A Multi-Omics, Machine Learning-Aware, Genome-Wide Metabolic Model of Bacillus Subtilis Refines the Gene Expression and Cell Growth Prediction.

Given the extensive heterogeneity and variability, understanding cellular functions and regulatory mechanisms through the analysis of multi-omics datasets becomes extremely challenging. Here, a comprehensive modeling framework of multi-omics machine learning and metabolic network models are proposed that covers various cellular biological processes across multiple scales. This model on an extensive normalized compendium of Bacillus subtilis is validated, which encompasses gene expression data from environmental perturbations, transcriptional regulation, signal transduction, protein translation, and growth measurements. Comparison with high-throughput experimental data shows that EM_iBsu1209-ME, constructed on this basis, can accurately predict the expression of 605 genes and the synthesis of 23 metabolites under different conditions. This study paves the way for the construction of comprehensive biological databases and high-performance multi-omics metabolic models to achieve accurate predictive analysis in exploring complex mechanisms of cell genotypes and phenotypes.

Integrating Deep Learning and Synthetic Biology: A Co-Design Approach for Enhancing Gene Expression via N-Terminal Coding Sequences.

N-terminal coding sequence (NCS) influences gene expression by impacting the translation initiation rate. The NCS optimization problem is to find an NCS that maximizes gene expression. The problem is important in genetic engineering. However, current methods for NCS optimization such as rational design and statistics-guided approaches are labor-intensive yield only relatively small improvements. This paper introduces a deep learning/synthetic biology codesigned few-shot training workflow for NCS optimization. Our method utilizes k-nearest encoding followed by word2vec to encode the NCS, then performs feature extraction using attention mechanisms, before constructing a time-series network for predicting gene expression intensity, and finally a direct search algorithm identifies the optimal NCS with limited training data. We took green fluorescent protein (GFP) expressed by Bacillus subtilis as a reporting protein of NCSs, and employed the fluorescence enhancement factor as the metric of NCS optimization. Within just six iterative experiments, our model generated an NCS (MLD62) that increased average GFP expression by 5.41-fold, outperforming the state-of-the-art NCS designs. Extending our findings beyond GFP, we showed that our engineered NCS (MLD62) can effectively boost the production of N-acetylneuraminic acid by enhancing the expression of the crucial rate-limiting GNA1 gene, demonstrating its practical utility. We have open-sourced our NCS expression database and experimental procedures for public use.

A synergistic bioelectrochemical-photocatalytic system for efficient uranium removal and recovery.

Bioelectrochemical system (BES) is a promising technology for uranium recovery, which also enables simultaneous electricity generation. However, the bioelectrochemical recovery of uranium is hindered by its slow process due to the low reduction potential provided by microorganisms. Herein, we developed an innovative bioelectrochemical-photocatalytic system (BEPS) that combines the advantages of BES and photocatalysis, achieving enhanced uranium removal and recovery. The photogenerated electrons in BEPS possess a more negative reduction potential and stronger reduction capability than microbial electrons in BES, significantly accelerating uranium reduction and deposition on the electrode surface. Moreover, the electrons from the bioanode combine with photogenerated holes through the external circuit, effectively inhibiting the recombination of charge carriers. The BEPS significantly enhances uranium removal efficiency, kinetic, and electricity generation through a synergistic coupling mechanism between the bioanode and photocathode. Notably, the UO2 deposited on the electrode surface exhibited a recovery efficiency of 98.21 ± 1.37%, and the regenerated electrode sustained its photoelectric response and uranium removal capabilities. Our findings highlight the potential of the BEPS as an effective technology for uranium recovery and electricity generation.

Enhancing cellular and enzymatic properties through in vivo continuous evolution.

