RESUMO
Although protein aggregation can cause cytotoxicity, such aggregates can also form to mitigate cytotoxicity from misfolded proteins, although the nature of these contrasting aggregates remains unclear. We previously found that overproduction (op) of a three green fluorescent protein-linked protein (3×GFP) induces giant aggregates and is detrimental to growth. Here, we investigated the mechanism of growth inhibition by 3×GFP-op using non-aggregative 3×MOX-op as a control in Saccharomyces cerevisiae. The 3×GFP aggregates were induced by misfolding, and 3×GFP-op had higher cytotoxicity than 3×MOX-op because it perturbed the ubiquitin-proteasome system. Static aggregates formed by 3×GFP-op dynamically trapped Hsp70 family proteins (Ssa1 and Ssa2 in yeast), causing the heat-shock response. Systematic analysis of mutants deficient in the protein quality control suggested that 3×GFP-op did not cause a critical Hsp70 depletion and aggregation functioned in the direction of mitigating toxicity. Artificial trapping of essential cell cycle regulators into 3×GFP aggregates caused abnormalities in the cell cycle. In conclusion, the formation of the giant 3×GFP aggregates itself is not cytotoxic, as it does not entrap and deplete essential proteins. Rather, it is productive, inducing the heat-shock response while preventing an overload to the degradation system.
Assuntos
Proteínas de Fluorescência Verde , Proteínas de Choque Térmico HSP70 , Agregados Proteicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteólise , Complexo de Endopeptidases do Proteassoma/metabolismo , Resposta ao Choque Térmico/genética , Dobramento de Proteína , Ciclo Celular/genética , Adenosina TrifosfatasesRESUMO
Pyrethrins are natural insecticides biosynthesised by Asteraceae plants, such as Tanacetum cinerariifolium and have a long history, dating back to ancient times. Pyrethrins are often used as low-persistence and safe insecticides to control household, horticultural, and agricultural insect pests. Despite its long history of use, pyrethrin biosynthesis remains a mystery, presenting a significant opportunity to improve yields and meet the growing demand for organic agriculture. To achieve this, both genetic modification and non-genetic methods, such as chemical activation and priming, are indispensable. Plants use pyrethrins as a defence against herbivores, but pyrethrin biosynthesis pathways are shared with plant hormones and signal molecules. Hence, the insight that pyrethrins may play broader roles than those traditionally expected is invaluable to advance the basic and applied sciences of pyrethrins.
Assuntos
Inseticidas , Piretrinas , Piretrinas/metabolismo , Inseticidas/metabolismo , Chrysanthemum cinerariifolium/metabolismo , Praguicidas/metabolismo , Animais , Vias BiossintéticasRESUMO
Overproduction of desired native or nonnative biochemical(s) in (micro)organisms can be achieved through metabolic engineering. Appropriate rewiring of cell metabolism is performed by making rational changes such as insertion, up-/down-regulation and knockout of genes and consequently metabolic reactions. Finding appropriate targets (including proper sets of reactions to be knocked out) for metabolic engineering to design optimal production strains has been the goal of a number of computational algorithms. We developed FastKnock, an efficient next-generation algorithm for identifying all possible knockout strategies (with a predefined maximum number of reaction deletions) for the growth-coupled overproduction of biochemical(s) of interest. We achieve this by developing a special depth-first traversal algorithm that allows us to prune the search space significantly. This leads to a drastic reduction in execution time. We evaluate the performance of the FastKnock algorithm using various Escherichia coli genome-scale metabolic models in different conditions (minimal and rich mediums) for the overproduction of a number of desired metabolites. FastKnock efficiently prunes the search space to less than 0.2% for quadruple- and 0.02% for quintuple-reaction knockouts. Compared to the classic approaches such as OptKnock and the state-of-the-art techniques such as MCSEnumerator methods, FastKnock found many more beneficial and important practical solutions. The availability of all the solutions provides the opportunity to further characterize, rank and select the most appropriate intervention strategy based on any desired evaluation index. Our implementation of the FastKnock method in Python is publicly available at https://github.com/leilahsn/FastKnock .
