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1.
Braz J Microbiol ; 55(1): 1023-1028, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38200375

RESUMO

The mechanism of colonisation of the chicken intestine by Salmonella remains poorly understood, while the severity of infections vary enormously depending on the serovar and the age of the bird. Several metabolism and virulence genes have been identified in Salmonella Heidelberg; however, information on their roles in infection, particularly in the chicken infection model, remains scarce. In the present publication, we investigated three Salmonella Heidelberg mutants containing deletions in misL, ssa, and pta-ackA genes by using signature-tagged mutagenesis. We found that mutations in these genes of S. Heidelberg result in an increase in fitness in the chicken model. The exception was perhaps the pta-ackA mutant where colonisation was slightly reduced (2, 7, 14, and 21 days post-infection) although some birds were still excreting at the end of the experiment. Our results suggest that for intestinal colonisation of the chicken caecum, substrate-level phosphorylation is likely to be more important than the MisL outer membrane protein or even the secretion system apparatus. These findings validate previous work that demonstrated the contribution of ackA and pta mutants to virulence in chickens, suggesting that the anaerobic metabolism genes such as pta-ackA could be a promising mitigation strategy to reduce S. Heidelberg virulence.


Assuntos
Galinhas , Salmonelose Animal , Animais , Fosforilação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fosfato Acetiltransferase/genética , Fosfato Acetiltransferase/metabolismo , Anaerobiose , Virulência , Salmonella , Salmonelose Animal/microbiologia
2.
PLoS One ; 18(5): e0283952, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37200262

RESUMO

The mechanisms of energy generation and carbon-source utilization in the syphilis spirochete Treponema pallidum have remained enigmatic despite complete genomic sequence information. Whereas the bacterium harbors enzymes for glycolysis, the apparatus for more efficient use of glucose catabolites, namely the citric-acid cycle, is apparently not present. Yet, the organism's energy needs likely exceed the modest output from glycolysis alone. Recently, building on our structure-function studies of T. pallidum lipoproteins, we proposed a "flavin-centric" metabolic lifestyle for the organism that partially resolves this conundrum. As a part of the hypothesis, we have proposed that T. pallidum contains an acetogenic energy-conservation pathway that catabolizes D-lactate, yielding acetate, reducing equivalents for the generation and maintenance of chemiosmotic potential, and ATP. We already have confirmed the D-lactate dehydrogenase activity in T. pallidum necessary for this pathway to operate. In the current study, we focused on another enzyme ostensibly involved in treponemal acetogenesis, phosphotransacetylase (Pta). This enzyme is putatively identified as TP0094 and, in this study, we determined a high-resolution (1.95 Å) X-ray crystal structure of the protein, finding that its fold comports with other known Pta enzymes. Further studies on its solution behavior and enzyme activity confirmed that it has the properties of a Pta. These results are consistent with the proposed acetogenesis pathway in T. pallidum, and we propose that the protein be referred to henceforth as TpPta.


Assuntos
Sífilis , Treponema pallidum , Humanos , Treponema pallidum/genética , Fosfato Acetiltransferase/metabolismo , Proteínas de Bactérias/metabolismo , Sífilis/microbiologia , Treponema/genética
3.
Microbiology (Reading) ; 168(9)2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36048631

RESUMO

Successful adaptation of Escherichia coli to constant environmental challenges demands the operation of a wide range of regulatory control mechanisms, some of which are global, while others are specific. Here, we show that the ability of acetate-negative phenotype strains of E. coli devoid of acetate kinase (AK) and phosphotransacetylase (PTA) to assimilate acetate when challenged at the end of growth on acetogenic substrates is explicable by the co-expression of acetyl CoA-synthetase (AcCoA-S) and acetate permease (AP). Furthermore, mRNA transcript measurements for acs and aceA, together with the enzymatic activities of their corresponding enzymes, acetyl CoA synthetase (AcCoA-S) and isocitrate lyase (ICL), clearly demonstrate that the expression of the two enzymes is inextricably linked and triggered in response to growth rate threshold signal (0.4 h-1± 0.03: n4). Interestingly, further restriction of carbon supply to the level of starvation led to the repression of acs (AcCoA-S), ackA (AK) and pta (PTA). Further, we provide evidence that the reaction sequence catalysed by PTA, AK and AcCoA-S is not in operation at low growth rates and that the reaction catalysed by AcCoA-S is not merely an ATP-dissipating reaction but rather advantageous, as it elevates the available free energy (ΔG°) in central metabolism. Moreover, the transcriptomic data reinforce the view that the expression of PEP carboxykinase is essential in gluconeogenic phenotypes.