Directed evolution seeks to evolve target genes at a rate far exceeding the natural mutation rate, thereby endowing cellular and enzymatic properties with desired traits. In vivo continuous directed evolution achieves these purposes by generating libraries within living cells, enabling a continuous cycle of mutant generation and selection, enhancing the exploration of gene variants. Continuous evolution has become powerful tools for unraveling evolution mechanism and improving cellular and enzymatic properties. This review categorizes current continuous evolution into three distinct classes: non-targeted chromosomal, targeted chromosomal, and extra-chromosomal hypermutation approaches. It also compares various continuous evolution strategies based on different principles, providing a reference for selecting suitable methods for specific evolutionary goals. Furthermore, this review discusses the two primary limitations for further widespread application of in vivo continuous evolution, which are lack of general applicability and insufficient mutagenic capability. We envision that developing generally applicable mutagenic components and methods to enhance mutation rates for in vivo continuous evolution are promising future directions for wide range applications of continuous evolution.

De novo engineering of programmable and multi-functional biomolecular condensates for controlled biosynthesis.

There is a growing interest in the creation of engineered condensates formed via liquid-liquid phase separation (LLPS) to exert precise cellular control in prokaryotes. However, de novo design of cellular condensates to control metabolic flux or protein translation remains a challenge. Here, we present a synthetic condensate platform, generated through the incorporation of artificial, disordered proteins to realize specific functions in Bacillus subtilis. To achieve this, the "stacking blocks" strategy is developed to rationally design a series of LLPS-promoting proteins for programming condensates. Through the targeted recruitment of biomolecules, our investigation demonstrates that cellular condensates effectively sequester biosynthetic pathways. We successfully harness this capability to enhance the biosynthesis of 2'-fucosyllactose by 123.3%. Furthermore, we find that condensates can enhance the translation specificity of tailored enzyme fourfold, and can increase N-acetylmannosamine titer by 75.0%. Collectively, these results lay the foundation for the design of engineered condensates endowed with multifunctional capacities.

Combined structure-based virtual screening and machine learning approach for the identification of potential dual inhibitors of ACC and DGAT2.

Acetyl-coenzyme A carboxylase (ACC) and diacylglycerol acyltransferase 2 (DGAT2) are recognized as potential therapeutic targets for nonalcoholic fatty liver disease (NAFLD). Inhibitors targeting ACC and DGAT2 have exhibited the capacity to reduce hepatic fat in individuals afflicted with NAFLD. However, there are no reports of dual inhibitors targeting ACC and DGAT2 for the treatment of NAFLD. Here, we aimed to identify potential dual inhibitors of ACC and DGAT2 using an integrated in silico approach. Machine learning-based virtual screening of commercial molecule databases yielded 395,729 hits, which were subsequently subjected to molecular docking aimed at both the ACC and DGAT2 binding sites. Based on the docking scores, nine compounds exhibited robust interactions with critical residues of both ACC and DGAT2, displaying favorable drug-like features. Molecular dynamics simulations (MDs) unveiled the substantial impact of these compounds on the conformational dynamics of the proteins. Furthermore, binding free energy assessments highlighted the notable binding affinities of specific compounds (V003-8107, G340-0503, Y200-1700, E999-1199, V003-6429, V025-4981, V006-1474, V025-0499, and V021-8916) to ACC and DGAT2. The compounds proposed in this study, identified using a multifaceted computational strategy, warrant experimental validation as potential dual inhibitors of ACC and DGAT2, with implications for the future development of novel drugs targeting NAFLD.

Modification of the Endoplasmic Reticulum to Enhance Ovalbumin Secretion in Saccharomyces cerevisiae.

Ovalbumin (OVA) is a high-quality protein for humans. Modifying microorganisms to produce proteins offers a solution to potential food protein shortages. In this study, OVA was expressed in Saccharomyces cerevisiae. Initially, screening signal peptides led to extracellular OVA reaching 3.4 mg/L using the INU1 signal peptide. Coexpressing Kar2 and PDI increased OVA production to 5.1 mg/L. Optimizing the expression levels of regulators OPI1, INO2, and INO4 expanded the endoplasmic reticulum membrane, raising yield to 5.5 mg/L. Combining both strategies increased OVA production to 6.2 mg/L, 82% higher than control. This strategy also enhanced secretion of other proteins. Finally, fed-batch fermentation in a 3-L bioreactor significantly boosted OVA production to 116.3 mg/L. This study provides insights for the heterologous synthesis of other high-quality proteins for future food applications.