Assuntos
Engenharia Metabólica , Modelos Biológicos , Algoritmos , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma , Redes e Vias MetabólicasRESUMO
Iron-sulfur clusters are ubiquitous cofactors required for fundamental biological processes. Structural and spectroscopic analysis of Fe-S proteins is often limited by low cluster occupancy in recombinantly produced proteins. In this work, we report a systematic comparison of different maturation strategies for three well-established [4Fe-4S] proteins. Aconitase B, HMBPP reductase (IspH), and quinolinate synthase (NadA) were used as model proteins as they have previously been characterized. The protein production strategies include expression of the gene of interest in BL21(DE3) cells, maturation of the apo protein using chemical or semi-enzymatic reconstitution, co-expression with two different plasmids containing the iron-sulfur cluster (isc) or sulfur formation (suf) operon, a cell strain lacking IscR, the transcriptional regulator of the ISC machinery, and an engineered "SufFeScient" derivative of BL21(DE3). Our results show that co-expression of a Fe-S biogenesis pathway influences the protein yield and the cluster content of the proteins. The presence of the Fe-S cluster is contributing to correct folding and structural stability of the proteins. In vivo maturation reduces the formation of Fe-S aggregates, which occur frequently when performing chemical reconstitution. Furthermore, we show that the in vivo strategies can be extended to the radical SAM protein ThnB, which was previously only maturated by chemical reconstitution. Our results shed light on the differences of in vitro and in vivo Fe-S cluster maturation and points out the pitfalls of chemical reconstitution.
Assuntos
Proteínas de Escherichia coli , Proteínas Ferro-Enxofre , Proteínas Ferro-Enxofre/metabolismo , Proteínas de Escherichia coli/metabolismo , Oxirredutases/metabolismo , Ferro/metabolismo , Enxofre/metabolismoRESUMO
Streptomyces lividans is a potent host for the extracellular overproduction of heterologous proteins. To further improve the usability and productivity of S. lividans, a dual gene expression vector of "pTSKr duet" containing two strong constitutive promoters, scmpPc and kasOp*, was constructed. The success in the overproduction of two secretory enzymes simultaneously without interference with each other indicated that the "pTSKr duet" vector can realize the coexpression of two genes simultaneously and independently. Further, using the two-gene coexpression vector, we screened the effects of the overexpression of five factors that possibly promote secretion on the extracellular overproduction of heterologous secretory proteins. Interestingly, the coexpression of a quality control regulator (CssR) promoted the overproduction level to 1.3-fold for a stable heterologous protein of SMTG (transglutaminase from S. mobaraensis), while other four factors limited the overproduction of SMTG at different degrees.
Assuntos
Streptomyces , Streptomyces/genética , Streptomyces lividans/genética , Vetores Genéticos , Proteínas de Bactérias/metabolismo , Transporte ProteicoRESUMO
Activation of IL-4R (IL-4 receptor) signaling in airway epithelial cells leads to airway hyperresponsiveness and mucus overproduction in asthma. CDH26 (cadherin-26), a cadherin implicated in the polarization of airway epithelial cells, is upregulated in asthma. However, the role of CDH26 in asthma remains unknown. In this study, we demonstrated that Cdh26 deficiency significantly reduced airway mucus overproduction, airway hyperresponsiveness, and airway eosinophilia in a murine model of allergic airway disease. Interestingly, allergen-induced Il-4Rα upregulation in airway epithelium was markedly reduced in Cdh26-/- mice. In cultured human bronchial epithelial cells, CDH26 knockdown inhibited IL-13, a ligand for IL-4R; induced IL-4Rα and IL-13Rα1 (IL-13 receptor α1) upregulation; and suppressed downstream Jak1 (Janus kinase 1) and Stat6 (signal transducer and activator of transcription 6) phosphorylation. Moreover, CDH26 knockdown inhibited IL-13-induced MUC5AC and eosinophilic chemokine expression. These results suggest that CDH26 plays a key role in epithelial IL-4R signaling activation and downstream effectors. In contrast, CDH26 overexpression amplified IL-13-activated IL-4R signaling in BEAS-2B cells. In the airway epithelium of patients with asthma, IL-4Rα expression was elevated, and CDH26 was the only cadherin that was upregulated among 11 cadherin family members. CDH26 expression was strongly correlated with epithelial IL-4Rα and MUC5AC expression, sputum eosinophilia, and fractional exhaled nitric oxide in patients with asthma. Taken together, we identified CDH26 as a key regulator of epithelial IL-4R signaling in asthma and a potential therapeutic target for IL-4R-mediated allergic diseases.
