Cardiac troponin T N-domain variant destabilizes the actin interface resulting in disturbed myofilament function.

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca2+ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca2+ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament's relaxed state resulting in an increased number of crossbridges during Ca2+ activation. Additionally, in the low Ca2+-relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca2+, and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.

An interaction between eIF4A3 and eIF3g drives the internal initiation of translation.

An RNA structure or modified RNA sequences can provide a platform for ribosome loading and internal translation initiation. The functional significance of internal translation has recently been highlighted by the discovery that a subset of circular RNAs (circRNAs) is internally translated. However, the molecular mechanisms underlying the internal initiation of translation in circRNAs remain unclear. Here, we identify eIF3g (a subunit of eIF3 complex) as a binding partner of eIF4A3, a core component of the exon-junction complex (EJC) that is deposited onto spliced mRNAs and plays multiple roles in the regulation of gene expression. The direct interaction between eIF4A3-eIF3g serves as a molecular linker between the eIF4A3 and eIF3 complex, thereby facilitating internal ribosomal entry. Protein synthesis from in vitro-synthesized circRNA demonstrates eIF4A3-driven internal translation, which relies on the eIF4A3-eIF3g interaction. Furthermore, our transcriptome-wide analysis shows that efficient polysomal association of endogenous circRNAs requires eIF4A3. Notably, a subset of endogenous circRNAs can express a full-length intact protein, such as ß-catenin, in an eIF4A3-dependent manner. Collectively, our results expand the understanding of the protein-coding potential of the human transcriptome, including circRNAs.

Short-term aerobic exercise prevents development of glucocorticoid myopathic features in aged skeletal muscle in a sex-dependent manner.

Older adults are vulnerable to glucocorticoid-induced muscle atrophy and weakness, with sex potentially influencing their susceptibility to those effects. Aerobic exercise can reduce glucocorticoid-induced muscle atrophy in young rodents. However, it is unknown whether aerobic exercise can prevent glucocorticoid myopathy in aged muscle. The objectives of this study were to define the extent to which sex influences the development of glucocorticoid myopathy in aged muscle, and to determine the extent to which aerobic exercise training protects against myopathy development. Twenty-four-month-old female (n = 30) and male (n = 33) mice were randomized to either sedentary or aerobic exercise groups. Within their respective groups, mice were randomized to either daily treatment with dexamethasone (DEX) or saline. Upon completing treatments, the contractile properties of the triceps surae complex were assessed in situ. DEX marginally lowered muscle mass and soluble protein content in both sexes, which was attenuated by aerobic exercise only in females. DEX increased sub-tetanic force and rate of force development only in females, which was not influenced by aerobic exercise. Muscle fatigue was higher in both sexes following DEX, but aerobic exercise prevented fatigue induction only in females. The sex-specific differences to muscle function in response to DEX treatment coincided with sex-specific changes to the content of proteins related to calcium handling, mitochondrial quality control, reactive oxygen species production, and glucocorticoid receptor in muscle. These findings define several important sexually dimorphic changes to aged skeletal muscle physiology in response to glucocorticoid treatment and define the capacity of short-term aerobic exercise to protect against those changes. KEY POINTS: There are sexually dimorphic effects of glucocorticoids on aged skeletal muscle physiology. Glucocorticoid-induced changes to aged muscle contractile properties coincide with sex-specific differences in the content of calcium handling proteins. Aerobic exercise prevents glucocorticoid-induced fatigue only in aged females and coincides with differences in the content of mitochondrial quality control proteins and glucocorticoid receptors.

Transcriptional and translational flux optimization at the key regulatory node for enhanced production of naringenin using acetate in engineered Escherichia coli.

