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1.
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
2.
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
3.
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
4.
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
5.
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 , Fosfato Acetiltransferase/genética , Fosfato Acetiltransferase/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Proteínas Quinases/metabolismo
6.
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
7.
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
8.
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
9.
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
10.
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
11.
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
12.
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
13.
Appl Environ Microbiol ; 83(8)2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28159797

RESUMO

For the efficient production of target metabolites from carbohydrates, syngas, or H2-CO2 by genetically engineered Moorella thermoacetica, the control of acetate production (a main metabolite of M. thermoacetica) is desired. Although propanediol utilization protein (PduL) was predicted to be a phosphotransacetylase (PTA) involved in acetate production in M. thermoacetica, this has not been confirmed. Our findings described herein directly demonstrate that two putative PduL proteins, encoded by Moth_0864 (pduL1) and Moth_1181 (pduL2), are involved in acetate formation as PTAs. To disrupt these genes, we replaced each gene with a lactate dehydrogenase gene from Thermoanaerobacter pseudethanolicus ATCC 33223 (T-ldh). The acetate production from fructose as the sole carbon source by the pduL1 deletion mutant was not deficient, whereas the disruption of pduL2 significantly decreased the acetate yield to approximately one-third that of the wild-type strain. The double-deletion (both pduL genes) mutant did not produce acetate but produced only lactate as the end product from fructose. These results suggest that both pduL genes are associated with acetate formation via acetyl-coenzyme A (acetyl-CoA) and that their disruption enables a shift in the homoacetic pathway to the genetically synthesized homolactic pathway via pyruvate.IMPORTANCE This is the first report, to our knowledge, on the experimental identification of PTA genes in M. thermoacetica and the shift of the native homoacetic pathway to the genetically synthesized homolactic pathway by their disruption on a sugar platform.


Assuntos
Acetatos/metabolismo , Fermentação , Engenharia Genética , Moorella/genética , Moorella/metabolismo , Acetilcoenzima A/metabolismo , Anaerobiose , Carbono/metabolismo , L-Lactato Desidrogenase/genética , Moorella/enzimologia , Fosfato Acetiltransferase/metabolismo , Propilenoglicóis/metabolismo , Thermoanaerobacter/genética
14.
Sci Rep ; 7: 42135, 2017 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-28186174

RESUMO

Escherichia coli excretes acetate upon growth on fermentable sugars, but the regulation of this production remains elusive. Acetate excretion on excess glucose is thought to be an irreversible process. However, dynamic 13C-metabolic flux analysis revealed a strong bidirectional exchange of acetate between E. coli and its environment. The Pta-AckA pathway was found to be central for both flux directions, while alternative routes (Acs or PoxB) play virtually no role in glucose consumption. Kinetic modelling of the Pta-AckA pathway predicted that its flux is thermodynamically controlled by the extracellular acetate concentration in vivo. Experimental validations confirmed that acetate production can be reduced and even reversed depending solely on its extracellular concentration. Consistently, the Pta-AckA pathway can rapidly switch from acetate production to consumption. Contrary to current knowledge, E. coli is thus able to co-consume glucose and acetate under glucose excess. These metabolic capabilities were confirmed on other glycolytic substrates which support the growth of E. coli in the gut. These findings highlight the dual role of the Pta-AckA pathway in acetate production and consumption during growth on glycolytic substrates, uncover a novel regulatory mechanism that controls its flux in vivo, and significantly expand the metabolic capabilities of E. coli.