Assuntos
Asma , Eosinofilia , Hipersensibilidade , Humanos , Camundongos , Animais , Interleucina-13 , Receptores de Interleucina-4 , Asma/metabolismo , Hipersensibilidade/metabolismo , CaderinasRESUMO
Adenosine, as a naturally occurring nucleoside, plays an important role in human health maintenance. In recent years, many studies have shown that adenosine has the effect of cancer inhibition, and some of its analogs have been successfully marketed as anticancer drugs. This report mainly describes the anti-colon cancer activities and mechanism of a novel halogenated adenosine analog named 5'-bromodeoxyadenosine (5'-BrDA). As a result, 5'-BrDA concentration-dependently inhibited colon cancer cells proliferation, induced autophagy without disruption of lysosomal stability, and promoted autophagy-independently cellular mitochondrial apoptosis by increasing the accumulation of reactive oxygen species. Furthermore, 5'-BrDA inhibited the tumor growth of colon cancer in CT26 inbred mice without affecting the body weight in vivo. Collectively, the above-mentioned mechanisms contributed to the anticancer activity of 5'-BrDA. It is rare to discover novel anticancer adenosine analogs during the past couple of decades. We believe that our work will enrich the understanding of adenosine analogs, also, pave the way for adenosine analogs product based anticancer drug development.
Assuntos
Adenosina/farmacologia , Neoplasias do Colo/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Células HCT116 , Células HT29 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Theoretical models were developed to propose a new mechanism enhancing mixed mating (reproduction by both outcrossing and selfing) in hermaphroditic plants; mixed mating can be maintained if there exists among-parent variation in early-acting inbreeding depression in embryos and parents can replace dead embryos by overproduction of ovules. In the two main models developed, the number of embryos produced is allowed to evolve, parents may overproduce embryos, and among-parent variation in early-acting inbreeding depression does not exist or exists. I found that mixed mating does not evolve if among-parent variation in early-acting inbreeding depression does not exist, whereas it evolves if it exists. If the degree of early-acting inbreeding depression in embryos is variable among parents, parents with the same selfing strategy suffer different effects of early-acting inbreeding depression. Specifically, overproduction of embryos may be insufficient when inbreeding depression is severe but wasteful when it is weak. Hence, it is advantageous to produce a moderate number of embryos to reduce waste of resources. Mixed mating is then advantageous to avoid great reductions in seed number caused by massive loss of selfed embryos in cases of severe inbreeding depression.