Evaluation of the efficacy of chlorhexidine-alcohol vs. aqueous/alcoholic iodine solutions for the prevention of surgical site infections: a systematic review and meta-analysis.

BACKGROUND: Surgical site infection (SSI) is the prevailing complication that occurs after surgery and significantly escalates healthcare expenses. Published meta-analyses and international standards vary in their recommendations for the most effective preoperative skin antiseptic solution and concentration. OBJECTIVE: The aim of this systematic review and meta-analysis is to assess the effectiveness of Chlorhexidine-alcohol compared to Aqueous/alcoholic iodine solutions in preventing post-operative surgical site infections. METHODS: A systematic search was conducted using four electronic databases (PubMed, EMBASE, Scopus, and Cochrane Library) to select publications published in peer-reviewed journals. The risk ratio (RR) was calculated, along with their 95% confidence intervals. We assessed heterogeneity using Cochrane Q and I2 statistics and the appropriate P-value. The analysis used RevMan 5.4. RESULTS: The current meta-analysis includes 14 Randomized controlled trials (RCTs) comparing either 2-2.5% chlorhexidine alcohol with aqueous/alcoholic iodine. It was demonstrated that the CAG-using group had an overall lower incidence of post-operative surgical site infections compared to the iodine-using group (RR=0.30, 95% CI=0.20 to 0.46, I2=95%, P<0.00001). It exhibits comparable efficacy across various surgical procedures, as evidenced by its RR of 0.25 [95% CI 0.15 to 0.41], I2=51%, and P<0.0001 for general surgery, RR=0.47 [95% CI 0.32 to 0.67], I2=82%, P=0.0002 for caesarean section and RR of 0.47 [95% CI 0.34 to 0.65], I2=76% and P<0.00001 for additional surgical procedures, including neurosurgery, orthopedic surgery etc. CONCLUSION: This meta-analysis suggests using either 2·0-2·5% Chlorhexidine in alcohol instead of aqueous, alcoholic iodine to prevent SSIs in adult patients undergoing surgery. Chlorhexidine in alcohol worked effectively for general surgery, cesarean sections, and other surgeries. Thus, preoperative skin cleansing with Chlorhexidine alcohol minimizes postoperative SSIs and bacterial colonization in diverse procedures.

Circular RNA Pum_0014 Targets miR-146a-5p/NF2 Axis to Regulate VEGF/PAK1 Pathway and Reduce H2O2-induced Cardiomyocyte Apoptosis.

Circular RNAs (circRNAs) have emerged as essential regulators in cardiovascular disease, including acute myocardial infarction (AMI). This study investigated the role of circRNA Pum1_0014 in myocardial infarction (MI) and its underlying mechanisms using an H9C2 cell model. Through Sanger sequencing, nucleic acid electrophoresis, RNase R, and transcriptional inhibition experiments, Pum1_0014 was identified as a novel circRNA. The cell localization of circRNA Pum1_0014 was detected by qPCR and fluorescence in situ hybridization, and the results revealed that circRNA Pum1_0014 is predominantly located in the cytoplasm. StarBase (URL: http://starbase.sysu.edu.cn/) and TargetScan (URL: https://www.targetscan.org/vert_80/) were used to predict circRNA Pum1_0014 targeting miRNAs and miRNA targeting mRNA, and the results identified miR-146a-5p as a potential target of Pum1_0014, which in turn targets NF2. The plasmid encoding the mutant circRNA Pum1_0014 or the 3'UTR mutant NF2 was constructed, and the interaction between Pum1_0014 and miR-146a-5p or miR-146a-5p and NF2 was detected by luciferase reporter gene assay. The results confirmed the interactions between Pum1_0014, miR-146a-5p, and NF2. In the MI cell model, upregulation of circRNA Pum1_0014 and NF2 and downregulation of miR-146a-5p were observed. Knockdown of circRNA Pum1_0014 inhibited NF2 expression and activated the VEGF/PAK1 pathway, reducing cardiomyocyte apoptosis. Conversely, inhibition of miR-146a-5p and overexpression of NF2 had opposite effects. These findings suggest that circRNA Pum1_0014 acts through the miR-146a-5p/NF2 axis to reduce cardiomyocyte apoptosis in MI via the VEGF/PAK1/NF2 pathway.