Assuntos
Acetato Quinase/metabolismo , Ácido Acético/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Glucose/metabolismo , Fosfato Acetiltransferase/metabolismo , Acetato Quinase/genética , Isótopos de Carbono , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fermentação , Marcação por Isótopo , Cinética , Redes e Vias Metabólicas/genética , Fosfato Acetiltransferase/genética , Especificidade por Substrato , Termodinâmica
15.
Metab Eng ; 40: 138-147, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28159643

RESUMO

Clostridium acetobutylicum possesses two homologous buk genes, buk (or buk1) and buk2, which encode butyrate kinases involved in the last step of butyrate formation. To investigate the contribution of buk in detail, an in-frame deletion mutant was constructed. However, in all the Δbuk mutants obtained, partial deletions of the upstream ptb gene were observed, and low phosphotransbutyrylase and butyrate kinase activities were measured. This demonstrates that i) buk (CA_C3075) is the key butyrate kinase-encoding gene and that buk2 (CA_C1660) that is poorly transcribed only plays a minor role; and ii) strongly suggests that a Δbuk mutant is not viable if the ptb gene is not also inactivated, probably due to the accumulation of butyryl-phosphate, which might be toxic for the cell. One of the ΔbukΔptb mutants was subjected to quantitative transcriptomic (mRNA molecules/cell) and fluxomic analyses in acidogenic, solventogenic and alcohologenic chemostat cultures. In addition to the low butyrate production, drastic changes in metabolic fluxes were also observed for the mutant: i) under acidogenic conditions, the primary metabolite was butanol and a new metabolite, 2-hydroxy-valerate, was produced ii) under solventogenesis, 58% increased butanol production was obtained compared to the control strain under the same conditions, and a very high yield of butanol formation (0.3gg-1) was reached; and iii) under alcohologenesis, the major product was lactate. Furthermore, at the transcriptional level, adhE2, which encodes an aldehyde/alcohol dehydrogenase and is known to be a gene specifically expressed in alcohologenesis, was surprisingly highly expressed in all metabolic states in the mutant. The results presented here not only support the key roles of buk and ptb in butyrate formation but also highlight the metabolic flexibility of C. acetobutylicum in response to genetic alteration of its primary metabolism.


Assuntos
Ácido Butírico/metabolismo , Clostridium acetobutylicum/fisiologia , Redes e Vias Metabólicas/fisiologia , Fosfato Acetiltransferase/metabolismo , Fosfotransferases (Aceptor do Grupo Carboxila)/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Engenharia Metabólica/métodos , Análise do Fluxo Metabólico/métodos , Mutação/genética , Fosfato Acetiltransferase/genética , Fosfotransferases (Aceptor do Grupo Carboxila)/genética
16.
Appl Environ Microbiol ; 83(7)2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28130304

RESUMO

In the dental caries pathogen Streptococcus mutans, phosphotransacetylase (Pta) and acetate kinase (Ack) convert pyruvate into acetate with the concomitant generation of ATP. The genes for this pathway are tightly regulated by multiple environmental and intracellular inputs, but the basis for differential expression of the genes for Pta and Ack in S. mutans had not been investigated. Here, we show that inactivation in S. mutans of ccpA or codY reduced the activity of the ackA promoter, whereas a ccpA mutant displayed elevated pta promoter activity. The interactions of CcpA with the promoter regions of both genes were observed using electrophoretic mobility shift and DNase protection assays. CodY bound to the ackA promoter region but only in the presence of branched-chain amino acids (BCAAs). DNase footprinting revealed that the upstream region of both genes contains two catabolite-responsive elements (cre1 and cre2) that can be bound by CcpA. Notably, the cre2 site of ackA overlaps with a CodY-binding site. The CcpA- and CodY-binding sites in the promoter region of both genes were further defined by site-directed mutagenesis. Some differences between the reported consensus CodY binding site and the region protected by S. mutans CodY were noted. Transcription of the pta and ackA genes in the ccpA mutant strain was markedly different at low pH relative to transcription at neutral pH. Thus, CcpA and CodY are direct regulators of transcription of ackA and pta in S. mutans that optimize acetate metabolism in response to carbohydrate, amino acid availability, and environmental pH.IMPORTANCE The human dental caries pathogen Streptococcus mutans is remarkably adept at coping with extended periods of carbohydrate limitation during fasting periods. The phosphotransacetylase-acetate kinase (Pta-Ack) pathway in S. mutans modulates carbohydrate flux and fine-tunes the ability of the organisms to cope with stressors that are commonly encountered in the oral cavity. Here, we show that CcpA controls transcription of the pta and ackA genes via direct interaction with the promoter regions of both genes and that branched-chain amino acids (BCAAs), particularly isoleucine, enhance the ability of CodY to bind to the promoter region of the ackA gene. A working model is proposed to explain how regulation of pta and ackA genes by these allosterically controlled regulatory proteins facilitates proper carbon flow and energy production, which are essential functions during infection and pathogenesis as carbohydrate and amino acid availability continually fluctuate.