As a key molecular scaffold for various flavonoids, naringenin is a value-added chemical with broad pharmaceutical applicability. For efficient production of naringenin from acetate, it is crucial to precisely regulate the carbon flux of the oxaloacetate-phosphoenolpyruvate (OAA-PEP) regulatory node through appropriate pckA expression control, as excessive overexpression of pckA can cause extensive loss of OAA and metabolic imbalance. However, considering the critical impact of pckA on naringenin biosynthesis, the conventional strategy of transcriptional regulation of gene expression is limited in its ability to cover the large and balanced solution space. To overcome this hurdle, in this study, pckA expression was fine-tuned at both the transcriptional and translational levels in a combinatorial expression library for the precise exploration of optimal naringenin production from acetate. Additionally, we identified the effects of regulating pckA expression by validating the correlation between phosphoenolpyruvate kinase (PCK) activity and naringenin production. As a result, the flux-optimized strain exhibited a 49.8-fold increase compared with the unoptimized strain, producing 122.12 mg/L of naringenin. Collectively, this study demonstrated the significance of transcriptional and translational flux rebalancing at the key regulatory node, proposing a pivotal metabolic engineering strategy for the biosynthesis of various flavonoids derived from naringenin using acetate. ONE-SENTENCE SUMMARY: In this study, transcriptional and translational regulation of pckA expression at the crucial regulatory node was conducted to optimize naringenin biosynthesis using acetate in E. coli.

Evaluation of the sensitivity of a measles diagnostic real-time RT-PCR assay incorporating recently observed priming mismatch variants, 2024.

We investigated a variant of measles virus that encodes three mismatches to the reverse priming site for a widely used diagnostic real-time RT-PCR assay; reduction of sensitivity was hypothesised. We examined performance of the assay in context of the variant using in silico data, synthetic RNA templates and clinical specimens. Sensitivity was reduced observed at low copy numbers for templates encoding the variant sequence. We designed and tested an alternate priming strategy, rescuing the sensitivity of the assay.

Synthesis, Antiproliferative Activity and Molecular Docking Analysis of Both Enantiomerically Pure Decursin Derivatives as Anticancer Agents.

Using (S)-decursinol isolated from root of Angelica gigas Nakai (AGN), we semi-synthesized and evaluated a series of both enantiomerically pure decursin derivatives for their antiproliferative activities against A549 human lung cancer cells. All synthesized compounds showed a broad spectrum of inhibitory activities against the growth of A549 cells. Especially, compound (S)-2d with (E)-(furan-3-yl)acryloyl group showed the most potent activity (IC50: 14.03 µM) against A549 cancer cells as compared with the reference compound, decursin (IC50: 43.55 µM) and its enantiomer, (R)-2d (IC50: 151.59 µM). Western blotting assays indicated that (S)-2d more strongly inhibited Janus kinase 1 (JAK1) and signal transducer and activator of transcription activation 3 (STAT3) phosphorylation than decursin in a dose-dependent manner, while having no effect on CXCR7 overexpression and total STAT3 level. In addition, (S)-2d induced cell cycle arrest at G1 phase and subsequent apoptotic cell death in A549 cancer cells. Our combined analysis of molecular docking studies and biological data suggests that the inhibition of JAK1 with (S)-2d resulted in loss of STAT3 phosphorylation and inhibition of cell growth in A549 cancer cells. These overall results strongly suggest that (S)-2d (MRC-D-004) as a novel JAK1 inhibitor may have therapeutic potential in the treatment of A549 human lung cancers by targeting the JAK1/STAT3 signaling pathway.

Riboswitch-guided chalcone synthase engineering and metabolic flux optimization for enhanced production of flavonoids.

Flavonoids are a group of secondary metabolites from plants that have received attention as high value-added pharmacological substances. Recently, a robust and efficient bioprocess using recombinant microbes has emerged as a promising approach to supply flavonoids. In the flavonoid biosynthetic pathway, the rate of chalcone synthesis, the first committed step, is a major bottleneck. However, chalcone synthase (CHS) engineering was difficult because of high-level conservation and the absence of effective screening tools, which are limited to overexpression or homolog-based combinatorial strategies. Furthermore, it is necessary to precisely regulate the metabolic flux for the optimum availability of malonyl-CoA, a substrate of chalcone synthesis. In this study, we engineered CHS and optimized malonyl-CoA availability to establish a platform strain for naringenin production, a key molecular scaffold for various flavonoids. First, we engineered CHS through synthetic riboswitch-based high-throughput screening of rationally designed mutant libraries. Consequently, the catalytic efficiency (kcat/Km) of the optimized CHS enzyme was 62% higher than that of the wild-type enzyme. In addition to CHS engineering, we designed genetic circuits using transcriptional repressors to fine-tune the malonyl-CoA availability. The best mutant with synergistic effects of the engineered CHS and the optimized genetic circuit produced 98.71 mg/L naringenin (12.57 mg naringenin/g glycerol), which is the highest naringenin concentration and yield from glycerol in similar culture conditions reported to date, a 2.5-fold increase compared to the parental strain. Overall, this study provides an effective strategy for efficient production of flavonoids.