Assuntos
Acetato-CoA Ligase , Escherichia coli , Acetato Quinase/genética , Acetato Quinase/metabolismo , Acetato-CoA Ligase/genética , Acetato-CoA Ligase/metabolismo , Acetatos/metabolismo , Acetilcoenzima A/metabolismo , Escherichia coli/metabolismo , Óperon , Fosfato Acetiltransferase/genética , Fosfato Acetiltransferase/metabolismo
4.
Microbiol Res ; 265: 127172, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36084615

RESUMO

In this study, it was found that reducing consumption of acetyl-CoA in mitochondria, peroxisome and lipid biosynthesis could not obviously enhance liamocin biosynthesis by engineered strains of Aureobasidium melanogenm 9-1, but decreased cell growth of the mutants. On the contrary, expression of heterologous PTA gene for phosphotransacetylase in PK pathway and native ALD gene for acetaldehyde dehydrogenase and ACS gene encoding acetyl-CoA synthetase in the PDH bypass pathway reduced liamocin biosynthesis. However, expression the PK gene for phosphoketolase, the PDC gene encoding pyruvate decarboxylase and VHb gene coding for Vitreoscilla hemoglobin (VHb) in the glucose derepression mutants could greatly enhance liamocin production. The resulting strain V33 could produce 55.38 g/L of liamocin and 25.10 g/L of cell dry weight from 117.27 g/L of glucose within 168 h of 10-liter fermentation, leading to the yield of 0.47 g/g of glucose, the productivity of 0.33 g/L/h and rate of glucose utilization of 0.70 ± 0.01 g/L/h. This was a new and efficient strategy for overproduction of liamocin by A. melanogenm.


Assuntos
Aureobasidium , Engenharia Metabólica , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Trifosfato de Adenosina , Glucose/metabolismo , Ligases , Lipídeos , Engenharia Metabólica/métodos , Fosfato Acetiltransferase , Piruvato Descarboxilase
5.
J Appl Microbiol ; 133(5): 2931-2940, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35938518

RESUMO

AIMS: Aromatic amines with diverse physical characteristics are often employed as antioxidants and precursors to pharmaceutical products. As the traditional chemical methods pose serious environmental pollution, there is an arising interest in biomanufacturing aromatic amines from renewable feedstocks. MATERIALS AND RESULTS: We report the establishment of a bacterial platform for synthesizing three types of aromatic amines, namely, tyramine, dopamine and phenylethylamine. First, we expressed aromatic amino acid decarboxylase from Enterococcus faecium (pheDC) in an Escherichia coli strain with increasing shikimate (SHK) pathway flux towards L-tyrosine. We found that glycerol served as a better carbon source than glucose, resulting in 940 ± 46 mg/L tyramine from 4% glycerol. Next, the genes of lactate dehydrogenase (ldhA), pyruvate formate lyase (pflB), phosphate acetyltransferase (pta) and alcohol dehydrogenase (adhE) were deleted to mitigate the fermentation by-product formation. The tyramine level was further increased to 1.965 ± 0.205 g/L in the shake flask, which was improved by 2.1 times compared with that of the parental strain. By using a similar strategy, we also managed to produce 703 ± 21 mg/L dopamine and 555 ± 50 mg/L phenethylamine. CONCLUSIONS: We demonstrated that the knockout of ldhA-pflB-pta-adhE is an effective strategy for improving aromatic amine productions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study achieved the highest aromatic amine titres in E. coli under shake flask reported to date.


Assuntos
Escherichia coli , Liases , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfato Acetiltransferase/metabolismo , Álcool Desidrogenase/genética , Glicerol/metabolismo , Dopamina/metabolismo , Fermentação , Glucose/metabolismo , Piruvatos/metabolismo , Descarboxilases de Aminoácido-L-Aromático/metabolismo , Tirosina/metabolismo , Tiramina , Fenetilaminas/metabolismo , Carbono/metabolismo , Preparações Farmacêuticas , Lactato Desidrogenases/metabolismo , Formiatos/metabolismo , Liases/metabolismo , Engenharia Metabólica
6.
Sci Total Environ ; 847: 157619, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-35901877

RESUMO

As an emerging pollutant, benzalkonium chlorides (BACs) potentially enriched in waste activated sludge (WAS). However, the microbial response mechanism under chronic effects of BACs on acidogenesis and methanogenesis in anaerobic digestion (AD) has not been clearly disclosed. This study investigated the AD (by-)products and microbial evolution under low to high BACs concentrations from bioreactor startup to steady running. It was found that BACs can lead to an increase of WAS hydrolysis and fermentation, but a disturbance to acidogenic bacteria also occurred at low BACs concentration. A noticeable inhibition to methanogenesis occurred when BAC concentration was up to 15 mg/g TSS. Metagenomic analysis revealed the key genes involved in acetic acid (HAc) biosynthesis (i.e. phosphate acetyltransferase, PTA), ß-oxidation pathway (acetyl-CoA C-acetyltransferase) and propionic acid (HPr) conversion was slightly promoted compared with control. Furthermore, BACs inhibited the acetotrophic methanogenesis (i.e. acetyl-CoA synthetase), especially BAC concentration was up to 15 mg/g TSS, thereby enhanced short chain fatty acids (SCFAs) accumulation. Overall, chronic stimulation of functional microorganisms with increasing concentrations of BACs impact WAS fermentation.


