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1.
Microb Cell Fact ; 23(1): 88, 2024 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-38519954

RESUMO

BACKGROUND: The halophilic bacterium Halomonas elongata is an industrially important strain for ectoine production, with high value and intense research focus. While existing studies primarily delve into the adaptive mechanisms of this bacterium under fixed salt concentrations, there is a notable dearth of attention regarding its response to fluctuating saline environments. Consequently, the stress response of H. elongata to salt shock remains inadequately understood. RESULTS: This study investigated the stress response mechanism of H. elongata when exposed to NaCl shock at short- and long-time scales. Results showed that NaCl shock induced two major stresses, namely osmotic stress and oxidative stress. In response to the former, within the cell's tolerable range (1-8% NaCl shock), H. elongata urgently balanced the surging osmotic pressure by uptaking sodium and potassium ions and augmenting intracellular amino acid pools, particularly glutamate and glutamine. However, ectoine content started to increase until 20 min post-shock, rapidly becoming the dominant osmoprotectant, and reaching the maximum productivity (1450 ± 99 mg/L/h). Transcriptomic data also confirmed the delayed response in ectoine biosynthesis, and we speculate that this might be attributed to an intracellular energy crisis caused by NaCl shock. In response to oxidative stress, transcription factor cysB was significantly upregulated, positively regulating the sulfur metabolism and cysteine biosynthesis. Furthermore, the upregulation of the crucial peroxidase gene (HELO_RS18165) and the simultaneous enhancement of peroxidase (POD) and catalase (CAT) activities collectively constitute the antioxidant defense in H. elongata following shock. When exceeding the tolerance threshold of H. elongata (1-13% NaCl shock), the sustained compromised energy status, resulting from the pronounced inhibition of the respiratory chain and ATP synthase, may be a crucial factor leading to the stagnation of both cell growth and ectoine biosynthesis. CONCLUSIONS: This study conducted a comprehensive analysis of H. elongata's stress response to NaCl shock at multiple scales. It extends the understanding of stress response of halophilic bacteria to NaCl shock and provides promising theoretical insights to guide future improvements in optimizing industrial ectoine production.


Assuntos
Diamino Aminoácidos , Halomonas , Cloreto de Sódio/farmacologia , Cloreto de Sódio/metabolismo , Halomonas/genética , Halomonas/metabolismo , Pressão Osmótica , Perfilação da Expressão Gênica , Peroxidases/metabolismo
2.
Int J Biol Macromol ; 261(Pt 2): 129838, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38307428

RESUMO

A novel α-amylase Amy03713 was screened and cloned from the starch utilization strain Vibrio alginolyticus LHF01. When heterologously expressed in Escherichia coli, Amy03713 exhibited the highest enzyme activity at 45 °C and pH 7, maintained >50 % of the enzyme activity in the range of 25-75 °C and pH 5-9, and sustained >80 % of the enzyme activity in 25 % (w/v) of NaCl solution, thus showing a wide range of adapted temperatures, pH, and salt concentrations. Halomonas bluephagenesis harboring amy03713 gene was able to directly utilize starch. With optimized amylase expression, H. bluephagenesis could produce poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB). When cultured for PHB production, recombinant H. bluephagenesis was able to grow up to a cell dry weight of 11.26 g/L, achieving a PHB titer of 6.32 g/L, which is the highest titer that has been reported for PHB production from starch in shake flasks. This study suggests that Amy03713 is an ideal amylase for PHA production using starch as the carbon source in H. bluephagenesis.