Assuntos
Depressão por Endogamia , Endogamia , Óvulo Vegetal , Plantas , ReproduçãoRESUMO
Clavulanic acid (CA) is a clinically important secondary metabolite used to treat infectious diseases. We aimed to decipher complex regulatory mechanisms acting in CA biosynthesis by analyzing transcriptome- and proteome-wide alterations in an industrial CA overproducer Streptomyces clavuligerus strain, namely DEPA and its wild-type counterpart NRRL3585. A total of 924 differentially expressed genes (DEGs) and 271 differentially produced proteins (DPPs) were obtained by RNA-seq and nanoLC-MS/MS analyses, respectively. In particular, CA biosynthetic genes, namely, car (cad), cas2, oat2, pah, bls, ceas2, orf12, and claR, a cluster situated regulatory (CSR) gene, were significantly upregulated as shown by RNA-seq. Enzymes of clavam biosynthesis were downregulated considerably in the DEPA strain, while the genes involved in the arginine biosynthesis, one of the precursors of CA pathway, were overexpressed. However, the biosynthesis of the other CA precursor, glyceraldehyde-3-phosphate (G3P), was not affected. CA overproduction in the DEPA strain was correlated with BldD, BldG, BldM, and BldN (AdsA) overrepresentation. In addition, TetR, WhiB, and Xre family transcriptional regulators were shown to be significantly overrepresented. Several uncharacterized/unknown proteins differentially expressed in the DEPA strain await further studies for functional characterization. Correlation analysis indicated an acceptable degree of consistency between the transcriptome and proteome data. The study represents the first integrative-omics analysis in a CA overproducer S. clavuligerus strain, providing insights into the critical control points and potential rational engineering targets for a purposeful increase of CA yields in strain improvement. KEY POINTS: â Transcriptome and proteome-wide alterations in industrial CA overproducer strain DEPA â An acceptable degree of consistency between the transcriptome and proteome data â New targets to be exploited for rational engineering.
Assuntos
Regulação Bacteriana da Expressão Gênica , Proteoma , Ácido Clavulânico , Proteoma/metabolismo , Streptomyces , Espectrometria de Massas em TandemRESUMO
Target proteins in biotechnological applications are highly diverse. Therefore, versatile flexible expression systems for their functional overproduction are required. In order to find the right heterologous gene expression strategy, suitable host-vector systems, which combine different genetic circuits, are useful. In this study, we designed a novel Bacillus subtilis expression toolbox, which allows the overproduction and secretion of potentially toxic enzymes. This toolbox comprises a set of 60 expression vectors, which combine two promoter variants, four strong secretion signals, a translation-enhancing downstream box, and three plasmid backbones. This B. subtilis toolbox is based on a tailor-made, clean deletion mutant strain, which is protease and sporulation deficient and exhibits reduced autolysis and secondary metabolism. The appropriateness of this alternative expression platform was tested for the overproduction of two difficult-to-produce eukaryotic model proteins. These included the sulfhydryl oxidase Sox from Saccharomyces cerevisiae, which forms reactive hydrogen peroxide and undesired cross-linking of functional proteins, and the human interleukin-1ß, a pro-inflammatory cytokine. For the best performing Sox and interleukin, overproducing and secreting variants of these new B. subtilis toolbox fermentation strategies were developed and tested. This study demonstrates the suitability of the prokaryotic B. subtilis host-vector system for the extracellular production of two eukaryotic proteins with biotechnological relevance. KEY POINTS: ⢠Construction of a versatile Bacillus subtilis gene expression toolbox. ⢠Verification of the toolbox by the secretory overproduction of two difficult-to-express proteins. ⢠Fermentation strategy for an acetoin-controlled overproduction of heterologous proteins.