Assuntos
Poluentes Ambientais , Esgotos , Acetilcoenzima A/metabolismo , Acetil-CoA C-Acetiltransferase/metabolismo , Anaerobiose , Compostos de Benzalcônio , Reatores Biológicos/microbiologia , Ácidos Graxos Voláteis/metabolismo , Fermentação , Ligases/metabolismo , Metano , Fosfato Acetiltransferase/metabolismo , Propionatos , Esgotos/microbiologia
7.
J Microbiol Biotechnol ; 31(10): 1393-1400, 2021 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-34584034

RESUMO

Acetone-butanol-ethanol (ABE) fermentation by the anaerobic bacterium Clostridium acetobutylicum has been considered a promising process of industrial biofuel production. Phosphotransbutyrylase (phosphate butyryltransferase, PTB) plays a crucial role in butyrate metabolism by catalyzing the reversible conversion of butyryl-CoA into butyryl phosphate. Here, we report the crystal structure of PTB from the Clostridial host for ABE fermentation, C. acetobutylicum, (CaPTB) at a 2.9 Å resolution. The overall structure of the CaPTB monomer is quite similar to those of other acyltransferases, with some regional structural differences. The monomeric structure of CaPTB consists of two distinct domains, the N- and C-terminal domains. The active site cleft was formed at the interface between the two domains. Interestingly, the crystal structure of CaPTB contained eight molecules per asymmetric unit, forming an octamer, and the size-exclusion chromatography experiment also suggested that the enzyme exists as an octamer in solution. The structural analysis of CaPTB identifies the substrate binding mode of the enzyme and comparisons with other acyltransferase structures lead us to speculate that the enzyme undergoes a conformational change upon binding of its substrate.


Assuntos
Proteínas de Bactérias/química , Clostridium acetobutylicum/enzimologia , Fosfato Acetiltransferase/química , Acetona/metabolismo , Acil Coenzima A , Sequência de Aminoácidos , Butanóis/metabolismo , Domínio Catalítico , Etanol/metabolismo , Fermentação , Estrutura Quaternária de Proteína
8.
Biochim Biophys Acta Proteins Proteom ; 1868(9): 140462, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32485238

RESUMO

Malic enzymes participate in key metabolic processes, the MaeB-like malic enzymes carry a catalytic inactive phosphotransacetylase domain whose function remains elusive. Here we show that acetyl-CoA directly binds and inhibits MaeB-like enzymes with a saturable profile under physiological relevant acetyl-CoA concentrations. A MaeB-like enzyme from the nitrogen-fixing bacterium Azospirillum brasilense, namely AbMaeB1, binds both acetyl-CoA and unesterified CoASH in a way that inhibition of AbMaeB1 by acetyl-CoA is relieved by increasing CoASH concentrations. Hence, AbMaeB1 senses the acetyl-CoA/CoASH ratio. We revisited E. coli MaeB regulation to determine the inhibitory constant for acetyl-CoA. Our data support that the phosphotransacetylase domain of MaeB-like enzymes senses acetyl-CoA to dictate the fate of carbon distribution at the phosphoenol-pyruvate / pyruvate / oxaloacetate metabolic node.


Assuntos
Acetilcoenzima A/metabolismo , Coenzima A/metabolismo , Malato Desidrogenase/metabolismo , Malatos/metabolismo , NADP/metabolismo , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Malato Desidrogenase/genética , Fosfato Acetiltransferase/metabolismo
9.
Lett Appl Microbiol ; 70(2): 64-70, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31665809