Assuntos
Halomonas , Ácidos Pentanoicos , Poli-Hidroxialcanoatos , Halomonas/genética , Halomonas/metabolismo , Carbono/metabolismo , Amido/metabolismo , Hidroxibutiratos/metabolismo , alfa-Amilases/genética , alfa-Amilases/metabolismo , Poliésteres/metabolismo
3.
Metab Eng ; 82: 238-249, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38401747

RESUMO

Ectoine, a crucial osmoprotectant for salt adaptation in halophiles, has gained growing interest in cosmetics and medical industries. However, its production remains challenged by stringent fermentation process in model microorganisms and low production level in its native producers. Here, we systematically engineered the native ectoine producer Halomonas bluephagenesis for ectoine production by overexpressing ectABC operon, increasing precursors availability, enhancing product transport system and optimizing its growth medium. The final engineered H. bluephagenesis produced 85 g/L ectoine in 52 h under open unsterile incubation in a 7 L bioreactor in the absence of plasmid, antibiotic or inducer. Furthermore, it was successfully demonstrated the feasibility of decoupling salt concentration with ectoine synthesis and co-production with bioplastic P(3HB-co-4HB) by the engineered H. bluephagenesis. The unsterile fermentation process and significantly increased ectoine titer indicate that H. bluephagenesis as the chassis of Next-Generation Industrial Biotechnology (NGIB), is promising for the biomanufacturing of not only intracellular bioplastic PHA but also small molecular compound such as ectoine.


Assuntos
Diamino Aminoácidos , Halomonas , Halomonas/genética , Diamino Aminoácidos/genética , Antibacterianos , Biopolímeros
4.
Curr Opin Biotechnol ; 85: 103064, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38262074

RESUMO

The use of extremophile organisms such as Halomomas spp. can eliminate the need for fermentation sterilization, significantly reducing process costs. Microbial fermentation is considered a pivotal strategy to reduce reliance on fossil fuel resources; however, sustainable processes continue to incur higher costs than their chemical industry counterparts. Most organisms require equipment sterilization to prevent contamination, a practice that introduces complexity and financial strain. Fermentations involving extremophile organisms can eliminate the sterilization process, relying instead on conditions that are conductive solely to the growth of the desired organism. This review discusses current challenges in pilot- and industrial-scale bioproduction when using the extremophile bacteria Halomomas spp. under nonsterile conditions.


Assuntos
Halomonas , Fermentação , Bactérias
5.
Environ Res ; 246: 118157, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38199468

RESUMO

Halomonas spp. are moderately halophilic bacteria with the ability to tolerate various heavy metals. However, the role of basic cellular metabolism, particularly amino acid metabolism, has not been investigated in Halomonas spp. under excess Mn(Ⅱ). The strain Halomonas sp. MNB13 was isolated from a deep-sea ferromanganese nodule and can tolerate 80 mM Mn(Ⅱ). To comprehensively explore the mechanisms underlying its resistance to excess Mn(Ⅱ), we conducted a comparative proteome analysis. The data revealed that both 10 mM and 50 mM Mn(Ⅱ) significantly up-regulated the expression of proteins involved in Mn(Ⅱ) transport (MntE), oxidative stress response (alkyl hydroperoxide reductase and the Suf system), and amino acid metabolism (arginine, cysteine, methionine, and phenylalanine). We further investigated the role of cysteine metabolism in Mn(Ⅱ) resistance by examining the function of its downstream product, H2S. Consistent with the up-regulation of cysteine desulfurase, we detected an elevated level of H2S in Halomonas sp. MNB13 cells under Mn(Ⅱ) stress, along with increased intracellular levels of H2O2 and O2•-. Upon exogenous addition of H2S, we observed a significant restoration of the growth of Halomonas sp. MNB13. Moreover, we identified decreased intracellular levels of H2O2 and O2•- in MNB13 cells, which coincided with a decreased formation of Mn-oxides during cultivation. In contrast, in cultures containing NaHS, the residual Mn(Ⅱ) levels were higher than in cultures without NaHS. Therefore, H2S improves Mn(Ⅱ) tolerance by eliminating intracellular reactive oxygen species rather than decreasing Mn(Ⅱ) concentration in solution. Our findings indicate that cysteine metabolism, particularly the intermediate H2S, plays a pivotal role in Mn(Ⅱ) resistance by mitigating the damage caused by reactive oxygen species. These findings provide new insights into the amino acid mechanisms associated with Mn(Ⅱ) resistance in bacteria.