Effects of daily versus weekly teriparatide for medication-related osteonecrosis of the jaw: A case-control study.

INTRODUCTION: Medication-related osteonecrosis of the jaw (MRONJ) is uncommon but can result in severe destruction of the jaw. This case-control study investigated the therapeutic effects of daily or weekly administration of teriparatide in the management of MRONJ using a cohort for osteonecrosis of the jaw. METHODS: Patients who were diagnosed with MRONJ and consented to teriparatide administration were assigned either to a group of daily injection or of weekly injection and completed a 4-week course of injection preoperatively and at least an 8-week course postoperatively. The control group received either the intraoperative rhBMP treatment (CG_BMP) or no additional perioperative treatment (CG_noBMP). The state of MRONJ was evaluated 2 months (T1) and 6 months (T2) postoperatively for all participants. RESULTS: Either group of daily injection (8.35 weeks ± 1.58; n = 17) or weekly injection (9.17 ± 3.79; n = 12) showed significantly faster healing than those of CG_BMP (14.40 ± 6.08; n = 25) or CG_noBMP (15.79 ± 9.79; n = 39). MRONJ was resolved completely in 24 out of 29 participants who completed the course of teriparatide injections, whereas 46.9% of CG showed delayed resolution. Multiple regression analysis indicated 7.50 times (95% CI, 1.77-31.82) more likelihood of complete resolution of MRONJ for participants with teriparatide injections. CONCLUSION: A course of daily or weekly administration of teriparatide injections may improve treatment outcomes for patients with MRONJ.

Translation mediated by the nuclear cap-binding complex is confined to the perinuclear region via a CTIF-DDX19B interaction.

Newly synthesized mRNA is translated during its export through the nuclear pore complex, when its 5'-cap structure is still bound by the nuclear cap-binding complex (CBC), a heterodimer of cap-binding protein (CBP) 80 and CBP20. Despite its critical role in mRNA surveillance, the mechanism by which CBC-dependent translation (CT) is regulated remains unknown. Here, we demonstrate that the CT initiation factor (CTIF) is tethered in a translationally incompetent manner to the perinuclear region by the DEAD-box helicase 19B (DDX19B). DDX19B hands over CTIF to CBP80, which is associated with the 5'-cap of a newly exported mRNA. The resulting CBP80-CTIF complex then initiates CT in the perinuclear region. We also show that impeding the interaction between CTIF and DDX19B leads to uncontrolled CT throughout the cytosol, consequently dysregulating nonsense-mediated mRNA decay. Altogether, our data provide molecular evidence supporting the importance of tight control of local translation in the perinuclear region.

The pioneer round of translation ensures proper targeting of ER and mitochondrial proteins.

The pioneer (or first) round of translation of newly synthesized mRNAs is largely mediated by a nuclear cap-binding complex (CBC). In a transcriptome-wide analysis of polysome-associated and CBC-bound transcripts, we identify RN7SL1, a noncoding RNA component of a signal recognition particle (SRP), as an interaction partner of the CBC. The direct CBC-SRP interaction safeguards against abnormal expression of polypeptides from a ribosome-nascent chain complex (RNC)-SRP complex until the latter is properly delivered to the endoplasmic reticulum. Failure of this surveillance causes abnormal expression of misfolded proteins at inappropriate intracellular locations, leading to a cytosolic stress response. This surveillance pathway also blocks protein synthesis through RNC-SRP misassembled on an mRNA encoding a mitochondrial protein. Thus, our results reveal a surveillance pathway in which pioneer translation ensures proper targeting of endoplasmic reticulum and mitochondrial proteins.

Oat (Avena sativa L.) Sprouts Restore Skin Barrier Function by Modulating the Expression of the Epidermal Differentiation Complex in Models of Skin Irritation.

