RESUMEN
Serratia marcescens is a Gram-negative bacterium of the Enterobacteriaceae family that can produce numbers of biologically active secondary metabolites. However, our understanding of the regulatory mechanisms behind secondary metabolites biosynthesis in S. marcescens remains limited. In this study, we identified an uncharacterized LysR family transcriptional regulator, encoding gene BVG90_12635, here we named psrA, that positively controlled prodigiosin synthesis in S. marcescens. This phenotype corresponded to PsrA positive control of transcriptional of the prodigiosin-associated pig operon by directly binding to a regulatory binding site (RBS) and an activating binding site (ABS) in the promoter region of the pig operon. We demonstrated that L-proline is an effector for the PsrA, which enhances the binding affinity of PsrA to its target promoters. Using transcriptomics and further experiments, we show that PsrA indirectly regulates pleiotropic phenotypes, including serrawettin W1 biosynthesis, extracellular polysaccharide production, biofilm formation, swarming motility and T6SS-mediated antibacterial activity in S. marcescens. Collectively, this study proposes that PsrA is a novel regulator that contributes to antibiotic synthesis, bacterial virulence, cell motility and extracellular polysaccharides production in S. marcescens and provides important clues for future studies exploring the function of the PsrA and PsrA-like proteins which are widely present in many other bacteria.
Asunto(s)
Proteínas Bacterianas/genética , Biopelículas , Prodigiosina/biosíntesis , Serratia marcescens/genética , Factores de Transcripción/genética , Proteínas Bacterianas/metabolismo , Depsipéptidos/biosíntesis , Movimiento , Operón , Polisacáridos Bacterianos/biosíntesis , Regiones Promotoras Genéticas , Serratia marcescens/metabolismo , Serratia marcescens/patogenicidad , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND AND PURPOSE: In ischemic stroke, breakdown of the blood-brain barrier (BBB) aggravates brain damage. Endothelial detachment contributes to BBB disruption and neurovascular dysfunction, but its regulation in stroke has yet to be clarified. We investigated the function of NMMHC IIA (nonmuscle myosin heavy chain IIA) in the endothelium on BBB breakdown and its potential mechanisms. METHODS: Endothelial conditional knockdown NMMHC IIA (Myh9ECKD) was constructed in vivo and in vitro, and its role was explored in middle cerebral artery occlusion/reperfusion-injured mice and oxygen-glucose deprivation/reoxygenation-injured brain microvascular endothelial cells. The degree of brain injury was analyzed using staining (2,3,5-triphenyltetrazolium chloride, hematoxylin, and eosin) and electron microscopy. BBB breakdown was investigated with leakage of Evans Blue dye and expression of TJs (tight junctions) and MMP (matrix metallopeptidase)-2/9. Transcriptomics for enrichment analysis was adopted to explore the potential downstream signaling pathways of NMMHC IIA involved in middle cerebral artery occlusion/reperfusion-induced BBB dysfunction. RESULTS: NMMHC IIA expression was upregulated in endothelial cells after cerebral ischemia/reperfusion injury. Myh9ECKD mice exhibited improvement in endothelial barrier hyperpermeability and TJs integrity stimulated by cerebral ischemia/reperfusion. Blebbistatin (NMMHC II inhibitor) treatment exerted the same effect. Transcriptomics showed that NMMHC IIA was involved in regulating various BBB-related genomic changes in the middle cerebral artery occlusion/reperfusion model, and NMMHC IIA was confirmed to significantly modulate Hippo and peroxisome proliferator-activated receptor gamma/nuclear factor-kappa B signaling pathways, which are closely related to BBB damage. CONCLUSIONS: Our findings provide some new insights into how NMMHC IIA contributes to maintaining the integrity of the cerebral endothelial barrier. NMMHC IIA could be a potential therapeutic target for ischemic stroke.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Isquemia Encefálica/tratamiento farmacológico , Células Endoteliales/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Daño por Reperfusión/metabolismo , Animales , Encéfalo/metabolismo , Supervivencia Celular , Masculino , Ratones , Ratones Endogámicos C57BL , Oxígeno/metabolismo , Permeabilidad , Transducción de Señal , Uniones Estrechas/metabolismoRESUMEN
Riboflavin is an essential nutrient for humans and animals, and its derivatives flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are cofactors in the cells. Therefore, riboflavin and its derivatives are widely used in the food, pharmaceutical, nutraceutical and cosmetic industries. Advances in biotechnology have led to a complete shift in the commercial production of riboflavin from chemical synthesis to microbial fermentation. In this review, we provide a comprehensive review of biotechnologies that enhance riboflavin production in microorganisms, as well as representative examples. Firstly, the synthesis pathways and metabolic regulatory processes of riboflavin in microorganisms; and the current strategies and methods of metabolic engineering for riboflavin production are systematically summarized and compared. Secondly, the using of systematic metabolic engineering strategies to enhance riboflavin production is discussed, including laboratory evolution, histological analysis and high-throughput screening. Finally, the challenges for efficient microbial production of riboflavin and the strategies to overcome these challenges are prospected.