Assuntos
Halomonas , Proteômica , Halomonas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Cisteína/metabolismo , Peróxido de Hidrogênio
6.
Carbohydr Res ; 536: 109019, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38211449

RESUMO

Lipopolysaccharide was obtained from the aerobic moderately halophilic bacterium Halomonas fontilapidosi KR26. The O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide and was examined by chemical methods and by 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, and 1H,13C HSQC, and HMBC experiments. The following structure of the linear tetrasaccharide repeating unit was deduced. →2)-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-ß-d-Galp-(1→.


Assuntos
Halomonas , Lipopolissacarídeos , Polissacarídeos/química , Espectroscopia de Ressonância Magnética , Antígenos O/química
7.
Syst Appl Microbiol ; 47(1): 126488, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38278082

RESUMO

Four vanillic acid-degrading bacterial strains, named LR5S13T, LR5S20, and M4R5S39T and LN1S58, were isolated from Kalidium cuspidatum rhizosphere and bulk soils, respectively. Phylogenetic analysis based on 16S rRNA gene as well as core genome revealed that LR5S13T and LR5S20 clustered closely with each other and with Halomonas ventosae Al12T, and that the two strains shared the highest similarities (both 99.3 %) with H. ventosae Al12T, in contrast, M4R5S39T and LN1S58 clustered together and with Halomonas heilongjiangensis 9-2T, and the two strains shared the highest similarities (99.4 and 99.2 %, respectively) with H. heilongjiangensis 9-2T. The average nucleotides identities based on BLAST (ANIb) and digital DNA-DNA hybridization (dDDH) values of strains LR5S13T to LR5S20, and M4R5S39T to LN1S58, were both higher than the threshold values for delineation of a species. The ANIb and dDDH values of the four strains to their closely relatives were lower than the threshold values. All four strains take phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol as the major polar lipids, Summed Feature 8, Summed Feature 3, and C16:0 as the major fatty acids. Based on the phylogenetic and phenotypic results, the four strains should be classified as two novel Halomonas species. Therefore, Halomonas rhizosphaerae sp. nov. (type strain LR5S13T = KCTC 8016T = CGMCC 1.62049T) and Halomonas kalidii (type strain M4R5S39T = KCTC 8015T = CGMCC 1.62047T) are proposed. The geographical distribution analysis based on 16S rRNA gene revealed that the two novel species are widely distributed across the globe, specifically in highly saline habits, especially in Central and Eastern Asia.


Assuntos
Halomonas , Hidroxibenzoatos , Halomonas/genética , Fosfolipídeos , Análise de Sequência de DNA , Filogenia , RNA Ribossômico 16S/genética , Ácidos Graxos/análise , DNA , DNA Bacteriano/genética , Técnicas de Tipagem Bacteriana , Hibridização de Ácido Nucleico
8.
Artigo em Inglês | MEDLINE | ID: mdl-38265421

RESUMO

Eight Gram-stain-negative bacterial strains were isolated from cheese rinds sampled in France. On the basis of 16S rRNA gene sequence analysis, all isolates were assigned to the genus Halomonas. Phylogenetic investigations, including 16S rRNA gene studies, multilocus sequence analysis, reconstruction of a pan-genome phylogenetic tree with the concatenated core-genome content and average nucleotide identity (ANI) calculations, revealed that they constituted three novel and well-supported clusters. The closest relative species, determined using the whole-genome sequences of the strains, were Halomonas zhanjiangensis for two groups of cheese strains, sharing 82.4 and 93.1 % ANI, and another cluster sharing 92.2 % ANI with the Halomonas profundi type strain. The strains isolated herein differed from the previously described species by ANI values <95 % and several biochemical, enzymatic and colony characteristics. The results of phenotypic, phylogenetic and chemotaxonomic analyses indicated that the isolates belonged to three novel Halomonas species, for which the names Halomonas citrativorans sp. nov., Halomonas casei sp. nov. and Halomonas colorata sp. nov. are proposed, with isolates FME63T (=DSM 113315T=CIRM-BIA2430T=CIP 111880T=LMG 32013T), FME64T (=DSM 113316T=CIRM-BIA2431T=CIP 111877T=LMG 32015T) and FME66T (=DSM 113318T=CIRM-BIA2433T=CIP 111876T=LMG 32014T) as type strains, respectively.