RESUMO

Escherichia coli generates acetate as an undesirable by-product that has several negative effects on protein expression, and the reduction of acetate accumulation by modifying genes of acetate synthesis pathway can improve the expression of recombinant proteins. In the present study, the effect of phosphotransacetylase (pta) or/and acetate kinase (ackA) deletion on glutamate dehydrogenase (GDH) expression was investigated. The results indicated that the disruptions of pta or/and ackA decreased the acetate accumulation and synthesis of per gram cell, and increased cell density, and GDH expression and synthesis of per gram cell. The pta gene was more important for acetate formation than the ackA gene. Using the strain with deletions of pta-ackA (SSGPA) for GDH expression, acetate accumulation (2·61 g l-1 ) and acetate synthesis of per gram cell (0·229 g g-1 ) were lowest, decreasing by 28·29 and 41·43% compared with those of the parental strain (SSG) respectively. The flux of acetate synthesis (6·6%) was decreased by 72·15% compared with that of SSG, and the highest cell density (11·38 g l-1 ), GDH expression (2·78 mg ml-1 ), and GDH formation of per gram cell (0·2442 mg mg-1 ) were obtained, which were 1·22-, 1·43- and 1·17-times higher than the parental strain respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Significance and Impact of the Study: Acetate is the key undesirable by-product in Escherichia coli cultivation, and both biomass and production of desired products are increased by the reduction of acetate accumulation. In the present study, the strains with deletions of pta or/and ackA were constructed to reduce the acetate accumulation and improve the GDH expression, and the highest expression level of GDH was obtained using the strain with lesion in pta-ackA that was 1·17-times higher than that of the parental strain. The construction strategy of recombinant E. coli for decreasing the acetate excretion can be used for high expression level of other desired products.


Assuntos
Acetato Quinase/genética , Acetatos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glutamato Desidrogenase/biossíntese , Fosfato Acetiltransferase/genética , Deleção de Genes , Glutamato Desidrogenase/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus suis/enzimologia , Streptococcus suis/genética
10.
Microb Cell Fact ; 18(1): 199, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31727065

RESUMO

BACKGROUND: Oleaginous yeasts are able to accumulate very high levels of neutral lipids especially under condition of excess of carbon and nitrogen limitation (medium with high C/N ratio). This makes necessary the use of two-steps processes in order to achieve high level of biomass and lipid. To simplify the process, the decoupling of lipid synthesis from nitrogen starvation, by establishing a cytosolic acetyl-CoA formation pathway alternative to the one catalysed by ATP-citrate lyase, can be useful. RESULTS: In this work, we introduced a new cytoplasmic route for acetyl-CoA (AcCoA) formation in Rhodosporidium azoricum by overexpressing genes encoding for homologous phosphoketolase (Xfpk) and heterologous phosphotransacetylase (Pta). The engineered strain PTAPK4 exhibits higher lipid content and produces higher lipid concentration than the wild type strain when it was cultivated in media containing different C/N ratios. In a bioreactor process performed on glucose/xylose mixture, to simulate an industrial process for lipid production from lignocellulosic materials, we obtained an increase of 89% in final lipid concentration by the engineered strain in comparison to the wild type. This indicates that the transformed strain can produce higher cellular biomass with a high lipid content than the wild type. The transformed strain furthermore evidenced the advantage over the wild type in performing this process, being the lipid yields 0.13 and 0.05, respectively. CONCLUSION: Our results show that the overexpression of homologous Xfpk and heterologous Pta activities in R. azoricum creates a new cytosolic AcCoA supply that decouples lipid production from nitrogen starvation. This metabolic modification allows improving lipid production in cultural conditions that can be suitable for the development of industrial bioprocesses using lignocellulosic hydrolysates.


Assuntos
Basidiomycota/metabolismo , Lignina/metabolismo , Lipídeos/biossíntese , Engenharia Metabólica/métodos , Acetilcoenzima A/metabolismo , Aldeído Liases/genética , Aldeído Liases/metabolismo , Bacillus subtilis/genética , Biomassa , Citoplasma/metabolismo , Proteínas Fúngicas/genética , Genes Bacterianos , Genes Fúngicos , Engenharia Genética , Recombinação Homóloga , Metabolismo dos Lipídeos/genética , Nitrogênio/metabolismo , Fosfato Acetiltransferase/genética , Fosfato Acetiltransferase/metabolismo , Proteínas Recombinantes , Transfecção
11.
J Biotechnol ; 301: 2-10, 2019 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-31158408