Asunto(s)
Flavina-Adenina Dinucleótido , Riboflavina , Vías Biosintéticas , Biotecnología , Ingeniería MetabólicaRESUMEN
Prodigiosin (PG), a red linear tripyrrole pigment normally secreted by Serratia marcescens, has received attention for its reported immunosuppressive, antimicrobial, and anticancer properties. Although several genes have been shown to be important for prodigiosin synthesis, information on the regulatory mechanisms behind this cellular process remains limited. In this work, we identified that the transcriptional regulator RcsB encoding gene BVG90_13250 (rcsB) negatively controlled prodigiosin biosynthesis in S. marcescens Disruption of rcsB conferred a remarkably increased production of prodigiosin. This phenotype corresponded to negative control of transcription of the prodigiosin-associated pig operon by RcsB, probably by binding to the promoter region of the prodigiosin synthesis positive regulator FlhDC. Moreover, using transcriptomics and further experiments, we revealed that RcsB also controlled some other important cellular processes, including swimming and swarming motilities, capsular polysaccharide production, biofilm formation, and acid resistance (AR), in S. marcescens Collectively, this work proposes that RcsB is a prodigiosin synthesis repressor in S. marcescens and provides insight into the regulatory mechanism of RcsB in cell motility, capsular polysaccharide production, and acid resistance in S. marcescensIMPORTANCE RcsB is a two-component response regulator in the Rcs phosphorelay system, and it plays versatile regulatory functions in Enterobacteriaceae However, information on the function of the RcsB protein in bacteria, especially in S. marcescens, remains limited. In this work, we illustrated experimentally that the RcsB protein was involved in diverse cellular processes in S. marcescens, including prodigiosin synthesis, cell motility, capsular polysaccharide production, biofilm formation, and acid resistance. Additionally, the regulatory mechanism of the RcsB protein in these cellular processes was investigated. In conclusion, this work indicated that RcsB could be a regulator for prodigiosin synthesis and provides insight into the function of the RcsB protein in S. marcescens.