Assuntos
Acetatos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Streptococcus mutans/genética , Streptococcus mutans/metabolismo , Acetato Quinase/genética , Aminoácidos de Cadeia Ramificada/metabolismo , Sítios de Ligação , Metabolismo dos Carboidratos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Cárie Dentária/microbiologia , Concentração de Íons de Hidrogênio , Mutagênese Sítio-Dirigida , Fosfato Acetiltransferase/genética , Fosfato Acetiltransferase/metabolismo , Regiões Promotoras Genéticas , Ácido Pirúvico/metabolismo , Transcrição Genética
17.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1862(3): 283-290, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27940001

RESUMO

Listeria monocytogenes, the causative agent of listeriosis, can build up to dangerous levels in refrigerated foods potentially leading to expensive product recalls. An important aspect of the bacterium's growth at low temperatures is its ability to increase the branched-chain fatty acid anteiso C15:0 content of its membrane at lower growth temperatures, which imparts greater membrane fluidity. Mutants in the branched-chain α-keto dehydrogenase (bkd) complex are deficient in branched-chain fatty acids (BCFAs,) but these can be restored by feeding C4 and C5 branched-chain carboxylic acids (BCCAs). This suggests the presence of an alternate pathway for production of acyl CoA precursors for fatty acid biosynthesis. We hypothesize that the alternate pathway is composed of butyrate kinase (buk) and phosphotransbutyrylase (ptb) encoded in the bkd complex which produce acyl CoA products by their sequential action through the metabolism of carboxylic acids. We determined the steady state kinetics of recombinant His-tagged Buk using 11 different straight-chain and BCCA substrates in the acyl phosphate forming direction. Buk demonstrated highest catalytic efficiency with pentanoate as the substrate. Low product formation observed with acetate (C2) and hexanoate (C6) as the substrates indicates that Buk is not involved in either acetate metabolism or long chain carboxylic acid activation. We were also able to show that Buk catalysis occurs through a ternary complex intermediate. Additionally, Buk demonstrates a strong preference for BCCAs at low temperatures. These results indicate that Buk may be involved in the activation and assimilation of exogenous carboxylic acids for membrane fatty acid biosynthesis.


Assuntos
Listeria monocytogenes/metabolismo , Fosfotransferases (Aceptor do Grupo Carboxila)/metabolismo , Acil Coenzima A/metabolismo , Ácidos Carboxílicos/metabolismo , Temperatura Baixa , Ácidos Graxos/metabolismo , Cinética , Lipogênese/fisiologia , Fluidez de Membrana/fisiologia , Fosfato Acetiltransferase/metabolismo , Especificidade por Substrato
18.
Biotechnol Lett ; 39(4): 529-533, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27999972