RESUMO

Diacetyl, an important flavor extensively used in the food industry, can be produced from the non-enzymatic oxidative decarboxylation of α-acetolactate in bacteria fermentation. In previous work, we obtained a strain of Bacillus sp. DL01-ΔalsD with low diacetyl accumulation. The strain was engineered and optimized for improving the production of diacetyl in this study. First, deletion of the gene encoding phosphotransacetylase (pta), by homologous recombination with high temperature sensitive shuttle plasmid vector pKS1, led to a reduction of acetate and 130% increase of diacetyl production in B. sp. DL01-ΔalsD-Δpta. Then overexpression of α-acetolactate synthase (ALS) from B. subtilis 168 in B. sp. DL01-ΔalsD-Δpta resulted in efficient diacetyl production with a titer of 5.43 g/L. To further increase diacetyl production, single factor and orthogonal experimental data were used to predict the optimal fermentation conditions by Back Propagation neural network. Optimal value of KLa (Dissolved oxygen volume coefficient) was 12.4 h-1 with fermentation parameters of aeration rate 0.66 vvm, agitation speed 179 rpm and temperature 35.7 ℃. A titer of 11.18 g/L diacetyl, the highest reported diacetyl production, was achieved by fed-batch fermentation at the optimal condition using the metabolic engineered strain of B. sp. DL01-ΔalsD-Δpta-als168. These results are of great importance as a new way for the efficient production of diacetyl by food-safety bacteria.


Assuntos
Bacillus , Diacetil , Engenharia Metabólica/métodos , Acetolactato Sintase/genética , Acetolactato Sintase/metabolismo , Bacillus/enzimologia , Bacillus/genética , Bacillus/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Reatores Biológicos/microbiologia , Diacetil/análise , Diacetil/metabolismo , Fermentação , Fosfato Acetiltransferase/genética , Fosfato Acetiltransferase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
12.
Mol Microbiol ; 111(5): 1182-1194, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30589958

RESUMO

The DevRS/DosT two-component system is essential for mycobacterial survival under hypoxia, a prevailing stress within granulomas. DevR (also known as DosR) is activated by an inducing stimulus, such as hypoxia, through conventional phosphorylation by its cognate sensor kinases, DevS (also known as DosS) and DosT. Here, we show that the DevR regulon is activated by acetyl phosphate under 'non-inducing' aerobic conditions when Mycobacterium tuberculosis devS and dosT double deletion strain is cultured on acetate. Overexpression of phosphotransacetylase caused a perturbation of the acetate kinase-phosphotransacetylase pathway, a decrease in the concentration of acetyl phosphate and dampened the aerobic induction response in acetate-grown bacteria. The operation of two pathways of DevR activation, one through sensor kinases and the other by acetyl phosphate, was established by an analysis of wild-type DevS and phosphorylation-defective DevSH395Q mutant strains under conditions partially mimicking a granulomatous-like environment of acetate and hypoxia. Our findings reveal that DevR can be phosphorylated in vivo by acetyl phosphate. Importantly, we demonstrate that acetyl phosphate-dependent phosphorylation can occur in the absence of DevR's cognate kinases. Based on our findings, we conclude that anti-mycobacterial therapy should be targeted to DevR itself and not to DevS/DosT kinases.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Organofosfatos/metabolismo , Proteínas Quinases/genética , Regulon , Acetatos/metabolismo , Aerobiose , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA , Fosfato Acetiltransferase/genética , Fosfato Acetiltransferase/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Proteínas Quinases/metabolismo
13.
J Biosci Bioeng ; 127(2): 256-264, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30150148

RESUMO

Many cyanophages, which infect cyanobacteria, most of possess putative sigma factors that have high amino acid sequence homology with the σ70-type sigma factor present in cyanobacteria, allowing them to obtain energy and metabolites for their own propagation. In this study, we aimed to modify the carbon metabolism of Synechococcus elongatus PCC 7942 by expressing putative sigma factors from Synechococcus phages to improve bioproduction. Four cyanophage-derived putative sigma factors-putative RpsD4 from Synechococcus phage S-CBS1, putative RpoD and putative RpoS from S-CBS2, and putative RpsD4 from S-CBS3-were selected for this purpose. These were introduced into S. elongatus PCC 7942, and their expression was controlled with a theophylline-dependent riboswitch. The expression of the putative RpoD from S-CBS2 and putative RpsD4 from S-CBS3 resulted in a significant decrease in the growth rate of S. elongatus PCC 7942. In addition, metabolome analysis showed a 3.2-fold increase in acetyl-CoA concentration with the expression of the putative RpoD from S-CBS2 and a 1.9-fold increase with the putative RpsD4 from S-CBS3. The results of RT-qPCR showed that several sugar metabolism genes were repressed by the putative RpoD and activated by the putative RpsD4. In particular, the engineered strain overexpressing the putative RpsD4 and expressing phosphate acetyltransferase succeeded in improving the productivity of the model target product acetate to 217% of its previous value. To the best of our knowledge, this study is the first to modify the metabolism of S. elongatus PCC 7942 by expressing their putative sigma factors from cyanophages.