Asunto(s)
Proteínas Bacterianas/genética , Prodigiosina/biosíntesis , Serratia marcescens/metabolismo , Regulación Bacteriana de la Expresión Génica , Operón , Serratia marcescens/genéticaRESUMEN
Prodigiosin, a secondary metabolite produced by Serratia marcescens, has attracted attention due to its immunosuppressive, antimicrobial, and anticancer properties. However, information on the regulatory mechanism behind prodigiosin biosynthesis in S. marcescens remains limited. In this work, a prodigiosin-hyperproducing strain with the BVG90_22495 gene disrupted (ZK66) was selected from a collection of Tn5G transposon insertion mutants. Using real-time quantitative PCR (RT-qPCR) analysis, ß-galactosidase assays, transcriptomics analysis, and electrophoretic mobility shift assays (EMSAs), the LysR-type regulator MetR encoded by the BVG90_22495 gene was found to affect prodigiosin synthesis, and this correlated with MetR directly binding to the promoter region of the prodigiosin-synthesis positive regulator PigP and hence negatively regulated the expression of the prodigiosin-associated pig operon. More analyses revealed that MetR regulated some other important cellular processes, including methionine biosynthesis, cell motility, H2O2 tolerance, heat tolerance, exopolysaccharide synthesis, and biofilm formation in S. marcescens Although MetR protein is highly conserved in many bacteria, we report here on the LysR-type regulator MetR exhibiting novel roles in negatively regulating prodigiosin synthesis and positively regulating heat tolerance, exopolysaccharide synthesis, and biofilm formation.IMPORTANCESerratia marcescens, a Gram-negative bacterium, is found in a wide range of ecological niches and can produce several secondary metabolites, including prodigiosin, althiomycin, and serratamolide. Among them, prodigiosin shows diverse functions as an immunosuppressant, antimicrobial, and anticancer agent. However, the regulatory mechanisms behind prodigiosin synthesis in S. marcescens are not completely understood. Here, we adapted a transposon mutant library to identify the genes related to prodigiosin synthesis, and the BVG90_22495 gene encoding the LysR-type regulator MetR was found to negatively regulate prodigiosin synthesis. The molecular mechanism of the metR mutant hyperproducing prodigiosin was investigated. Additionally, we provided evidence supporting new roles for MetR in regulating methionine biosynthesis, cell motility, heat tolerance, H2O2 tolerance, and exopolysaccharide synthesis in S. marcescens Collectively, this work provides novel insight into regulatory mechanisms of prodigiosin synthesis and uncovers novel roles for the highly conserved MetR protein in regulating prodigiosin synthesis, heat tolerance, exopolysaccharide (EPS) synthesis, and biofilm formation.
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Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Peróxido de Hidrógeno/metabolismo , Metionina/biosíntesis , Prodigiosina/biosíntesis , Serratia marcescens/fisiología , Termotolerancia/genética , Transactivadores/genética , Antibacterianos/biosíntesis , Proteínas Bacterianas/metabolismo , Serratia marcescens/genética , Transactivadores/metabolismoRESUMEN
Members of the bacterial genus Aeromonas are important aquatic pathogens that cause severe fish diseases. Here, we characterize a novel lytic phage, Aeromonas virus phiA8-29, isolated from the alimentary tract of a freshwater fish. Transmission electron microscopy showed that phiA8-29 has a long contractile tail and thus can be classified as a member of the family Myoviridae. The phage genome was identified as a double-stranded DNA molecule of 144,974 bp containing 185 open reading frames and nine tRNA-encoding genes. Comparative genomic analysis revealed that the phiA8-29 genome has little similarity to any of the currently sequenced Aeromonas phage genomes. Our data indicate that phiA8-29 is a novel lytic Myoviridae phage that does not belong to any of the known genera.
Asunto(s)
Aeromonas/virología , Bacteriófagos/aislamiento & purificación , Myoviridae/aislamiento & purificación , Bacteriófagos/clasificación , Bacteriófagos/genética , Bacteriófagos/ultraestructura , Genoma Viral , Myoviridae/clasificación , Myoviridae/genética , Myoviridae/ultraestructura , Sistemas de Lectura Abierta , Filogenia , Proteínas Virales/genéticaRESUMEN
Biofilm formation is an important virulence factor which is controlled by complex regulatory circuits in Pseudomonas aeruginosa. In this work, a biofilm hyper-producing strain, P2-7, was selected from a collection of transposon insertion mutants in which the PA2121 gene was disrupted. PA2121 was predicted as a putative LysR-type regulator. Analyses showed that it was involved in early biofilm formation, mature biofilm development, and colony morphology. Quantitative measurements revealed that PA2121 repressed biosynthesis of extracellular polysaccharides (alginate, psl and pel). Furthermore, it was observed that PA2121 was self-regulated, highly expressed in the early phase of biofilm development, and subject to the negative regulation by a biofilm synthesis regulator SrpA that binds directly to the PA2121 gene promoter. Collectively, this study proposes that PA2121 is a novel biofilm synthesis repressor (BsrA) in P. aeruginosa.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas , Pseudomonas aeruginosa/fisiología , Factores de Transcripción/metabolismo , Alginatos/metabolismo , Regulación Bacteriana de la Expresión Génica , Polisacáridos Bacterianos/biosíntesisRESUMEN
Phage O4 of Pseudomonas aeruginosa was previously visualized as a short-tailed virus using a transmission electron microscope. In this work, the O4 genome was characterized to be a linear dsDNA molecule comprising 50509 bp with 76 predicted genes located in five clusters. Mass spectrometry showed that the O4 virion contains 6 putative structural proteins, 2 putative enzymes, and 7 hypothetical proteins. By analyzing a Tn5G transposon mutation library, eight genes, wbpR, wbpV, wbpO, wbpT, wbpS, wbpL, galU, and wzy, were identified and confirmed responsible for the phage-resistant phenotype; all of them are related to the synthesis of O-specific antigen (OSA) of lipopolysaccharide (LPS), indicating that OSA is the receptor for the adsorption of phage O4. Comparative genomic analysis revealed that the phage O4 genome shares little similarity to any known podovirus, indicating that phage O4 is classifiable as a novel member of the Podoviridae family.