Oats (Avena sativa L.) are used as therapeutic plants, particularly in dermatology. Despite numerous studies on their skin moisturization, anti-inflammation, and antioxidation effects, the precise molecular mechanisms of these effects are only partially understood. In this study, the efficacy of oat sprouts in the treatment of allergic contact dermatitis (ACD) was investigated, and their specific phytoconstituents and exact mechanisms of action were identified. In the in vivo ACD model, by stimulating the mitogen-activated protein kinase signaling pathway, oat sprouts increased the expression levels of proteins associated with skin barrier formation, which are produced during the differentiation of keratinocytes. In addition, in a lipopolysaccharide-induced skin irritation model using HaCaT, steroidal saponins (avenacoside B and 26-deglucoavenacoside B) and a flavonoid (isovitexin-2-o-arabinoside) of oat sprouts regulated the genetic expression of the same proteins located on the adjacent locus of human chromosomes known as the epidermal differentiation complex (EDC). Furthermore, oat sprouts showed immunomodulatory functions. These findings suggest the potential for expanding the use of oat sprouts as a treatment option for various diseases characterized by skin barrier disruption.

Sarcospan Deficiency Increases Oxidative Stress and Arrhythmias in Hearts after Acute Ischemia-Reperfusion Injury.

The protein sarcospan (SSPN) is an integral member of the dystrophin-glycoprotein complex (DGC) and has been shown to be important in the heart during the development and the response to acute stress. In this study, we investigated the role of SSPN in the cardiac response to acute ischemia-reperfusion (IR) injury in SSPN-deficient (SSPN-/-) mice. First, the hemodynamic response of SSPN-/- mice was tested and was similar to SSPN+/+ (wild-type) mice after isoproterenol injection. Using the in situ Langendorff perfusion method, SSPN-/- hearts were subjected to IR injury and found to have increased infarct size and arrhythmia susceptibility compared to SSPN+/+. Ca2+ handling was assessed in single cardiomyocytes and diastolic Ca2+ levels were increased after acute ß-AR stimulation in SSPN+/+ but not SSPN-/-. It was also found that SSPN-/- cardiomyocytes had reduced Ca2+ SR content compared to SSPN+/+ but similar SR Ca2+ release. Next, we used qRT-PCR to examine gene expression of Ca2+ handling proteins after acute IR injury. SSPN-/- hearts showed a significant decrease in L-type Ca2+ channels and a significant increase in Ca2+ release channel (RyR2) expression. Interestingly, under oxidizing conditions reminiscent of IR, SSPN-/- cardiomyocytes, had increased H2O2-induced reactive oxygen species production compared to SSPN+/+. Examination of oxidative stress proteins indicated that NADPH oxidase 4 and oxidized CAMKII were increased in SSPN-/- hearts after acute IR injury. These results suggest that increased arrhythmia susceptibility in SSPN-/- hearts post-IR injury may arise from alterations in Ca2+ handling and a reduced capacity to regulate oxidative stress pathways.

Therapeutic Efficacy of YM155 to Regulate an Epigenetic Enzyme in Major Subtypes of RCC.

Renal cell carcinoma (RCC) is the most common type of kidney cancer and includes more than 10 subtypes. Compared to the intensively investigated clear cell RCC (ccRCC), the underlying mechanisms and treatment options of other subtypes, including papillary RCC (pRCC) and chromogenic RCC (chRCC), are limited. In this study, we analyzed the public databases for ccRCC, pRCC, and chRCC and found that BIRC5 was commonly overexpressed in a large cohort of pRCC and chRCC patients as well as ccRCC and was closely related to the progression of RCCs. We investigated the potential of BIRC5 as a therapeutic target for these three types of RCCs. Loss and gain of function studies showed the critical role of BIRC5 in cancer growth. YM155, a BIRC5 inhibitor, induced a potent tumor-suppressive effect in the three types of RCC cells and xenograft models. To determine the mechanism underlying the anti-tumor effects of YM155, we examined epigenetic modifications in the BIRC5 promoter and found that histone H3 lysine 27 acetylation (H3K27Ac) was highly enriched on the promoter region of BIRC5. Chromatin-immunoprecipitation analysis revealed that H3K27Ac enrichment was significantly decreased by YM155. Immunohistochemistry of xenografted tissue showed that overexpression of BIRC5 plays an important role in malignancy in RCC. Furthermore, high expression of P300 was significantly associated with the progression of RCC. Our findings demonstrate the P300-H3K27Ac-BIRC5 cascade in three types of RCC and provide a therapeutic path for future research on RCC.