Assuntos
Bacteriófagos/fisiologia , Carbono/metabolismo , Engenharia Metabólica/métodos , Fator sigma/genética , Synechococcus/genética , Synechococcus/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Dióxido de Carbono/metabolismo , Redes e Vias Metabólicas/genética , Técnicas Microbiológicas/métodos , Organismos Geneticamente Modificados , Fosfato Acetiltransferase/genética , Fosfato Acetiltransferase/metabolismo , Fator sigma/metabolismo , Synechococcus/crescimento & desenvolvimento , Transformação Bacteriana/fisiologia
14.
J Microbiol Biotechnol ; 28(12): 2009-2018, 2018 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-30304917

RESUMO

Leuconostoc mesenteroides can be used to produce mannitol by fermentation, but the mannitol productivity is not high. Therefore, in this study modify the chromosome of Leuconostoc mesenteroides by genetic methods to obtain high-yield strains of mannitol production. In this study, gene knock-out strains and gene knock-in strains were constructed by a two-step homologous recombination method. The mannitol productivity of the pat gene (which encodes phosphate acetyltransferase) deleteon strain (Δpat::amy), fk gene (which encodes fructokinase) deleteon strain (Δfk::amy) and stpk gene (which encodes serine-threonine protein kinase) deleteon strain (Δstpk::amy) were all increased compared to the wild type, and the productivity of mannitol for each strain was 84.8%, 83.5% and 84.1% respectively. The mannitol productivity of the mdh gene (which encodes mannitol dehydrogenase) knock-in strains (Δpat::mdh, Δfk::mdh and Δstpk::mdh) was increased to a higher level than that of the single-gene deletion strains, and the productivity of mannitol for each was 96.5%, 88% and 93.2%, respectively. The multi-mutant strain ΔdtsΔldhΔpat::mdhΔstpk::mdhΔfk::mdh had mannitol productivity of 97.3%. This work shows that multi-gene knock-out and gene knock-in strains have the greatest impact on mannitol production, with mannitol productivity of 97.3% and an increase of 24.7% over wild type. This study used the methods of gene knock-out and gene knock-in to genetically modify the chromosome of Leuconostoc mesenteroides. It is of great significance that we increased the ability of Leuconostoc mesenteroides to produce mannitol and revealed its broad development prospects.


Assuntos
Técnicas de Introdução de Genes/métodos , Técnicas de Inativação de Genes/métodos , Genes Bacterianos/genética , Leuconostoc mesenteroides/genética , Leuconostoc mesenteroides/metabolismo , Manitol/metabolismo , Cromossomos Bacterianos , DNA Bacteriano , Fermentação , Frutoquinases/genética , Deleção de Genes , Recombinação Homóloga , Manitol Desidrogenases/genética , Fosfato Acetiltransferase/genética , Proteínas Proto-Oncogênicas c-akt/genética , Recombinação Genética
15.
Microb Cell Fact ; 17(1): 102, 2018 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-29970091

RESUMO

BACKGROUND: High production cost of bioplastics polyhydroxyalkanoates (PHA) is a major obstacle to replace traditional petro-based plastics. To address the challenges, strategies towards upstream metabolic engineering and downstream fermentation optimizations have been continuously pursued. Given that the feedstocks especially carbon sources account up to a large portion of the production cost, it is of great importance to explore low cost substrates to manufacture PHA economically. RESULTS: Escherichia coli was metabolically engineered to synthesize poly-3-hydroxybutyrate (P3HB), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) using acetate as a main carbon source. Overexpression of phosphotransacetylase/acetate kinase pathway was shown to be an effective strategy for improving acetate assimilation and biopolymer production. The recombinant strain overexpressing phosphotransacetylase/acetate kinase and P3HB synthesis operon produced 1.27 g/L P3HB when grown on minimal medium supplemented with 10 g/L yeast extract and 5 g/L acetate in shake flask cultures. Further introduction succinate semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, and CoA transferase lead to the accumulation of P3HB4HB, reaching a titer of 1.71 g/L with a 4-hydroxybutyrate monomer content of 5.79 mol%. When 1 g/L of α-ketoglutarate or citrate was added to the medium, P3HB4HB titer increased to 1.99 and 2.15 g/L, respectively. To achieve PHBV synthesis, acetate and propionate were simultaneously supplied and propionyl-CoA transferase was overexpressed to provide 3-hydroxyvalerate precursor. The resulting strain produced 0.33 g/L PHBV with a 3-hydroxyvalerate monomer content of 6.58 mol%. Further overexpression of propionate permease improved PHBV titer and 3-hydroxyvalerate monomer content to 1.09 g/L and 10.37 mol%, respectively. CONCLUSIONS: The application of acetate as carbon source for microbial fermentation could reduce the consumption of food and agro-based renewable bioresources for biorefineries. Our proposed metabolic engineering strategies illustrate the feasibility for producing polyhydroxyalkanoates using acetate as a main carbon source. Overall, as an abundant and renewable resource, acetate would be developed into a cost-effective feedstock to achieve low cost production of chemicals, materials, and biofuels.