Assuntos
Acetoína , Bacillus subtilis , Microrganismos Geneticamente Modificados , Acetoína/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fermentação , Plasmídeos , Regiões Promotoras GenéticasRESUMO
Biofilm-producing bacteria can decrease Cd uptake in vegetables, but mechanisms underlying this effect are poorly characterized. In this study, two mutant strains B12ΔYwcc and B12ΔSlrR were constructed from a biofilm-producing Bacillus subtilis strain B12. Then, the impacts of strain B12 and its high biofilm-producing mutant strain B12ΔYwcc and low biofilm-producing mutant strain B12ΔSlrR on Cd availability and uptake in Chinese cabbage and the related mechanisms were investigated in the Cd-polluted soil. Strain B12 and its mutants B12ΔYwcc and B12ΔSlrR increased the dry biomasses of edible tissues by 54%-130% compared with the controls. Strain B12 and its mutant B12ΔYwcc reduced the soil available Cd content by 36%-50% and root and edible tissue Cd contents by 23%-50% compared with the controls. Furthermore, the mutant strain B12ΔYwcc reduced the edible tissue Cd content by 40% and increased the polysaccharide content by 23%, invertase activity by 139%, and gene copies of the cumA by 4.5-fold, epsA by 7.1-fold, and cadA by 4.3-fold, which were involved in Cd adsorption in the rhizosphere soils, respectively, compared with strain B12. The polysaccharide content and cumA, epsA, and cadA gene copy numbers showed significantly reverse correlations with the available Cd content. Notably, the mutant strain B12ΔYwcc showed better ability to colonize the vegetable root surface than strain B12. These findings demonstrated that the biofilm-overproducing mutant strain B12ΔYwcc increased the polysaccharide production and Cd-immobilizing related cumA, epsA, and cadA gene copies, resulting in lower Cd availability and accumulation in Chinese cabbage in the Cd-polluted soil.
Assuntos
Brassica , Poluentes do Solo , Bacillus subtilis/genética , Biofilmes , Cádmio/análise , Cádmio/toxicidade , China , Polissacarídeos , Solo , Poluentes do Solo/análise , VerdurasRESUMO
The clerodane and ent-kaurane diterpenoids are two typical categories of diterpenoid natural products with complicated polycyclic carbon skeletons and significant pharmacological activities. Despite exciting advances in organic chemistry, access to these skeletons is still highly challenging. Using synthetic biology to engineer microbes provides an innovative alternative to bypass synthetic challenges. In this study, we constructed two truncated artificial pathways to efficiently produce terpentetriene and ent-kaurene, two representative clerodane and ent-kaurane diterpenes, in Escherichia coli. Both pathways depend on the exogenous addition of isoprenoid alcohol to reinforce the supply of IPP and DMAPP via two sequential phosphorylation reactions. Optimization of these constructs provided terpentetriene and ent-kaurene titers of 66 ± 4 mg/L and 113 ± 7 mg/L, respectively, in shake-flask fermentation. The truncated pathways to overproduce clerodane and ent-kaurane skeletons outlined here may provide an attractive route to prepare other privileged diterpene scaffolds.
RESUMO
Amyloidoses are a group of diseases associated with the formation of pathological protein fibrils with cross-ß structures. Approximately 5-10% of the cases of these diseases are determined by amyloidogenic mutations, as well as by transmission of infectious amyloids (prions) between organisms. The most common group of so-called sporadic amyloidoses is associated with abnormal aggregation of wild-type proteins. Some sporadic amyloidoses are known to be induced only against the background of certain pathologies, but in some cases the cause of amyloidosis is unclear. It is assumed that these diseases often occur by accident. Here we present facts and hypotheses about the association of sporadic amyloidoses with vascular pathologies, trauma, oxidative stress, cancer, metabolic diseases, chronic infections and COVID-19. Generalization of current data shows that all sporadic amyloidoses can be regarded as a secondary event occurring against the background of diseases provoking a cellular stress response. Various factors causing the stress response provoke protein overproduction, a local increase in the concentration or modifications, which contributes to amyloidogenesis. Progress in the treatment of vascular, metabolic and infectious diseases, as well as cancers, should lead to a significant reduction in the risk of sporadic amyloidoses.