Evaluation of wound healing effects of micronized acellular dermal matrix in combination with negative pressure wound therapy: In vivo study.

Acellular dermal matrix (ADM) grafts can provide coverage for full-thickness skin defects and substitute for dermal defects. We tested the effectiveness of micronized ADM (mADM) as a dressing material, combined with negative pressure wound therapy (NPWT), for managing superficial wounds. We compared the wound healing effect of mADM in combination with NPWT with those of gelatin and mADM applied with a foam dressing. These therapeutic materials were applied to 36 cm2 excisional wounds in a porcine full-thickness skin defect model. Wound healing kinetics and new tissue formation were assessed 10 days after the initial treatment by measuring the wound area. Collagen deposition and neovascularization were histologically evaluated. Compared with the other two groups, mADM plus NPWT combination group had a significantly larger wound area at the baseline (P = .0040), but the smallest on the 7th day (P = .0093). In addition, collagen formation and neovascularization were more histologically promoted than in the other two groups. mADM showed better results than the gelatin group but less collagen and revascularization than the combination group, and there was no significant difference in wound area. Our results show that the combination of mADM and NPWT has a synergistic wound healing effect.

Cell Proliferation Receptor-Enhanced 3D High-Throughput Screening Model for Optimized Drug Efficacy Evaluation in Breast Cancer Cells.

A higher correlation of epidermal growth factor receptor (EGFR)-targeting drugs has been reported with the use of the cell proliferation receptor-enhanced three-dimensional high-throughput screening model (CPRE 3D-HTS model) compared with two-dimensional (2D) cell-based HTS. A greater expression of differential human EGFR 2 (HER2) protein between HER2-positive and HER2-negative cell lines was observed in breast cancer (BC) cell lines cultured using the CPRE 3D-HTS model compared with 2D-cultured cells. When using 2D-cultured cells, properties such as the expression of the cell proliferation receptor are lost as the cells attach to the bottom of the well plate. In an effort to solve this problem, the CPRE 3D-HTS model expressing high cell proliferation receptors was optimized by the selection of alginate as the extracellular matrix. Results from the use of the CPRE 3D-HTS model showed higher drug resistance with increased expression of drug resistance-related proteins. Of particular interest, a higher correlation of HER2-targeted drugs was observed with the use of the CPRE 3D-HTS model. In order to validate this higher correlation of target drugs observed in the CPRE 3D-HTS model, the results of Western blot analysis and high content imaging analysis were analyzed, which confirmed that 3D-cultured BC cell lines showed a greater difference in the expression of HER2-positive and HER2-negative BC cell lines than 2D-cultured cells. Thus, the use of CPRE 3D-HTS using a 384-pillar plate resulted in increased accuracy when screening HER2-targeted drugs in BC, and it is a very useful platform for analyzing the efficacy of targeted drugs by enhancing the expression of HER2.

Single polysome analysis of mRNP.

Eukaryotic translation is a complex process that involves the interplay of various translation factors to convert genetic information into a specific amino acid chain. According to an elegant model of eukaryotic translation initiation, the 3' poly(A) tail of an mRNA, which is occupied by poly(A)-binding proteins (PABPs), communicates with the 5'-cap bound by eIF4E to enhance translation. Although the circularization of mRNA resulting from the communication is widely understood, it has yet to be directly observed. To explore mRNA circularization in translation, we analyzed the level of colocalization of eIF4E, eIF4G, and PABP on individual mRNAs in polysomal and subpolysomal fractions using single polysome analysis. Our results show that the three tested proteins barely coexist in mRNA in either polysomal or subpolysomal fractions, implying that the closed-loop structure generated by the communication between eIF4E, eIF4G, and PAPB may be transient during translation.

Targeted PMP22 TATA-box editing by CRISPR/Cas9 reduces demyelinating neuropathy of Charcot-Marie-Tooth disease type 1A in mice.