A comparative study of acupuncture combined with rehabilitation gymnastics on postoperative anal function of lower rectal cancer.

BACKGROUND: From the anal function, inflammatory response and other indicators, acupuncture combined with rehabilitation gymnastics was applied to patients with cancer undergoing low resection, aiming to improve the prognosis of patients. AIM: To explore the effects of acupuncture combined with rehabilitation gymnastics on anal function after lower rectal cancer surgery. METHODS: From January 2020 to December 2022, 128 patients who underwent rectal cancer surgery in the Department of Oncology of Hebei Provincial Hospital of Traditional Chinese Medicine Hospital were selected and divided into two groups using the random number table method, with 64 patients in each group. Patients in the control group were not treated with acupuncture or rehabilitation gymnastics and served as blank controls. Patients in the study group were treated with acupuncture and rehabilitation gymnastics from the 7th postoperative day. The anal incontinence scores, changes in serum interleukin-4, interleukin-6, and interleukin-10 Levels, and serum motilin, 5-hydroxytryptamine, and vasoactive intestinal peptide levels were compared. RESULTS: There were no significant differences in serum interleukin-4, interleukin-6, and interleukin-10 Levels between the groups before treatment (P > 0.05). After treatment, these levels were better than those of the control group (P < 0.05). There was no significant difference in the anal incontinence scores between the groups before and 7 d after surgery (P > 0.05). Anal incontinence scores in the study group were lower than those in the control group at 14 d, 21 d, and 28 d postoperatively (P < 0.05). There were no significant differences in serum motilin, 5-hydroxytryptamine, or vasoactive intestinal peptide levels between the groups before treatment (P > 0.05). After treatment, these levels were higher in the study group than in the control group, and vasoactive intestinal peptide level was lower in the study group than in the control group (P < 0.05). CONCLUSION: Acupuncture combined with rehabilitation gymnastics can promote the recovery of anal function and reduce the inflammatory response in patients with lower rectal cancer after surgery.

Evaluating the clinical application and effect of acupuncture therapy in anal function rehabilitation after low-tension rectal cancer surgery.

BACKGROUND: According to the indexes of serum and anal function, acupuncture therapy was applied to patients with low rectal cancer in order to avoid the occurrence of anal incontinence and reduce complications. AIM: To explore the clinical application and evaluate the effect of acupuncture therapy for anal function rehabilitation after low-tension rectal cancer surgery. METHODS: From the anorectal surgery cases, we selected 120 patients who underwent colorectal cancer surgery between January 2020 and December 2022 and randomly divided them into a control group (n = 60), observation group (n = 60), and control group after surgery for lifestyle intervention (including smoking cessation and exercise), dietary factor adjustment, anal movement, and oral loperamide treatment. The serum levels of motilin, 5-hydroxytryptamine, and vasoactive intestinal peptide (VIP), Wexner score for anal incontinence, and incidence of complications were compared between groups. RESULTS: After treatment, the VIP and 5-hydroxytryptamine levels in the observation group were lower than those in the control group (P < 0.05). The motilin level was higher than that in the control group (P < 0.05). Postoperative anal incontinence was better in the observation group than in the control group (P < 0.05). The incidence of complications in the observation group was 6.67%, which was significantly lower than that in the control group (21.67%; P < 0.05). CONCLUSION: Acupuncture therapy has a positive effect on the rehabilitation of anal function after low-tension rectal cancer surgery; it can effectively help to improve the serum indices of patients, avoid the occurrence of anal incontinence, and reduce the incidence of complications. Popularizing and applying it will be valuable.