Assuntos
Amiloidose/etiologia , Estresse Fisiológico , Lesões Encefálicas/complicações , Doenças Transmissíveis/complicações , Humanos , Doenças Metabólicas/complicações , Neoplasias/complicações , Estresse Oxidativo , Doenças Vasculares/complicaçõesRESUMO
The mitochondrial respiratory chain is the main site of reactive oxygen species (ROS) production in the cell. Although mitochondria possess a powerful antioxidant system, an excess of ROS cannot be completely neutralized and cumulative oxidative damage may lead to decreasing mitochondrial efficiency in energy production, as well as an increasing ROS excess, which is known to cause a critical imbalance in antioxidant/oxidant mechanisms and a "vicious circle" in mitochondrial injury. Due to insufficient energy production, chronic exposure to ROS overproduction consequently leads to the oxidative damage of life-important biomolecules, including nucleic acids, proteins, lipids, and amino acids, among others. Different forms of mitochondrial dysfunction (mitochondriopathies) may affect the brain, heart, peripheral nervous and endocrine systems, eyes, ears, gut, and kidney, among other organs. Consequently, mitochondriopathies have been proposed as an attractive diagnostic target to be investigated in any patient with unexplained progressive multisystem disorder. This review article highlights the pathomechanisms of mitochondriopathies, details advanced analytical tools, and suggests predictive approaches, targeted prevention and personalization of medical services as instrumental for the overall management of mitochondriopathy-related cascading pathologies.
Assuntos
Metabolismo Energético , Mitocôndrias/patologia , Doenças Mitocondriais/patologia , Estresse Oxidativo , Animais , Carcinogênese/patologia , Humanos , Mitocôndrias/metabolismo , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/metabolismo , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Medicina de Precisão , Espécies Reativas de Oxigênio/metabolismoRESUMO
Tumor necrosis factor-alpha (TNF-α) is an inflammatory cytokine that plays a major role in immune regulation, homeostatic function, and cellular organization. The present study was undertaken to overproduce recombinant human TNF-α (rhTNF-α) in Escherichia coli (E.coli) in high cell density culture. The use of a codon-optimized gene and strong promoter-based (T7) expression system, choice of Terrific Broth (TB) as medium, and subsequent optimization of culture conditions in shake flasks resulted in production of 0.95 g/L insoluble rhTNF-α comprising upto 50% of total cellular protein (TCP) The protein yield further increased upto 1.26 g/L in 1 L TB medium batch culture in bioreactor with the controlled temperature, pH, and dissolved oxygen. In a series of chemostats operated at dilution rates of 0.2 h-1, 0.3 h-1, 0.4 h-1 and 0.5 h-1 the specific growth rate (µ) positively correlated with specific yield (Yp/x) and a maximum yield of 164 mg/g DCW was obtained at µ = 0.4 h-1 within 4 h post-induction. A fed-batch cultivation in TB with an exponential feeding profile (µ = â¼0.4 h-1) of concentrated feed resulted in an accumulation of 5.5 g/L of rhTNF-α within 14 h of cultivation which accounted for â¼29% of TCP.
Assuntos
Biotecnologia/métodos , Escherichia coli/metabolismo , Proteínas Recombinantes/química , Fator de Necrose Tumoral alfa/química , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Meios de Cultura , Humanos , Concentração de Íons de Hidrogênio , Cinética , Oxigênio/metabolismo , Regiões Promotoras Genéticas , Proteínas/química , Solubilidade , TemperaturaRESUMO
Methylobacterium sp. CLZ was isolated from soil contaminated with chemical wastewater. This strain simultaneously synthesizes Pyrroloquinoline quinone (PQQ), Coenzyme Q10 (CoQ10), and carotenoids by utilizing methanol as a carbon source. Comparative genomic analysis was performed for five Methylobacterium strains. As per the outcomes, the Methylobacterium CLZ strain showed the smallest genome size and the lowest number of proteins. Thus, it can serve as an ideal cell model for investigating the biological process of Methylobacterium and constructing genetically engineered Methylobacterium. The Methylobacterium CLZ strain's pqqL gene, which does not occur in other Methylobacterium strains but plays a crucial role in PQQ synthesis. This was a surprising finding for the study of PQQ biosynthesis in Methylobacterium. Methylobacterium sp. NI91 strain was generated by random mutagenesis of CLZ strain, and NI91 strain showed a 72.44% increase in PQQ yield. The mutation in the mxaJ gene involved in the methanol dehydrogenase (MDH) synthesis was identified through comparative genomic analysis of the whole genome of mutant strain NI91 and wild-type strain CLZ. The mxaJ gene was found to be upregulated in the NI91 strain. Thus, the up-regulation of the mxaJ gene could be correlated with the high yield of PQQ, and it could provide valuable clues for strain engineering to improve PQQ production.