Assuntos
Queijo , Halomonas , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , DNA Bacteriano/genética , Técnicas de Tipagem Bacteriana , Composição de Bases , Ácidos Graxos/química , Nucleotídeos
9.
Int J Biol Macromol ; 254(Pt 1): 127475, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37863147

RESUMO

Polyhydroxybutyrate (PHB) is a well-known biodegradable bioplastic synthesized by microorganisms and can be produced from volatile fatty acids (VFAs). Among VFAs acetate can be utilized by Halomonas sp. YLGW01 for growth and PHB production. In this study, Halomonas sp. JJY01 was developed through introducing acetyl-CoA acetyltransferase (atoAD) with LacIq-Ptrc promoter into Halomonas sp. YLGW01. The effect of expression of atoAD on acetate was investigated by comparison with acetate consumption and PHB production. Shake-flask study showed that Halomonas sp. JJY01 increased acetate consumption rate, PHB yield and PHB production (0.27 g/L/h, 0.075 g/g, 0.72 g/L) compared to the wild type strain (0.17 g/L/h, 0.016 g/g, 0.11 g/L). In 10 L fermenter scale fed-batch fermentation, the growth of Halomonas sp. JJY01 resulted in higher acetate consumption rate, PHB yield and PHB titer (0.55 g/L/h, 0.091 g/g, 4.6 g/L) than wild type strain (0.35 g/L/h, 0.067 h/h, 2.9 g/L). These findings demonstrate enhanced acetate utilization and PHB production through the introduction of atoAD in Halomonas strains.


Assuntos
Halomonas , Hidroxibutiratos , Hidroxibutiratos/metabolismo , Halomonas/genética , Halomonas/metabolismo , Acetil-CoA C-Acetiltransferase/metabolismo , Poli-Hidroxibutiratos , Acetatos/metabolismo , Poliésteres/metabolismo
10.
Lab Med ; 55(1): 80-87, 2024 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-37210212

RESUMO

OBJECTIVE: The aim of this study was to identify the species of a Halomonas strain isolated from a neonatal blood sample and to understand the potential pathogenicity and characteristic genes of the strain. METHODS: The genomic DNA of strain 18071143 (identified as Halomonas by matrix-assisted laser desorption-ionization time of flight-mass spectrometry and the 16S ribosomal RNA (rRNA) gene sequence) was sequenced using Nanopore PromethION platforms. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were calculated using the complete genome sequences of the strain. Comparative genomic analyses were performed on strain 18071143 and 3 strains of Halomonas (Halomonas stevensii S18214, Halomonas hamiltonii KCTC 22154, and Halomonas johnsoniae KCTC 22157) that were associated with human infections and had high genomic similarity to strain 18071143. RESULTS: Phylogenetic, ANI, and dDDH similarity analyses based on genome sequence indicated that strain 18071143 belonged to the species H stevensii. Similarities exist between strain 18071143 and the other 3 Halomonas strains in terms of gene structure and protein function. Nonetheless, strain 18071143 has greater potential for DNA replication, recombination, repair, and horizontal transfer. CONCLUSION: Whole-genome sequencing holds great promise for accurate strain identification in clinical microbiology. In addition, the results of this study provide data for understanding Halomonas from the perspective of pathogenic bacteria.


Assuntos
Halomonas , Recém-Nascido , Humanos , Halomonas/genética , Ácidos Graxos/química , Análise de Sequência de DNA , Filogenia , Genômica , DNA , RNA Ribossômico 16S/genética , DNA Bacteriano/genética , DNA Bacteriano/química
11.
Bioresour Technol ; 394: 130175, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38086463

RESUMO

Polyhydroxyalkanoates (PHA) have emerged as a promising bio-compound in the industrial application due to their potential to replace conventional petroleum-based plastics with sustainable bioplastics. This study focuses on Halomonas sp. YJPS3-3, a halophilic bacterium, and presents a novel approach to enhance PHA production by exploiting its salt tolerance toward PHA biosynthesis. Through gamma irradiation-induced mutants with enhanced salt tolerance from 15% NaCl to 20% NaCl, mutant halo6 showing a significant 11% increase in PHA yield, was achieved. Moreover, the mutants displayed not only higher PHA content but also remarkable cell morphology with elongation. In addition, this research unravels the genetic determinants behind the elevated PHA content and identifies a corresponding shift in fatty acid composition favoring PHA accumulation. This novel mutant obtained from gamma irradiation with enhanced salt tolerance in halophilic bacteria opens up new avenues not only for the bioplastic industry but also for applications in the production of high-value metabolites.


