRNA engineered cellular electrospun membrane complexes promotes mouse skin wound repair.

Artificial skin substitutes are one of the most promising areas of wound healing research; however, graft survival largely depends on how the treatment is performed. Early angiogenesis is essential for wound healing and graft survival and vascular endothelial growth factor A (VEGFA) is an important cytokine that stimulates angiogenesis. Here, we first investigated the effects of different ratios of collagen (BC) and gelatin blended with poly (l-lactide-co-caprolactone) (PLCL) on nanofibrous membranes. The Young's modulus and cell proliferation were significantly higher in the 50% BC group than that in all other groups. Then, cellular electrospun membrane complexes (CEMC) were successfully constructed from nanoscaffolds and fibroblasts extracted from human foreskin and engineered with controlled autocrine VEGFA by transfecting VEGFA modified mRNA (modRNA). Engineered CEMC significantly promoted wound healing in vivo and contributed to stable vascular network formation in the grafted area, thereby increasing the survival rate of the engineered skin. This study provides a potential solution for wound healing while establishing the value of different RNA modification methods for various engineered skins in the future, thereby advancing engineered skin development.

Energy Level Engineering in Gold Nanoclusters for Exceptionally Bright NIR Electrochemiluminescence at a Low Trigger Potential.

Electrochemiluminescence (ECL) is a widely used light output mechanism from electrochemical excitation. Understanding the intrinsic essence for ideal ECL generation remains a fundamental challenge. Here, based on the molecular orbital theory, we reported an energy level engineering strategy to regulate the ECL performance by using ligand-protected gold nanoclusters (AuNCs) as luminophores and N,N-diisopropylethylamine (DIPEA) as a coreactant. The energy level matching between the AuNCs and DIPEA effectively promoted their electron transfer reactions, thus improving the excitation efficiency and reducing the trigger potential. Simultaneously, the narrow band gap of the AuNCs further enabled enhanced emission efficiency. Using the energy level engineering theory developed here, a dual-enhanced strategy was proposed, and ß-CD-AuNCs were designed to further verify this mechanism. The ß-CD-AuNCs/DIPEA system resulted in highly stable near-infrared ECL with an unprecedented ECL efficiency (145-fold higher than that of the classic Ru(bpy)32+/tetra-n-butylammonium perchlorate system) and a low trigger potential of 0.48 V. A visual NIR-ECL based on this ECL system was successfully realized by an infrared camera. This work provides an original mechanistic understanding for designing efficient ECL systems, which promises to be a harbinger for broad applicability of this strategy for other ECL systems and ECL sensing platforms.

Enhanced adipose-derived stem cells with IGF-1-modified mRNA promote wound healing following corneal injury.

The cornea serves as an important barrier structure to the eyeball and is vulnerable to injuries, which may lead to scarring and blindness if not treated promptly. To explore an effective treatment that could achieve multi-dimensional repair of the injured cornea, the study herein innovatively combined modified mRNA (modRNA) technologies with adipose-derived mesenchymal stem cells (ADSCs) therapy, and applied IGF-1 modRNA (modIGF1)-engineered ADSCs (ADSCmodIGF1) to alkali-burned corneas in mice. The therapeutic results showed that ADSCmodIGF1 treatment could achieve the most extensive recovery of corneal morphology and function when compared not only with simple ADSCs but also IGF-1 protein eyedrops, which was reflected by the healing of corneal epithelium and limbus, the inhibition of corneal stromal fibrosis, angiogenesis and lymphangiogenesis, and also the repair of corneal nerves. In vitro experiments further proved that ADSCmodIGF1 could more significantly promote the activity of trigeminal ganglion cells and maintain the stemness of limbal stem cells than simple ADSCs, which were also essential for reconstructing corneal homeostasis. Through a combinatorial treatment regimen of cell-based therapy with mRNA technology, this study highlighted comprehensive repair in the damaged cornea and showed the outstanding application prospect in the treatment of corneal injury.

Engineering a conduction-consistent cardiac patch with graphene oxide modified butterfly wings and human pluripotent stem cell-derived cardiomyocytes.

Engineering a conduction-consistent cardiac patch has direct implications to biomedical research. However, there is difficulty in obtaining and maintaining a system that allows researchers to study physiologically relevant cardiac development, maturation, and drug screening due to the issues around inconsistent contractions of cardiomyocytes. Butterfly wings have special nanostructures arranged in parallel, which could help generate the alignment of cardiomyocytes to better mimic the natural heart tissue structure. Here, we construct a conduction-consistent human cardiac muscle patch by assembling human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on graphene oxide (GO) modified butterfly wings. We also show this system functions as a versatile model to study human cardiomyogenesis by assembling human induced pluripotent stem cell-derived cardiac progenitor cells (hiPSC-CPCs) on the GO modified butterfly wings. The GO modified butterfly wing platform facilitated the parallel orientation of hiPSC-CMs, enhanced relative maturation as well as improved conduction consistency of the cardiomyocytes. In addition, GO modified butterfly wings enhanced the proliferation and maturation characteristics of the hiPSC-CPCs. In accordance with data obtained from RNA-sequencing and gene signatures, assembling hiPSC-CPCs on GO modified butterfly wings stimulated the differentiation of the progenitors into relatively mature hiPSC-CMs. These characteristics and capabilities of GO modified butterfly wings make them an ideal platform for heart research and drug screening.

Analysis of microRNA-199a-3p expression in CD4+ T cells of systemic lupus erythematosus.