Charcot-Marie-Tooth 1A (CMT1A) is the most common inherited neuropathy without a known therapy, which is caused by a 1.4 Mb duplication on human chromosome 17, which includes the gene encoding the peripheral myelin protein of 22 kDa (PMP22). Overexpressed PMP22 protein from its gene duplication is thought to cause demyelination and subsequently axonal degeneration in the peripheral nervous system (PNS). Here, we targeted TATA-box of human PMP22 promoter to normalize overexpressed PMP22 level in C22 mice, a mouse model of CMT1A harboring multiple copies of human PMP22. Direct local intraneural delivery of CRISPR/Cas9 designed to target TATA-box of PMP22 before the onset of disease, downregulates gene expression of PMP22 and preserves both myelin and axons. Notably, the same approach was effective in partial rescue of demyelination even after the onset of disease. Collectively, our data present a proof-of-concept that CRISPR/Cas9-mediated targeting of TATA-box can be utilized to treat CMT1A.

Evaluation of Commercial Corrosion Sensors for Real-Time Monitoring of Pipe Wall Thickness under Various Operational Conditions.

In this study, we investigated the performance and reliability of commercial corrosion sensors for monitoring the integrity of piping systems in various fluid environments as an alternative to ultrasonic transducers. To this end, we investigated pipes' wall-thinning using commercial electrical resistance (ER), linear polarization resistance (LPR), and ultrasonic transducer (UT) sensors under various operating environments. A pilot-scale closed-loop test bed was built to simulate a real pipeline flow situation, from which the sensor data were collected and analyzed. Experimental results indicate that, in the case of the LPR sensor, it is challenging to accurately measure the corrosion rate when a specific measure exceeds the threshold in a severe corrosion environment. In contrast, the ER sensor could measure metal loss under all conditions and reflect the corresponding characteristics. The metal loss (about 0.25 mm) of the real pipe after the experiment was confirmed to be equal to the metal loss (0.254 mm) measured by the sensor. Furthermore, the regression analysis revealed a high correlation between the results obtained from the ER and UT sensors. Thus, evaluating the remaining thickness of the piping system using the commercial ER sensor is deemed to be effective and reliable.

Factors and Pathways Modulating Endothelial Cell Senescence in Vascular Aging.

Aging causes a progressive decline in the structure and function of organs. With advancing age, an accumulation of senescent endothelial cells (ECs) contributes to the risk of developing vascular dysfunction and cardiovascular diseases, including hypertension, diabetes, atherosclerosis, and neurodegeneration. Senescent ECs undergo phenotypic changes that alter the pattern of expressed proteins, as well as their morphologies and functions, and have been linked to vascular impairments, such as aortic stiffness, enhanced inflammation, and dysregulated vascular tone. Numerous molecules and pathways, including sirtuins, Klotho, RAAS, IGFBP, NRF2, and mTOR, have been implicated in promoting EC senescence. This review summarizes the molecular players and signaling pathways driving EC senescence and identifies targets with possible therapeutic value in age-related vascular diseases.

Multi-level rebalancing of the naringenin pathway using riboswitch-guided high-throughput screening.

Recombinant microbes have emerged as promising alternatives to natural sources of naringenin-a key molecular scaffold for flavonoids. In recombinant strains, expression levels of the pathway genes should be optimized at both transcription and the translation stages to precisely allocate cellular resources and maximize metabolite production. However, the optimization of the expression levels of naringenin generally relies on evaluating a small number of variants from libraries constructed by varying transcription efficiency only. In this study, we introduce a systematic strategy for the multi-level optimization of biosynthetic pathways. We constructed a multi-level combinatorial library covering both transcription and translation stages using synthetic T7 promoter variants and computationally designed 5'-untranslated regions. Furthermore, we identified improved strains through high-throughput screening based on a synthetic naringenin riboswitch. The most-optimized strain obtained using this approach exhibited a 3-fold increase in naringenin production, compared with the parental strain in which only the transcription efficiency was modulated. Furthermore, in a fed-batch bioreactor, the optimized strain produced 260.2 mg/L naringenin, which is the highest concentration reported to date using glycerol and p-coumaric acid as substrates. Collectively, this work provides an efficient strategy for the expression optimization of the biosynthetic pathways.