Assuntos
Halomonas , Poli-Hidroxialcanoatos , Poli-Hidroxibutiratos , Ácido 3-Hidroxibutírico/metabolismo , Tolerância ao Sal , Cloreto de Sódio/farmacologia , Cloreto de Sódio/metabolismo , Poli-Hidroxialcanoatos/metabolismo , Biopolímeros/metabolismo , Halomonas/genética , Halomonas/metabolismo
12.
Metab Eng ; 81: 227-237, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38072357

RESUMO

5-Aminovaleric acid (5-AVA), 5-hydroxyvalerate (5HV), copolymer P(3HB-co-5HV) of 3-hydroxybutyrate (3HB) and 5HV were produced from L-lysine as a substrate by recombinant Halomonas bluephagenesis constructed based on codon optimization, deletions of competitive pathway and L-lysine export protein, and three copies of davBA genes encoding L-lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) inserted into its genome to form H. bluephagenesis YF117ΔgabT1+2, which produced 16.4 g L-1 and 67.4 g L-1 5-AVA in flask cultures and in 7 L bioreactor, respectively. It was able to de novo synthesize 5-AVA from glucose by L-lysine-overproducing H. bluephagenesis TD226. Corn steep liquor was used instead of yeast extract for cost reduction during the 5-AVA production. Using promoter engineering based on Pporin mutant library for downstream genes, H. bluephagenesis YF117 harboring pSEVA341-Pporin42-yqhDEC produced 6 g L-1 5HV in shake flask growth, while H. bluephagenesis YF117 harboring pSEVA341-Pporin42-yqhDEC-Pporin278-phaCRE-abfT synthesized 42 wt% P(3HB-co-4.8 mol% 5HV) under the same condition. Thus, H. bluephagenesis was successfully engineered to produce 5-AVA and 5HV in supernatant and intracellular P(3HB-co-5HV) utilizing L-lysine as the substrate.


Assuntos
Halomonas , Engenharia Metabólica , Lisina/genética , Lisina/metabolismo , Halomonas/genética , Halomonas/metabolismo , Ácido 3-Hidroxibutírico/metabolismo , Poliésteres/metabolismo , Porinas/genética , Porinas/metabolismo
13.
Appl Environ Microbiol ; 90(1): e0190523, 2024 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-38112419

RESUMO

A moderately halophilic eubacterium, Halomonas elongata, has been used as cell factory to produce fine chemical 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine), which functions as a major osmolyte protecting the cells from high-salinity stress. To explore the possibility of using H. elongata to biosynthesize other valuable osmolytes, an ectoine-deficient salt-sensitive H. elongata deletion mutant strain KA1 (ΔectABC), which only grows well in minimal medium containing up to 3% NaCl, was subjected to an adaptive mutagenesis screening in search of mutants with restored salt tolerance. Consequently, we obtained a mutant, which tolerates 6% NaCl in minimal medium by overproducing L-glutamic acid (Glu). However, this Glu-overproducing (GOP) strain has a lower tolerance level than the wild-type H. elongata, possibly because the acidity of Glu interferes with the pH homeostasis of the cell and hinders its own cellular accumulation. Enzymatic decarboxylation of Glu to γ-aminobutyric acid (GABA) by a Glu decarboxylase (GAD) could restore cellular pH homeostasis; therefore, we introduced an engineered salt-inducible HopgadBmut gene, which encodes a wide pH-range GAD mutant, into the genome of the H. elongata GOP strain. We found that the resulting H. elongata GOP-Gad strain exhibits higher salt tolerance than the GOP strain by accumulating high concentration of GABA as an osmolyte in the cell (176.94 µmol/g cell dry weight in minimal medium containing 7% NaCl). With H. elongata OUT30018 genetic background, H. elongata GOP-Gad strain can utilize biomass-derived carbon and nitrogen compounds as its sole carbon and nitrogen sources, making it a good candidate for the development of GABA-producing cell factories.IMPORTANCEWhile the wild-type moderately halophilic H. elongata can synthesize ectoine as a high-value osmolyte via the aspartic acid metabolic pathway, a mutant H. elongata GOP strain identified in this work opens doors for the biosynthesis of alternative valuable osmolytes via glutamic acid metabolic pathway. Further metabolic engineering to install a GAD system into the H. elongata GOP strain successfully created a H. elongata GOP-Gad strain, which acquired higher tolerance to salt stress by accumulating GABA as a major osmolyte. With the ability to assimilate biomass-derived carbon and nitrogen sources and thrive in high-salinity environment, the H. elongata GOP-Gad strain can be used in the development of sustainable GABA-producing cell factories.