OBJECTIVES: Accumulating evidence have suggested microRNAs (miRNAs) play important roles in the pathogenesis of systemic lupus erythematosus (SLE). Here we aimed to explore aberrant expression of miRNAs in CD4+ T cells from SLE patients and their potential function in SLE pathogenesis. METHODS: First, next-generation sequencing was performed on CD4+ T cells from four SLE patients and three healthy controls (HCs). Candidate miRNAs were then validated in CD4+ T cells from 97 patients with SLE, 16 patients with rheumatoid arthritis, and 12 HCs using qRT-PCR. Then the relationship between the candidate miRNA and clinical characteristics was analyzed. Bioinformatics analysis and validation of the target genes of the candidate miRNA were performed. RESULTS: A total of 66 upregulated miRNAs and 70 downregulated miRNAs were found between SLE and normal CD4+ T cells samples. miR-199a-3p was identified significant upregulation in the CD4+ T cells of lupus patients. High expression of miR-199a-3p was correlated with several clinical characteristics including low C3 level, positive anti-dsDNA antibody, high ESR level, active lupus nephritis, and active disease activity. When distinguishing active LN from non-LN or active lupus from stable lupus, the AUCs of miR-199a-3p were 0.68 and 0.70, respectively. And the expression of miR-199a-3p, involved in JAK-STAT signaling pathway, was negatively correlated with the STAM expression in CD4+ T cells of SLE. CONCLUSION: Our study suggested a novel and promising role of miR-199a-3p in CD4+ T cells for SLE. Further studies are needed to precisely determine the function of miR-199a-3p in this disease. Key Points • Aberrant expression of miRNAs in CD4+ T cells and their potential function in SLE pathogenesis remained unclear. • miR-199a-3p in CD4+ T cells plays a novel role in the pathogenesis of SLE and serves as a potential target for SLE.

Safe and Effective Delivery of mRNA Using Modified PEI-Based Lipopolymers.

Chemically modified mRNA (modRNA) has proven to be a versatile tool for the treatment of various cancers and infectious diseases due to recent technological advancements. However, a safe and effective delivery system to overcome the complex extracellular and intracellular barriers is required in order to achieve higher therapeutic efficacy and broaden clinical applications. Here, we explored All-Fect and Leu-Fect C as novel transfection reagents derived from lipopolymers, which demonstrated excellent biocompatibility, efficient delivery capabilities, and a robust ability to escape the lysosomes. These properties directly increase mRNA stability by preventing mRNA degradation by nucleases and simultaneously promote efficient gene translation in vitro and in vivo. The modRNA delivered with lipopolymer vectors sustained effective transfection in mouse hearts following direct intramyocardial injection, as well as in major organs (liver and spleen) after systemic administration. No observable immune reactions or systemic toxicity were detected following the systemic administration of lipopolymer-mRNA complexes to additional solid organs. This study identified commercial reagents for the effective delivery of modRNA and may help facilitate the advancement of gene-based interventions involving the safe and effective delivery of nucleic acid drug substances.

Engineering a conduction-consistent cardiac patch with rGO/PLCL electrospun nanofibrous membranes and human iPSC-derived cardiomyocytes.

The healthy human heart has special directional arrangement of cardiomyocytes and a unique electrical conduction system, which is critical for the maintenance of effective contractions. The precise arrangement of cardiomyocytes (CMs) along with conduction consistency between CMs is essential for enhancing the physiological accuracy of in vitro cardiac model systems. Here, we prepared aligned electrospun rGO/PLCL membranes using electrospinning technology to mimic the natural heart structure. The physical, chemical and biocompatible properties of the membranes were rigorously tested. We next assembled human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on electrospun rGO/PLCL membranes in order to construct a myocardial muscle patch. The conduction consistency of cardiomyocytes on the patches were carefully recorded. We found that cells cultivated on the electrospun rGO/PLCL fibers presented with an ordered and arranged structure, excellent mechanical properties, oxidation resistance and effective guidance. The addition of rGO was found to be beneficial for the maturation and synchronous electrical conductivity of hiPSC-CMs within the cardiac patch. This study verified the possibility of using conduction-consistent cardiac patches to enhance drug screening and disease modeling applications. Implementation of such a system could one day lead to in vivo cardiac repair applications.

Generalized Granuloma Annulare Associated with Sjogren's Syndrome: A Case Report.

Granuloma annulare (GA) is an uncommon disease in dermatology. Here, we report a case of generalized GA combined with Sjogren's syndrome (SS) in a 65-year-old woman. To our knowledge, generalized GA combined with SS has not been reported before.

A Review of the Release Profiles and Efficacies of Chemotherapy Drug-Loaded Electrospun Membranes.

Electrospun fibrous membranes loaded with chemotherapy drugs have been broadly studied, many of which have had promising data demonstrating therapeutic effects on cancer cell inhibition, tumor size reduction, the life extension of tumor-bearing animals, and more. Nevertheless, their drug release profiles are difficult to predict since their degradation pattern varies with crystalline polymers. In addition, there is room for improving their release performances, optimizing the release patterns, and achieving better therapeutic outcomes. In this review, the key factors affecting electrospun membrane drug release profiles have been systematically reviewed. Case studies of the release profiles of typical chemotherapy drugs are carried out to determine the preferred polymer choices and techniques to achieve the expected prolonged or enhanced release profiles. The therapeutic effects of these electrospun, chemo-drug-loaded membranes are also discussed. This review aims to assist in the design of future drug-loaded electrospun materials to achieve preferred release profiles with enhanced therapeutic efficacies.

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.