Assuntos
Acetatos/metabolismo , Escherichia coli/metabolismo , Engenharia Metabólica , Poli-Hidroxialcanoatos/biossíntese , Ácido 3-Hidroxibutírico/biossíntese , Acetato Quinase/genética , Técnicas de Cultura Celular por Lotes , Biopolímeros/biossíntese , Carbono/metabolismo , Escherichia coli/genética , Fermentação , Fosfato Acetiltransferase/genética , Plásticos
16.
Metab Eng ; 48: 243-253, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29906505

RESUMO

Clostridium ljungdahlii has emerged as an attractive candidate for the bioconversion of synthesis gas (CO, CO2, H2) to a variety of fuels and chemicals through the Wood-Ljungdahl pathway. However, metabolic engineering and pathway elucidation in this microbe is limited by the lack of genetic tools to downregulate target genes. To overcome this obstacle, here we developed an inducible CRISPR interference (CRISPRi) system for C. ljungdahlii that enables efficient (> 94%) transcriptional repression of several target genes, both individually and in tandem. We then applied CRISPRi in a strain engineered for 3-hydroxybutyrate (3HB) production to examine targets for increasing carbon flux toward the desired product. Downregulating phosphotransacetylase (pta) with a single sgRNA led to a 97% decrease in enzyme activity and a 2.3-fold increase in titer during heterotrophic growth. However, acetate production still accounted for 40% of the carbon flux. Repression of aldehyde:ferredoxin oxidoreductase (aor2), another potential route for acetate production, led to a 5% reduction in acetate flux, whereas using an additional sgRNA targeted to pta reduced the enzyme activity to 0.7% of the wild-type level, and further reduced acetate production to 25% of the carbon flux with an accompanying increase in 3HB titer and yield. These results demonstrate the utility of CRISPRi for elucidating and controlling carbon flow in C. ljungdahlii.


Assuntos
Ácido 3-Hidroxibutírico , Sistemas CRISPR-Cas , Carbono/metabolismo , Clostridium , Engenharia Metabólica , Ácido 3-Hidroxibutírico/biossíntese , Ácido 3-Hidroxibutírico/genética , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clostridium/genética , Clostridium/metabolismo , Fosfato Acetiltransferase/genética , Fosfato Acetiltransferase/metabolismo
17.
J Ind Microbiol Biotechnol ; 45(5): 357-367, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29460214

RESUMO

L-tryptophan (L-trp) is a precursor of various bioactive components and has great pharmaceutical interest. However, due to the requirement of several precursors and complex regulation of the pathways involved, the development of an efficient L-trp production strain is challenging. In this study, Escherichia coli (E. coli) strain KW001 was designed to overexpress the L-trp operator sequences (trpEDCBA) and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (aroG fbr ). To further improve the production of L-trp, pyruvate kinase (pykF) and the phosphotransferase system HPr (ptsH) were deleted after inactivation of repression (trpR) and attenuation (attenuator) to produce strain KW006. To overcome the relatively slow growth and to increase the transport rate of glucose, strain KW018 was generated by combinatorial regulation of glucokinase (galP) and galactose permease (glk) expression. To reduce the production of acetic acid, strain KW023 was created by repressive regulation of phosphate acetyltransferase (pta) expression. In conclusion, strain KW023 efficiently produced 39.7 g/L of L-trp with a conversion rate of 16.7% and a productivity of 1.6 g/L/h in a 5 L fed-batch fermentation system.


Assuntos
Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Triptofano/biossíntese , Proteínas de Escherichia coli/metabolismo , Fermentação , Glucoquinase/metabolismo , Glucose/metabolismo , Proteínas de Transporte de Monossacarídeos , Fosfato Acetiltransferase/metabolismo , Piruvato Quinase/metabolismo
18.
PLoS One ; 13(1): e0189144, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29381705

RESUMO

Succinate is a precursor of multiple commodity chemicals and bio-based succinate production is an active area of industrial bioengineering research. One of the most important microbial strains for bio-based production of succinate is the capnophilic gram-negative bacterium Actinobacillus succinogenes, which naturally produces succinate by a mixed-acid fermentative pathway. To engineer A. succinogenes to improve succinate yields during mixed acid fermentation, it is important to have a detailed understanding of the metabolic flux distribution in A. succinogenes when grown in suitable media. To this end, we have developed a detailed stoichiometric model of the A. succinogenes central metabolism that includes the biosynthetic pathways for the main components of biomass-namely glycogen, amino acids, DNA, RNA, lipids and UDP-N-Acetyl-α-D-glucosamine. We have validated our model by comparing model predictions generated via flux balance analysis with experimental results on mixed acid fermentation. Moreover, we have used the model to predict single and double reaction knockouts to maximize succinate production while maintaining growth viability. According to our model, succinate production can be maximized by knocking out either of the reactions catalyzed by the PTA (phosphate acetyltransferase) and ACK (acetyl kinase) enzymes, whereas the double knockouts of PEPCK (phosphoenolpyruvate carboxykinase) and PTA or PEPCK and ACK enzymes are the most effective in increasing succinate production.