Combinatorial metabolic engineering of Bacillus subtilis for menaquinone-7 biosynthesis.

Menaquinone-7 (MK-7), a form of vitamin K2, supports bone health and prevents arterial calcification. Microbial fermentation for MK-7 production has attracted widespread attention because of its low cost and short production cycles. However, insufficient substrate supply, unbalanced precursor synthesis, and low catalytic efficiency of key enzymes severely limited the efficiency of MK-7 synthesis. In this study, utilizing Bacillus subtilis BSAT01 (with an initial MK-7 titer of 231.0 mg/L) obtained in our previous study, the glycerol metabolism pathway was first enhanced to increase the 3-deoxy-arabino-heptulonate 7-phosphate (DHAP) supply, which led to an increase in MK-7 titer to 259.7 mg/L. Subsequently, a combination of knockout strategies predicted by the genome-scale metabolic model etiBsu1209 was employed to optimize the central carbon metabolism pathway, and the resulting strain showed an increase in MK-7 production from 259.7 to 318.3 mg/L. Finally, model predictions revealed the methylerythritol phosphate pathway as the major restriction pathway, and the pathway flux was increased by heterologous introduction (Introduction of Dxs derived from Escherichia coli) and fusion expression (End-to-end fusion of two enzymes by a linker peptide), resulting in a strain with a titer of 451.0 mg/L in a shake flask and 474.0 mg/L in a 50-L bioreactor. This study achieved efficient MK-7 synthesis in B. subtilis, laying the foundation for large-scale MK-7 bioproduction.

Protein O-fucosyltransferase 1 promotes PD-L1 stability to drive immune evasion and directs liver cancer to immunotherapy.

BACKGROUND AND AIMS: The immunosuppressive tumor microenvironment (TME) plays an essential role in cancer progression and immunotherapy response. Despite the considerable advancements in cancer immunotherapy, the limited response to immune checkpoint blockade (ICB) therapies in patients with hepatocellular carcinoma (HCC) remains a major challenge for its clinical implications. Here, we investigated the molecular basis of the protein O-fucosyltransferase 1 (POFUT1) that drives HCC immune evasion and explored a potential therapeutic strategy for enhancing ICB efficacy. METHODS: De novo MYC/Trp53-/- liver tumor and the xenograft tumor models were used to evaluate the function of POFUT1 in immune evasion. Biochemical assays were performed to elucidate the underlying mechanism of POFUT1-mediated immune evasion. RESULTS: We identified POFUT1 as a crucial promoter of immune evasion in liver cancer. Notably, POFUT1 promoted HCC progression and inhibited T-cell infiltration in the xenograft tumor and de novo MYC/Trp53-/- mouse liver tumor models. Mechanistically, we demonstrated that POFUT1 stabilized programmed death ligand 1 (PD-L1) protein by preventing tripartite motif containing 21-mediated PD-L1 ubiquitination and degradation independently of its protein-O-fucosyltransferase activity. In addition, we further demonstrated that PD-L1 was required for the tumor-promoting and immune evasion effects of POFUT1 in HCC. Importantly, inhibition of POFUT1 could synergize with anti-programmed death receptor 1 therapy by remodeling TME in the xenograft tumor mouse model. Clinically, POFUT1 high expression displayed a lower response rate and worse clinical outcome to ICB therapies. CONCLUSIONS: Our findings demonstrate that POFUT1 functions as a novel regulator of tumor immune evasion and inhibition of POFUT1 may be a potential therapeutic strategy to enhance the efficacy of immune therapy in HCC.

Exploring new frontiers: a rare case of catheter ablation for persistent atrial fibrillation in a patient with cor triatriatum sinister guided by intracardiac echocardiography.