Assuntos
Diamino Aminoácidos , Halomonas , Tolerância ao Sal , Ácido Glutâmico/metabolismo , Halomonas/genética , Engenharia Metabólica , Salinidade , Cloreto de Sódio/metabolismo , Carbono/metabolismo , Nitrogênio/metabolismo , Ácido gama-Aminobutírico/metabolismo
14.
Environ Microbiol Rep ; 16(1): e13225, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38146695

RESUMO

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters produced by a wide range of microorganisms, including extremophiles. These unique microorganisms have gained interest in PHA production due to their ability to utilise low-cost carbon sources under extreme conditions. In this study, Halomonas alkaliantarctica was examined with regards to its potential to produce PHAs using crude glycerol from biodiesel industry as the only carbon source. We found that cell dry mass concentration was not dependent on the applying substrate concentration. Furthermore, our data confirmed that the analysed halophile was capable of metabolising crude glycerol into poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer within 24 h of the cultivation without addition of any precursors. Moreover, crude glycerol concentration affects the repeat units content in the purified PHAs copolymers and their thermal properties. Nevertheless, a differential scanning calorimetric and thermogravimetric analysis showed that the analysed biopolyesters have properties suitable for various applications. Overall, this study described a promising approach for the valorisation of crude glycerol as a future strategy of industrial waste management to produce high value microbial biopolymers.


Assuntos
Glicerol , Halomonas , Ácidos Pentanoicos , Poli-Hidroxialcanoatos , Poli-Hidroxibutiratos , Biocombustíveis , Poli-Hidroxialcanoatos/química , Hidroxibutiratos , Carbono
15.
ACS Synth Biol ; 13(1): 61-67, 2024 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-38100561

RESUMO

Halomonas bluephagenesis is a halophilic bacterium capable of efficiently producing polyhydroxyalkanoates and other valuable chemicals through high salinity open fermentation, offering an appealing platform for next-generation industrial biotechnology. Various techniques have been developed to engineer Halomonas bluephagenesis, each with its inherent shortcomings. Genome editing methods often entail complex and time-consuming processes, while flexible expression systems relying on plasmids necessitate the use of antibiotics. In this study, we developed a stable recombinant plasmid vector, pHbPBC, based on a novel hbpB/hbpC toxin-antitoxin system found within the endogenous plasmid of Halomonas bluephagenesis. Remarkably, pHbPBC exhibited exceptional stability during 7 days of continuous subculture, eliminating the need for antibiotics or other selection pressures. This stability even rivaled genomic integration, all while achieving higher levels of heterologous expression. Our research introduces a novel approach for genetically modifying and harnessing nonmodel halophilic bacteria, contributing to the advancement of next-generation industrial biotechnology.