Asunto(s)
Genoma Viral , Lisogenia/fisiología , Podoviridae/genética , Fagos Pseudomonas/genética , Pseudomonas aeruginosa/virología , Receptores Virales/metabolismo , Proteínas Virales/genética , ADN/genética , ADN/metabolismo , Elementos Transponibles de ADN , ADN Viral/genética , ADN Viral/metabolismo , Ontología de Genes , Anotación de Secuencia Molecular , Antígenos O/química , Antígenos O/metabolismo , Filogenia , Podoviridae/clasificación , Podoviridae/metabolismo , Fagos Pseudomonas/clasificación , Fagos Pseudomonas/metabolismo , Receptores Virales/química , Análisis de Secuencia de ADN , Proteínas Virales/metabolismo , Virión/genética , Virión/metabolismoRESUMEN
Bacterial growth phase has been reported affecting phage infection. To underpin the related mechanism, infection efficiency of Pseudomonas aeruginosa phage K5 is characterized. When infecting the logarithmic cells, phage K5 produced significantly more infection centers than the stationary cells, well concordant with the viable cell ratio in the different growth phases. Additionally, the burst size decreased dramatically in the stationary cells, implying that the physiological state of the viable cells contributed to the productivity of phage K5, and it was consistent with the expression variation of the phage RNA polymerase. Quorum sensing inhibitor penicillic acid was applied and could significantly improve the viable cell proportion and the infection center numbers, but had less effect on the corresponding burst sizes. Moreover, the effect of penicillic acid and the quorum sensing regulator mutants on the production of phage C11 was also analyzed. Taken together, our data suggest that quorum sensing is involved in the defense of phage K5 infection by influencing the viable cell population and their physiological state, and it is an efficient and intrinsic pathway allowing bacteria to resist phage attacks in natural environment.
Asunto(s)
Interacciones Huésped-Parásitos , Fagos Pseudomonas/crecimiento & desarrollo , Pseudomonas aeruginosa/fisiología , Pseudomonas aeruginosa/virología , Percepción de QuorumRESUMEN
Phage genomic information and the nature of host-phage interactions are important for phage applications. In this study, Pseudomonas aeruginosa phage K5 is characterized as a linear double-stranded genomic DNA molecule of 93,754 bp with identical 1182-bp direct terminal repeats. Comparative genomic analysis reveals that phage K5 is highly homologous to the "PaP1-like" phages. Thirteen mutants resistant to phage K5 are screened in a transposon mutant library. The disrupted genetic loci are identified as gene Y880_RS05480 encoding a putative O-antigen polymerase Wzy and gene wapH encoding a glycosyltransferase. The mutants are confirmed by the complementation experiment. The production of biofilm and the profile of lipopolysaccharide (LPS) are further analyzed in the Y880_RS05480 mutant. Our data indicate that LPS is the receptor of phage K5.