Assuntos
Proteínas de Bactérias/genética , Genômica/métodos , Methylobacterium/genética , Cofator PQQ/biossíntese , Carotenoides/metabolismo , Regulação Bacteriana da Expressão Gênica , Tamanho do Genoma , Methylobacterium/isolamento & purificação , Methylobacterium/metabolismo , Mutagênese , Microbiologia do Solo , Ubiquinona/análogos & derivados , Ubiquinona/biossíntese , Águas Residuárias/microbiologiaRESUMO
Structural and spectroscopic analysis of iron-sulfur [Fe-S] cluster-containing proteins is often limited by the occupancy and yield of recombinantly produced proteins. Here we report that Escherichia coli BL21(DE3), a strain routinely used to overproduce [Fe-S] cluster-containing proteins, has a nonfunctional Suf pathway, one of two E. coli [Fe-S] cluster biogenesis pathways. We confirmed that BL21(DE3) and commercially available derivatives carry a deletion that results in an in-frame fusion of sufA and sufB genes within the sufABCDSE operon. We show that this fusion protein accumulates in cells but is inactive in [Fe-S] cluster biogenesis. Restoration of an intact Suf pathway combined with enhanced suf operon expression led to a remarkable (â¼3-fold) increase in the production of the [4Fe-4S] cluster-containing BchL protein, a key component of the dark-operative protochlorophyllide oxidoreductase complex. These results show that this engineered "SufFeScient" derivative of BL21(DE3) is suitable for enhanced large-scale synthesis of an [Fe-S] cluster-containing protein.IMPORTANCE Large quantities of recombinantly overproduced [Fe-S] cluster-containing proteins are necessary for their in-depth biochemical characterization. Commercially available E. coli strain BL21(DE3) and its derivatives have a mutation that inactivates the function of one of the two native pathways (Suf pathway) responsible for cluster biogenesis. Correction of the mutation, combined with sequence changes that elevate Suf protein levels, can increase yield and cluster occupancy of [Fe-S] cluster-containing enzymes, facilitating the biochemical analysis of this fascinating group of proteins.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Adenosina Trifosfatases/genética , Vias Biossintéticas/genética , Vias Biossintéticas/fisiologia , Proteínas de Transporte/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas Ferro-Enxofre/genética , Óperon/genéticaRESUMO
Controlling metabolism of engineered microbes is important to modulate cell growth and production during a bioprocess. For example, external parameters such as light, chemical inducers, or temperature can act on metabolism of production strains by changing the abundance or activity of enzymes. Here, we created temperature-sensitive variants of an essential enzyme in arginine biosynthesis of Escherichia coli (argininosuccinate synthetase, ArgG) and used them to dynamically control citrulline overproduction and growth of E. coli. We show a method for high-throughput enrichment of temperature-sensitive ArgG variants with a fluorescent TIMER protein and flow cytometry. With 90 of the thus derived ArgG variants, we complemented an ArgG deletion strain showing that 90% of the strains exhibit temperature-sensitive growth and 69% of the strains are auxotrophic for arginine at 42 °C and prototrophic at 30 °C. The best temperature-sensitive ArgG variant enabled precise and tunable control of cell growth by temperature changes. Expressing this variant in a feedback-dysregulated E. coli strain allowed us to realize a two-stage bioprocess: a 33 °C growth-phase for biomass accumulation and a 39 °C stationary-phase for citrulline production. With this two-stage strategy, we produced 3 g/L citrulline during 45 h cultivation in a 1-L bioreactor. These results show that temperature-sensitive enzymes can be created en masse and that they may function as metabolic valves in engineered bacteria.