Assuntos
Halomonas , Poli-Hidroxialcanoatos , Sistemas Toxina-Antitoxina , Halomonas/genética , Halomonas/metabolismo , Poli-Hidroxialcanoatos/metabolismo , Biotecnologia/métodos , Antibacterianos/metabolismo
16.
Metab Eng ; 81: 249-261, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38159902

RESUMO

Predictability and robustness are challenges for bioproduction because of the unstable intracellular synthetic activities. With the deeper understanding of the gene expression process, fine-tuning has become a meaningful tool for biosynthesis optimization. This study characterized several gene expression elements and constructed a multiple inducible system that responds to ten different small chemical inducers in halophile bacterium Halomonas bluephagenesis. Genome insertion of regulators was conducted for the purpose of gene cluster stabilization and regulatory plasmid simplification. Additionally, dynamic ranges of the multiple inducible systems were tuned by promoter sequence mutations to achieve diverse scopes for high-resolution gene expression control. The multiple inducible system was successfully employed to precisely control chromoprotein expression, lycopene and poly-3-hydroxybutyrate (PHB) biosynthesis, resulting in colorful bacterial pictures, optimized cell growth, lycopene and PHB accumulation. This study demonstrates a desirable approach for fine-tuning of rational and efficient gene expressions, displaying the significance for metabolic pathway optimization.


Assuntos
Halomonas , Poliésteres , Poliésteres/metabolismo , Halomonas/genética , Halomonas/metabolismo , Licopeno/metabolismo , Biotecnologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Engenharia Metabólica/métodos
17.
Sci Rep ; 13(1): 22289, 2023 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-38097607

RESUMO

Currently, the global demand for polyhydroxyalkanoates (PHAs) is significantly increasing. PHAs are produced by several bacteria that are an alternative source of synthetic polymers derived from petrochemical refineries. This study established a simple and more feasible process of PHA production by Halomonas alkaliantarctica using dairy waste as the only carbon source. The data confirmed that the analyzed halophile could metabolize cheese whey (CW) and cheese whey mother liquor (CWML) into biopolyesters. The highest yield of PHAs was 0.42 g/L in the cultivation supplemented with CWML. Furthermore, it was proved that PHA structure depended on the type of by-product from cheese manufacturing, its concentration, and the culture time. The results revealed that H. alkaliantarctica could produce P(3HB-co-3HV) copolymer in the cultivations with CW at 48 h and 72 h without adding of any precursors. Based on the data obtained from physicochemical and thermal analyses, the extracted copolymer was reported to have properties suitable for various applications. Overall, this study described a promising approach for valorizing of dairy waste as a future strategy of industrial waste management to produce high value microbial biopolymers.


Assuntos
Halomonas , Poli-Hidroxialcanoatos , Poli-Hidroxialcanoatos/química , Biopolímeros , Resíduos Industriais , Proteínas do Soro do Leite
18.
BMC Genomics ; 24(1): 696, 2023 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-37986038

RESUMO

BACKGROUND: Isabel Island is a Mexican volcanic island primarily composed of basaltic stones. It features a maar known as Laguna Fragatas, which is classified as a meromictic thalassohaline lake. The constant deposition of guano in this maar results in increased levels of phosphorus, nitrogen, and carbon. The aim of this study was to utilize high-quality genomes from the genus Halomonas found in specialized databases as a reference for genome mining of moderately halophilic bacteria isolated from Laguna Fragatas. This research involved genomic comparisons employing phylogenetic, pangenomic, and metabolic-inference approaches. RESULTS: The Halomonas genus exhibited a large open pangenome, but several genes associated with salt metabolism and homeostatic regulation (ectABC and betABC), nitrogen intake through nitrate and nitrite transporters (nasA, and narGI), and phosphorus uptake (pstABCS) were shared among the Halomonas isolates. CONCLUSIONS: The isolated bacteria demonstrate consistent adaptation to high salt concentrations, and their nitrogen and phosphorus uptake mechanisms are highly optimized. This optimization is expected in an extremophile environment characterized by minimal disturbances or abrupt seasonal variations. The primary significance of this study lies in the dearth of genomic information available for this saline and low-disturbance environment. This makes it important for ecosystem conservation and enabling an exploration of its biotechnological potential. Additionally, the study presents the first two draft genomes of H. janggokensis.