Assuntos
Actinobacillus/metabolismo , Técnicas de Silenciamento de Genes , Ácido Succínico/metabolismo , Actinobacillus/enzimologia , Actinobacillus/genética , Biomassa , Meios de Cultura , Fermentação , Modelos Biológicos , Fosfato Acetiltransferase/genética , Fosfato Acetiltransferase/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo
19.
Enzyme Microb Technol ; 102: 1-8, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28465055

RESUMO

In this study, L-lactic acid production was investigated from metabolically engineered strain of E. aerogenes ATCC 29007. The engineered strain E. aerogenes SUMI01 (Δpta) was generated by the deletion of phosphate acetyltransferase (pta) gene from the chromosome of E. aerogenes ATCC 29007 and deletion was confirmed by colony PCR. Under the optimized fermentation conditions, at 37°C and pH 6 for 84h, the L-lactic acid produced by engineered strain E. aerogenes SUMI01 (Δpta) in flask fermentation using 100g/L mannitol as the carbon source was 40.05g/L as compared to that of the wild type counterpart 20.70g/L. At the end of the batch fermentation in bioreactor the production of L-lactic acid reached to 46.02g/L and yield was 0.41g/g by utilizing 112.32g/L mannitol. This is the first report regarding the production of L-lactic acid from Enterobacter species. We believe that this result may provide valuable guidelines for further engineering Enterobacter strain for the improvement of L-lactic acid production.


Assuntos
Enterobacter aerogenes/genética , Enterobacter aerogenes/metabolismo , Ácido Láctico/biossíntese , Proteínas de Bactérias/genética , Reatores Biológicos/microbiologia , Fermentação , Deleção de Genes , Genes Bacterianos , Microbiologia Industrial , Cinética , Manitol/metabolismo , Engenharia Metabólica , Fosfato Acetiltransferase/genética
20.
J Ind Microbiol Biotechnol ; 44(8): 1245-1260, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28536840

RESUMO

Genetic research enables the evolution of novel biochemical reactions for the production of valuable chemicals from environmentally-friendly raw materials. However, the choice of appropriate microorganisms to support these reactions, which must have strong robustness and be capable of a significant product output, is a major difficulty. In the present study, the complete genome of the Clostridium tyrobutyricum strain CCTCC W428, a hydrogen- and butyric acid-producing bacterium with increased oxidative tolerance was analyzed. A total length of 3,011,209 bp of the C. tyrobutyricum genome with a GC content of 31.04% was assembled, and 3038 genes were discovered. Furthermore, a comparative clustering of proteins from C. tyrobutyricum CCTCC W428, C. acetobutylicum ATCC 824, and C. butyricum KNU-L09 was conducted. The results of genomic analysis indicate that butyric acid is produced by CCTCC W428 from butyryl-CoA through acetate reassimilation via CoA transferase, instead of the well-established phosphotransbutyrylase-butyrate kinase pathway. In addition, we identified ten proteins putatively involved in hydrogen production and 21 proteins associated with CRISPR systems, together with 358 ORFs related to ABC transporters and transcriptional regulators. Enzymes, such as oxidoreductases, HNH endonucleases, and catalase, were also found in this species. The genome sequence illustrates that C. tyrobutyricum has several desirable traits, and is expected to be suitable as a platform for the high-level production of bulk chemicals as well as bioenergy.


Assuntos
Proteínas de Bactérias/genética , Clostridium tyrobutyricum/genética , Genoma Bacteriano , Acil Coenzima A/genética , Acil Coenzima A/metabolismo , Proteínas de Bactérias/metabolismo , Biotecnologia , Ácido Butírico/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Clostridium tyrobutyricum/metabolismo , Meios de Cultura/química , DNA Bacteriano/genética , Hidrogênio/metabolismo , Microbiologia Industrial , Fosfato Acetiltransferase/genética , Fosfato Acetiltransferase/metabolismo , Fosfotransferases (Aceptor do Grupo Carboxila)/genética , Fosfotransferases (Aceptor do Grupo Carboxila)/metabolismo , Análise de Sequência de DNA
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