Assuntos
Argininossuccinato Sintase/genética , Argininossuccinato Sintase/metabolismo , Citrulina/biossíntese , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Arginina , Biomassa , Citometria de Fluxo , Glucose/metabolismo , Plasmídeos/genética , Proteômica , TemperaturaRESUMO
Cytokine storm is an important cause of acute respiratory distress syndrome and multiple organ failure. Excessive secretion and accumulation of mucins on the surface of airway cause airway obstruction and exacerbate lung infections. MUC5AC and MUC5B are the main secreted mucins and overexpressed in various inflammatory responses. S-allylmercaptocysteine, a water-soluble organic sulfur compound extracted from garlic, has anti-inflammatory and anti-oxidative effects for various pulmonary diseases. The aim of this work was to investigate the therapeutic effects of SAMC on mucin overproduction and inflammation in 16HBE cells and LPS-induced ARDS mice. Results show that SAMC treatment ameliorated inflammatory cell infiltration and lung histopathological changes in the LPS-induced ARDS mice. SAMC also inhibited the expressions of MUC5AC and MUC5B, decreased the production of pro-inflammatory markers (IL-6, TNF-α, CD86 and IL-12) and increased the production of anti-inflammatory markers (IL-10, CD206 and TGF-ß). These results confirm that SAMC had potential beneficial effects on suppressed hyperinflammation and mucin overexpression. Furthermore, SAMC exerted the therapeutic effects through the inhibition of phosphorylation of MAPKs and PI3K-Akt signaling pathways in the 16HBE cells and mice. Overall, our results demonstrate the effects of SAMC on the LPS-induced mucin overproduction and inflammation both in the 16HBE cells and mice.
Assuntos
Anti-Inflamatórios/farmacologia , Cisteína/análogos & derivados , Pulmão/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mucina-5AC/metabolismo , Mucina-5B/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Síndrome do Desconforto Respiratório/tratamento farmacológico , Animais , Linhagem Celular , Cisteína/farmacologia , Citocinas/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos , Pulmão/enzimologia , Pulmão/patologia , Camundongos Endogâmicos BALB C , Mucina-5AC/genética , Mucina-5B/genética , Fosforilação , Síndrome do Desconforto Respiratório/induzido quimicamente , Síndrome do Desconforto Respiratório/enzimologia , Síndrome do Desconforto Respiratório/genética , Transdução de Sinais , Regulação para CimaRESUMO
Overproduction of recombinant secretory proteins triggers numerous physiological perturbations. Depending on a given heterologous protein characteristics, the producer cell is faced with different challenges which lead to varying responses in terms of its physiology and the target protein production rate. In the present study, we used steady-state-maintained Yarrowia lipolytica cells to investigate the impact of different heterologous proteins on the physiological behavior of the host cells. Such an approach allowed to uncouple the impact of the overproduction of a particular protein from the phenomena that result from growth phase or are caused by the heterogeneity of the analyzed populations. Altogether, eight variants of recombinant strains, individually overproducing heterologous proteins of varying molecular weight (27-65 kDa) and reporting activity (enzymatic and fluorescent) were subjected to chemostat cultivations. The steady-state-maintained cells were analyzed in terms of the substrate utilization, biomass and metabolites production, as well as the reporter protein synthesis. Simplified distribution of carbon and nitrogen between the respective products, as well as expression analysis of the heterologous genes were conducted. The here-obtained data suggest that using a more transcriptionally active promoter results in channeling more C flux towards the target protein, giving significantly higher specific amounts and production rates of the target polypeptide, at the cost of biomass accumulation, and with no significant impact on the polyols production. The extent of the reporter protein's post-translational modifications, i.e., the number of disulfide bonds and glycosylation pattern, strongly impacts the synthesis process. Specific responses in terms of the protein formation kinetics, the gene expression levels, and transcript-to-protein linearity were observed.Key Points⢠Eight expression systems, producing different reporter proteins were analyzed.⢠The cells were maintained in steady-state by continuous chemostat culturing.⢠Protein- and promoter-specific effects were observed.