Assuntos
Halomonas , Halomonas/genética , Halomonas/metabolismo , Lagos/microbiologia , Filogenia , Ecossistema , Genômica , Nitrogênio/metabolismo , Fósforo/metabolismo , RNA Ribossômico 16S/genética , DNA Bacteriano/genética
19.
Microb Cell Fact ; 22(1): 211, 2023 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-37838676

RESUMO

BACKGROUND: Halophiles possess several unique properties and have broad biotechnological applications including industrial biotechnology production. Halomonas spp., especially Halomonas bluephagenesis, have been engineered to produce various biopolyesters such as polyhydroxyalkanoates (PHA), some proteins, small molecular compounds, organic acids, and has the potential to become a chassis cell for the next-generation of industrial biotechnology (NGIB) owing to its simple culture, fast growth, contamination-resistant, low production cost, and high production value. An efficient genome editing system is the key for its engineering and application. However, the efficiency of the established CRISPR-Cas-homologous recombination (HR) gene editing tool for large DNA fragments was still relatively low. In this study, we firstly report a CRISPR-Cas9 gene editing system combined with a non-homologous end joining (NHEJ) repair system for efficient large DNA fragment deletion in Halomonas bluephagenesis. RESULTS: Three different NHEJ repair systems were selected and functionally identified in Halomonas bluephagenesis TD01. The NHEJ system from M. tuberculosis H37Rv (Mt-NHEJ) can functionally work in H. bluephagenesis TD01, resulting in base deletion of different lengths for different genes and some random base insertions. Factors affecting knockout efficiencies, such as the number and position of sgRNAs on the DNA double-strands, the Cas9 protein promoter, and the interaction between the HR and the NHEJ repair system, were further investigated. Finally, the optimized CRISPR-Cas9-NHEJ editing system was able to delete DNA fragments up to 50 kb rapidly with high efficiency of 31.3%, when three sgRNAs on the Crick/Watson/Watson DNA double-strands and the arabinose-induced promoter Para for Cas9 were used, along with the background expression of the HR repair system. CONCLUSIONS: This was the first report of CRISPR-Cas9 gene editing system combined with a non-homologous end joining (NHEJ) repair system for efficient large DNA fragment deletion in Halomonas spp. These results not only suggest that this editing system is a powerful genome engineering tool for constructing chassis cells in Halomonas, but also extend the application of the NHEJ repair system.


Assuntos
Edição de Genes , Halomonas , Sistemas CRISPR-Cas , Halomonas/genética , RNA Guia de Sistemas CRISPR-Cas , DNA
20.
Carbohydr Polym ; 320: 121203, 2023 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-37659791

RESUMO

Based on stimuli in the biological milieu, macrophages can undergo classical activation into the M1 pro-inflammatory (anti-cancer) phenotype or to the alternatively activated M2 anti-inflammatory one. Drug-free biomaterials have emerged as a new therapeutic strategy to modulate macrophage phenotype. Among them, polysaccharides polarize macrophages to M1 or M2 phenotypes based on the surface receptors they bind. Levan, a fructan, has been proposed as a novel biomaterial though its interaction with macrophages has been scarcely explored. In this study, we investigate the interaction of non-hydrolyzed and hydrolyzed Halomonas levan and its sulfated derivative with human macrophages in vitro. Viability studies show that these levans are cell compatible. In addition, RNA-sequencing analysis reveals the upregulation of pro-inflammatory pathways. These results are in good agreement with real time-quantitative polymerase chain reaction that indicates higher expression levels of C-X-C Motif Chemokine Ligand 8 and interleukin-6 genes and the M2-to-M1 reprogramming of these cells upon levan treatment. Finally, cytokine release studies confirm that hydrolyzed levans increase the secretion of pro-inflammatory cytokines and reprogram IL-4-polarized macrophages to the M1 state. Overall findings indicate that Halomonas levans trigger a classical macrophage activation and pave the way for their application in therapeutic interventions requiring a pro-inflammatory phenotype.


Assuntos
Halomonas , Transcriptoma , Humanos , Perfilação da Expressão Gênica , Frutanos/farmacologia , Materiais Biocompatíveis , Citocinas/genética , Macrófagos
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