Asunto(s)
Genoma Viral , Antígenos O/metabolismo , Fagos Pseudomonas/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/virología , Receptores Virales/genética , Secuencia de Aminoácidos , Biopelículas/crecimiento & desarrollo , Elementos Transponibles de ADN , ADN Viral/genética , Prueba de Complementación Genética , Glicosiltransferasas/genética , Hexosiltransferasas/genética , Interacciones Huésped-Patógeno , Mutación , Antígenos O/química , Proteínas Asociadas a Pancreatitis , Pseudomonas aeruginosa/fisiología , Análisis de Secuencia de ADNRESUMEN
Branched-chain amino acids (BCAAs) such as L-valine, L-leucine, and L-isoleucine are widely used in food and feed. To comply with sustainable development goals, commercial production of BCAAs has been completely replaced with microbial fermentation. However, the efficient production of BCAAs by microorganisms remains a serious challenge due to their staggered metabolic networks and cell growth. To overcome these difficulties, systemic metabolic engineering has emerged as an effective and feasible strategy for the biosynthesis of BCAA. This review firstly summarizes the research advances in the microbial synthesis of BCAAs and representative engineering strategies. Second, systematic methods, such as high-throughput screening, adaptive laboratory evolution, and omics analysis, can be used to analyses the synthesis of BCAAs at the whole-cell level and further improve the titer of target chemicals. Finally, new tools and engineering strategies that may increase the production output and development direction of the microbial production of BCAAs are discussed.
Asunto(s)
Aminoácidos de Cadena Ramificada , Isoleucina , Aminoácidos de Cadena Ramificada/metabolismo , Leucina/metabolismo , Valina , Ingeniería MetabólicaRESUMEN
L-serine is a high-value amino acid widely used in the food, medicine, and cosmetic industries. However, the low yield of L-serine has limited its industrial production. In this study, a cellular factory for efficient synthesis of L-serine was obtained by engineering the serine hydroxymethyltransferases (SHMT). Firstly, after screening the SHMT from Alcanivorax dieselolei by genome mining, a mutant AdSHMTE266M with high thermal stability was identified through rational design. Subsequently, an iterative saturating mutant library was constructed by using coevolutionary analysis, and a mutant AdSHMTE160L/E193Q with enzyme activity 1.35 times higher than AdSHMT was identified. Additionally, the target protein AdSHMTE160L/E193Q/E266M was efficiently overexpressed by improving its mRNA stability. Finally, combining the substrate addition strategy and system optimization, the optimized strain BL21/pET28a-AdSHMTE160L/E193Q/E266M-5'UTR-REP3S16 produced 106.06 g/L L-serine, which is the highest production to date. This study provides new ideas and insights for the engineering design of SHMT and the industrial production of L-serine.
Asunto(s)
Escherichia coli , Glicina Hidroximetiltransferasa , Escherichia coli/metabolismo , Glicina Hidroximetiltransferasa/genética , Glicina Hidroximetiltransferasa/química , Glicina Hidroximetiltransferasa/metabolismo , Serina/genética , Serina/metabolismo , Ingeniería MetabólicaRESUMEN
Aromatic amino acids (AAA) and derived compounds have enormous commercial value with extensive applications in the food, chemical and pharmaceutical fields. Microbial production of AAA and derived compounds is a promising prospect for its environmental friendliness and sustainability. However, low yield and production efficiency remain major challenges for realizing industrial production. With the advancement of synthetic biology, microbial production of AAA and derived compounds has been significantly facilitated. In this review, a comprehensive overview on the current progresses, challenges and corresponding solutions for AAA and derived compounds biosynthesis is provided. The most cutting-edge developments of synthetic biology technology in AAA and derived compounds biosynthesis, including CRISPR-based system, genetically encoded biosensors and synthetic genetic circuits, were highlighted. Finally, future prospects of modern strategies conducive to the biosynthesis of AAA and derived compounds are discussed. This review offers guidance on constructing microbial cell factory for aromatic compound using synthetic biology technology.