RESUMO

OBJECTIVES: To reduce the unpleasant odor during 1-deoxynojirimycin (DNJ) production, the genes of leucine dehydrogenase (bcd) and phosphate butryltransferase (ptb) were deleted from Bacillus amyloliquefaciens HZ-12, and the concentrations of branched-chain short fatty acids (BCFAs) and DNJ were compared. RESULTS: By knockout of the ptb gene, 1.01 g BCFAs kg-1 was produced from fermented soybean by HZ-12Δptb. This was a 56% decrease compared with that of HZ-12 (2.27 g BCFAs kg-1). Moreover, no significant difference was found in the DNJ concentration (0.7 g kg-1). After further deletion of the bcd gene from HZ-12Δptb, no BCFAs was detected in fermented soybeans with HZ-12ΔptbΔbcd, while the DNJ yield decreased by 26% compared with HZ-12. CONCLUSIONS: HZ-12Δptb had decreased BCFAs formation but also maintained the stable DNJ yield, which contributed to producing DNJ-rich products with decreased unpleasant smell.


Assuntos
1-Desoxinojirimicina/metabolismo , Bacillus amyloliquefaciens/metabolismo , Ácidos Graxos/biossíntese , Microbiologia de Alimentos , Engenharia Metabólica , Bacillus amyloliquefaciens/genética , Cromatografia Gasosa , Cromatografia Líquida de Alta Pressão , Regulação para Baixo , Fermentação , Expressão Gênica , Técnicas de Inativação de Genes , Genes Bacterianos , Leucina Desidrogenase/metabolismo , Odorantes/prevenção & controle , Fosfato Acetiltransferase/metabolismo , Soja/metabolismo
19.
J Biosci Bioeng ; 123(1): 39-45, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27613406

RESUMO

Cyanobacteria engineered for production of biofuels and biochemicals from carbon dioxide represent a promising area of research in relation to a sustainable economy. Previously, we have succeeded in producing isopropanol from cellular acetyl-CoA by means of Synechococcus elongatus PCC 7942 into which a synthetic metabolic pathway was introduced. The isopropanol production by this synthetic metabolic pathway requires acetate; therefore, the cells grown under photosynthetic conditions have to be transferred to a dark and anaerobic conditions to produce acetate. In this study, we achieved acetate production under photosynthetic conditions by S. elongatus PCC 7942 into which we introduced the pta gene encoding phosphate acetyltransferase from Escherichia coli. The metabolic modification (via pta introduction) of the isopropanol-producing strain enabled production of isopropanol under photosynthetic conditions. During 14 days of production, the titer of isopropanol reached 0.55 mM (33.1 mg/l) with an intermediate product, acetone, at 0.21 mM (12.2 mg/l).


Assuntos
2-Propanol/metabolismo , Engenharia Metabólica , Fotossíntese , Synechococcus/genética , Synechococcus/metabolismo , Biocombustíveis , Escherichia coli/enzimologia , Escherichia coli/genética , Fosfato Acetiltransferase/genética
20.
ACS Synth Biol ; 5(11): 1284-1289, 2016 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-27700053

RESUMO

We demonstrate metabolic enzyme ligation using a transpeptidase (Staphylococcal sortase A) in the microbial cytoplasm for the redirection of metabolic flux through metabolic channeling. Here, sortase A expression was controlled by the lac promoter to trigger metabolic channeling by the addition of isopropyl-ß-d-thiogalactopyranoside (IPTG). We tested covalent linking of pyruvate-formate lyase and phosphate acetyltransferase by sortase A-mediated ligation and evaluated the production of acetate. The time point of addition of IPTG was not critical for facilitating metabolic enzyme ligation, and acetate production increased upon expression of sortase A. These results show that sortase A-mediated enzyme ligation enhances an acetate-producing flux in E. coli. We have validated that sortase A-mediated enzyme ligation offers a metabolic channeling approach to redirect a central flux to a desired flux.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica , Acetatos/metabolismo , Acetiltransferases/metabolismo , Aminoaciltransferases/genética , Proteínas de Bactérias/genética , Cisteína Endopeptidases/genética , Isopropiltiogalactosídeo/metabolismo , Engenharia Metabólica , Fosfato Acetiltransferase/metabolismo , Plasmídeos/genética , Regiões Promotoras Genéticas
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