BACKGROUND: Cor triatriatum sinister (CTS) is an uncommon congenital cardiac anomaly. Atrial fibrillation (AF) is commonly the initial symptom in patients with CTS, occurring in approximately 32% of the cases. The complexity of performing AF catheter ablation, particularly in cases with persistent AF, increases in patients with CTS due to its unique structural challenges. CASE PRESENTATION: We report the treatment course of a 60-year-old male patient diagnosed with CTS, who underwent catheter ablation of drug-refractory, persistent AF. The complex anatomical structure of the condition made catheter ablation of AF challenging. To navigate these challenges, we performed comprehensive assessments using transthoracic echocardiography and transesophageal echocardiography, along with cardiac computed tomography angiography, prior to treatment initiation. The intricate anatomy of CTS was further clarified during the procedure via intracardiac echocardiography (ICE). Additionally, the complexity of catheter manipulation was further reduced with the aid of the VIZIGO sheath and the vein of Marshall ethanol infusion to achieve effective mitral isthmus blockage, thereby circumventing the impact of the CTS membrane. CONCLUSIONS: This case underscores the complexity and potential of advanced ablation techniques in managing cardiac arrhythmias associated with unusual cardiac anatomies. During the procedure, ICE facilitated detailed modeling of the left atrium, including the membranous structure and its openings, thus providing a clearer understanding of CTS. It is noteworthy that the membrane within the CTS may serve as a potential substrate for arrhythmias, which warrants further validation through larger sample studies.

[The expression and secretion of human lactoferrin in Bacillus subtilis].

Human lactoferrin (HLF), an essential nutrient found in breast milk, possesses antibacterial, anti-inflammatory, and immune-enhancing properties. In this study, the effects of three constitutive promoters (P21, P43, and Pveg) and three inducible promoters (Pgrac100, PxylA, and Ptet*) on the expression of HLF were compared using Bacillus subtilis G601 as the host strain. The results showed that the highest expression of HLF, reaching 651.57 µg/L, was achieved when regulated by the Ptet* promoter. Furthermore, the combinational optimization of ribosome binding site (RBS) and signal peptides was investigated, and the optimal combination of RBS6 and SPyycP resulted in increased HLF expression to 1 099.87 µg/L, with 498.68 µg/L being secreted extracellularly. To further enhance HLF secretion, the metal cations-related gene dltD was knocked out, leading to an extracellular HLF level of 637.28 µg/L. This study successfully demonstrated the secretory expression of HLF in B. subtilis through the selection and optimization of expression elements, laying the foundation for the development of efficient B. subtilis cell factories for lactoprotein synthesis.

CPT1A loss disrupts BCAA metabolism to confer therapeutic vulnerability in TP53-mutated liver cancer.

Driver genomic mutations in tumors define specific molecular subtypes that display distinct malignancy competence, therapeutic resistance and clinical outcome. Although TP53 mutation has been identified as the most common mutation in hepatocellular carcinoma (HCC), current understanding on the biological traits and therapeutic strategies of this subtype has been largely unknown. Here, we reveal that fatty acid ß oxidation (FAO) is remarkable repressed in TP53 mutant HCC and which links to poor prognosis in HCC patients. We further demonstrate that carnitine palmitoyltransferase 1 (CPT1A), the rate-limiting enzyme of FAO, is universally downregulated in liver tumor tissues, and which correlates with poor prognosis in HCC and promotes HCC progression in the de novo liver tumor and xenograft tumor models. Mechanically, hepatic Cpt1a loss disrupts lipid metabolism and acetyl-CoA production. Such reduction in acetyl-CoA reduced histone acetylation and epigenetically reprograms branched-chain amino acids (BCAA) catabolism, and leads to the accumulation of cellular BCAAs and hyperactivation of mTOR signaling. Importantly, we reveal that genetic ablation of CPT1A renders TP53 mutant liver cancer mTOR-addicted and sensitivity to mTOR inhibitor AZD-8055 treatment. Consistently, Cpt1a loss in HCC directs tumor cell therapeutic response to AZD-8055. CONCLUSION: Our results show genetic evidence for CPT1A as a metabolic tumor suppressor in HCC and provide a therapeutic approach for TP53 mutant HCC patients.