Asunto(s)
Aminoácidos Aromáticos , Biología Sintética , Biología Sintética/métodos , Aminoácidos Aromáticos/biosíntesis , Ingeniería Metabólica/métodos , Técnicas Biosensibles/métodos , Bacterias/metabolismo , Bacterias/genéticaRESUMEN
The coevolution of bacteria and bacteriophages has created a great diversity of mechanisms by which bacteria fight phage infection, and an equivalent diversity of mechanisms by which phages subvert bacterial immunity. Effective and continuous evolution by phages is necessary to deal with coevolving bacteria. In this study, to better understand the connection between phage genes and host range, we examine the isolation and genomic characterization of two bacteriophages, JNUWH1 and JNUWD, capable of infecting Escherichia coli. Sourced from factory fermentation pollutants, these phages were classified within the Siphoviridae family through TEM and comparative genomic analysis. Notably, the phages exhibited a viral burst size of 500 and 1,000 PFU/cell, with latent periods of 15 and 20 min, respectively. They displayed stability over a pH range of 5 to 10, with optimal activity at 37°C. The complete genomes of JNUWH1 and JNUWD were 44,785 bp and 43,818 bp, respectively. Phylogenetic analysis revealed their close genetic relationship to each other. Antibacterial assays demonstrated the phages' ability to inhibit E. coli growth for up to 24 h. Finally, through laboratory-driven adaptive evolution, we successfully identified strains for both JNUWH1 and JNUWD with mutations in receptors specifically targeting lipopolysaccharides (LPS) and the lptD gene. Overall, these phages hold promise as additives in fermentation products to counter E. coli, offering potential solutions in the context of evolving bacterial resistance.
RESUMEN
The supramolecular crystals, Mn(15-crown-5)(MnCl4)(DMF), (1; 15-crown-5 = 1,4,7,10,13-pentaoxacyclopentadecane), were synthesized via a self-assembly strategy under ambient conditions. Comprehensive characterization of the crystals involved microanalysis for C, H, and N elements, thermogravimetric (TG) analysis, differential scanning calorimetry (DSC) and single-crystal X-ray diffraction techniques. The results reveal that 1 undergoes a two-step thermotropic and isostructural phase transition at around 217 K and 351 K upon heating. All three phases belong to the same space group (P212121) with analogous cell parameters. These two phase transitions primarily involve the thermally activated ring rotational dynamics of the 15-crown-5 molecule, with only the transition at ca. 351 K being associated with a dielectric anomaly. 1 exhibits intense luminescence with a peak at â¼600 nm and a high quantum yield of 68%. The mechanisms underlying this intense luminescence are likely linked to low-symmetry ligand fields. Additionally, 1 displays phase transition-induced luminescence enhancement behavior, and the possible mechanism is further discussed.
RESUMEN
L-theanine is a natural non-protein amino acid with wide applications. Thus, a high yield of L-theanine production is required on an industrial scale. Herein, an efficient L-theanine-producing strain of Corynebacterium glutamicum was constructed by combining protein and metabolic engineering. Firstly, a γ-glutamylmethylamide synthetase from Paracoccus aminovorans (PaGMAS) was isolated and engineered by computer-aided design, the resulting mutant E179K/N105R improved L-theanine yield by 36.61 %. Subsequently, to increase carbon flux towards L-theanine production, the gene ggt which degrades L-theanine, the gene alaT which participated in L-alanine synthesis, and the gene NCgl1221 which encodes glutamate-exporting protein were deleted. Finally, ppk gene was overexpressed to enhance intracellular ATP production. The reprogramed strain produced 44.12 g/L L-theanine with a yield of 57.11 % and productivity of 1.16 g/L/h, which is the highest L-theanine titer reported by Corynebacterium glutamicum. This study provides an efficient and economical biosynthetic pathway for the industrial production of L-theanine.
Asunto(s)
Corynebacterium glutamicum , Glutamatos , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Ingeniería Metabólica/métodos , Fermentación , Ácido Glutámico/metabolismoRESUMEN
The application of synthetic biology tools to modulate gene expression to increase yield has been thoroughly demonstrated as an effective and convenient approach in industrial production. In this study, we employed a high-throughput screening strategy to identify a 5' UTR sequence from the genome of B. subtilis 168. This sequence resulted in a 5.8-fold increase in the expression level of EGFP. By utilizing the 5' UTR sequence to overexpress individual genes within the rib operon, it was determined that the genes ribD and ribAB serve as rate-limiting enzymes in the riboflavin synthesis pathway. Constructing a 5' UTR library to regulate EGFP expression resulted in a variation range in gene expression levels exceeding 100-fold. Employing the same 5' UTR library to regulate the expression of EGFP and mCherry within the operon led to a change in the expression ratio of these two genes by over 10,000-fold. So, employing a 5' UTR library to modulate the expression of the rib operon gene and construct a synthetic rib operon resulted in a 2.09-fold increase in riboflavin production. These results indicate that the 5' UTR sequence identified and characterized in this study can serve as a versatile synthetic biology toolkit for achieving complex metabolic network reconstruction. This toolkit can facilitate the fine-tuning of gene expression to produce target products.
RESUMEN
L-Carnosine has various physiological functions and is widely used in cosmetics, medicine, food additives, and other fields. However, the yield of L-Carnosine obtained by biological methods is far from the level of industrial production. Herein, a cell factory for efficient synthesis of L-Carnosine was constructed based on transporter engineering and protein engineering. Firstly, a dipeptidase (SmpepD) was screened from Serratia marcescens through genome mining to construct a cell factory for synthesizing L-Carnosine. Subsequently, through rationally designed SmPepD, a double mutant T168S/G148D increased the L-Carnosine yield by 41.6% was obtained. Then, yeaS, a gene encoding the exporter of L-histidine, was deleted to further increase the production of L-Carnosine. Finally, L-Carnosine was produced by one-pot biotransformation in a 5 L bioreactor under optimized conditions with a yield of 133.2 mM. This study represented the highest yield of L-Carnosine synthesized in microorganisms and provided a biosynthetic pathway for the industrial production of L-Carnosine.
Asunto(s)
Carnosina , Carnosina/genética , Carnosina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Reactores Biológicos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ingeniería de Proteínas , Ingeniería Metabólica/métodosRESUMEN
α-Arbutin is extensively used in cosmetic industries. The lack of highly active enzymes and the cytotoxicity of hydroquinone limit the biosynthesis of α-arbutin. In this study, a whole-cell biocatalytic approach based on enzyme engineering and engineered cell modification was identified as effective in enhancing α-arbutin production. First, a sucrose phosphorylase (SPase) mutant with higher enzyme activity was obtained by experimental screening. Next, to avoid the oxidation of hydroquinone, we established an anaerobic process to improve the robustness of the cells by knocking out lytC, sdpC, and skfA in Bacillus subtilis and overcoming the inhibitory effect of a high concentration of hydroquinone. Finally, the engineered strain was used for biotransformation in a 5 L fermenter with batch feeding for 24 h. The final yield of α-arbutin achieved was 129.6 g/L, which may provide a basis for the large-scale industrial production of α-arbutin.
Asunto(s)
Arbutina , Hidroquinonas , BiotransformaciónRESUMEN
Two haloargentate hybrids, [Me-dabco]Ag2X3 (Me-dabco = 1-methyl-1,4-diazabicyclo-[2.2.2]octan-1-ium, X = I (1) or Br (2)), with the same formula but different structures have been synthesized by a slow evaporation method and characterized by microanalysis, infrared spectroscopy, thermogravimetric, and powder X-ray diffraction techniques. Hybrid 1 consists of completely isolated [Ag4I6]2- clusters, while hybrid 2 exhibits a complicated one-dimensional (1D) chain structure formed by four different configurations of neutral chains and two dissimilar configurations of anionic chains. Hybrid 2 undergoes two reversible order-disorder phase transitions, while hybrid 1 displays one reversible and one irreversible structural phase transition. Both 1 and 2 displayed step-like dielectric anomalies in the vicinity of the phase transition temperature. The corresponding dielectric constants in the high dielectric states are approximately 13 and 6 times higher than those in the low dielectric states for 1 and 2, respectively. Interestingly, the subtle change of halides from I- to Br- significantly affects the aggregated structure of haloargentate, the phase transition, and dielectric behaviors, revealing the typical 'butterfly effect' with the ion radii of halides in these two haloargentate hybrids.