Formation of iron-rich encrustation layer on anammox granules for high load stress resistance: Performance, advantages, and mechanisms.

Anaerobic ammonia oxidation (anammox) is a cost-effective technology but its performance can be seriously inhibited by high load stress. This study has created an innovative iron-rich encrustation layer (IEL) on the surface of anammox granules (AnGS) through the addition of a certain amount of nano zero-valent iron. The IEL was formed through the aggregation of a gel network and the binding of iron species with extracellular polymeric substances (EPS), resulting in a significant increase in settling ability, EPS secretion, and heme content. Metagenomic analysis indicated a notable rise in the functional genes associated with nitrogen andiron metabolism in IEL AnGS. Under high load stress, the ammonia removal performance of AnGS without IEL severely declined. In contrast, IEL AnGS exhibited excellent ammonia removal efficiency of over 90%. The IEL served as a protective barrier for AnGS, effectively mitigating the strong shear forces, thereby enhancing their resistance to high load stress.

Combinatorial Engineering of Escherichia coli for Enhancing 3-Fucosyllactose Production.

3-Fucosyllactose (3-FL) is an important fucosylated human milk oligosaccharide (HMO) with biological functions such as promoting immunity and brain development. Therefore, the construction of microbial cell factories is a promising approach to synthesizing 3-FL from renewable feedstocks. In this study, a combinatorial engineering strategy was used to achieve efficient de novo 3-FL production in Escherichia coli. α-1,3-Fucosyltransferase (futM2) from Bacteroides gallinaceum was introduced into E. coli and optimized to create a 3-FL-producing chassis strain. Subsequently, the 3-FL titer increased to 5.2 g/L by improving the utilization of the precursor lactose and down-regulating the endogenous competitive pathways. Furthermore, a synthetic membraneless organelle system based on intrinsically disordered proteins was designed to spatially regulate the pathway enzymes, producing 7.3 g/L 3-FL. The supply of the cofactors NADPH and GTP was also enhanced, after which the 3-FL titer of engineered strain E26 was improved to 8.2 g/L in a shake flask and 10.8 g/L in a 3 L fermenter. In this study, we developed a valuable approach for constructing an efficient 3-FL-producing cell factory and provided a versatile workflow for other chassis cells and HMOs.

Genome-wide characterization of Remorin gene family and their responsive expression to abiotic stresses and plant hormone in Brassica napus.

KEY MESSAGE: Remorin proteins could be positively related to salt and osmotic stress resistance in rapeseed. Remorins (REMs) play a crucial role in adaptations to adverse environments. However, their roles in abiotic stress and phytohormone responses in oil crops are still largely unknown. In this study, we identified 47 BnaREM genes in the B.napus genome. Phylogenetic relationship and synteny analysis revealed that they were categorized into 5 distinct groups and have gone through 55 segmental duplication events under purifying selection. Gene structure and conserved domains analysis demonstrated that they were highly conserved and all BnaREMs contained a conserved Remorin_C domain, with a variable N-terminal region. Promoter sequence analysis showed that BnaREM gene promoters contained various hormones and stress-related cis-acting elements. Transcriptome data from BrassicaEDB database exhibited that all BnaREMs were ubiquitously expressed in buds, stamens, inflorescences, young leaves, mature leaves, roots, stems, seeds, silique pericarps, embryos and seed coats. The qRT-PCR analysis indicated that most of them were responsive to ABA, salt and osmotic treatments. Further mutant complementary experiments revealed that the expression of BnaREM1.3-4C-1 in the Arabidopsis rem1.3 mutant restored the retarded growth phenotype and the ability to resistance to salt and osmotic stresses. Our findings provide fundamental information on the structure and evolutionary relationship of the BnaREM family genes in rapeseed, and reveal the potential function of BnaREM1.3-4C-1 in stress and hormone response.