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Microb Cell Fact ; 18(1): 151, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31484572


BACKGROUND: Escherichia coli (E. coli) is a bacteria that is widely employed in many industries for the production of high interest bio-products such as recombinant proteins. Nevertheless, the use of E. coli for recombinant protein production may entail some disadvantages such as acetate overflow. Acetate is accumulated under some culture conditions, involves a decrease in biomass and recombinant protein production, and its metabolism is related to protein lysine acetylation. Thereby, the carbon and nitrogen sources employed are relevant factors in cell host metabolism, and the study of the central metabolism of E. coli and its regulation is essential for optimizing the production of biomass and recombinant proteins. In this study, our aim was to find the most favourable conditions for carrying out recombinant protein production in E. coli BL21 using two different approaches, namely, manipulation of the culture media composition and the deletion of genes involved in acetate metabolism and Nε-lysine acetylation. RESULTS: We evaluated protein overexpression in E. coli BL21 wt and five mutant strains involved in acetate metabolism (Δacs, ΔackA and Δpta) and lysine acetylation (ΔpatZ and ΔcobB) grown in minimal medium M9 (inorganic ammonium nitrogen source) and in complex TB7 medium (peptide-based nitrogen source) supplemented with glucose (PTS carbon source) or glycerol (non-PTS carbon source). We observed a dependence of recombinant protein production on acetate metabolism and the carbon and nitrogen source employed. The use of complex medium supplemented with glycerol as a carbon source entails an increase in protein production and an efficient use of resources, since is a sub-product of biodiesel synthesis. Furthermore, the deletion of the ackA gene results in a fivefold increase in protein production with respect to the wt strain and a reduction in acetate accumulation. CONCLUSION: The results showed that the use of diverse carbon and nitrogen sources and acetate metabolism knockout strains can redirect E. coli carbon fluxes to different pathways and affect the final yield of the recombinant protein bioprocess. Thereby, we obtained a fivefold increase in protein production and an efficient use of the resources employing the most suitable strain and culture conditions.

Acetatos/metabolismo , Carbono/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Nitrogênio/metabolismo , Proteínas Recombinantes/biossíntese , Acetatos/química , Acetilação , Carbono/química , Meios de Cultura/química , Proteínas de Escherichia coli/biossíntese , Lisina/metabolismo , Nitrogênio/química , Engenharia de Proteínas , Processamento de Proteína Pós-Traducional
Biochim Biophys Acta Gen Subj ; 1863(6): 1040-1049, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30928490


BACKGROUND: The superfamily of adenylating enzymes is a large family of enzymes broadly distributed from bacteria to humans. Acetyl-CoA synthetase (Acs), member of this family, is a metabolic enzyme with an essential role in Escherichia coli (E. coli) acetate metabolism, whose catalytic activity is regulated by acetylation/deacetylation in vivo. METHODS: In this study, the kinetics and thermodynamic parameters of deacetylated and acetylated E. coli Acs were studied for the adenylating step. Moreover, the role of the T264, K270, D500 and K609 residues in catalysis and ATP-binding was also determined by Isothermal titration calorimetry. RESULTS: The results showed that native Acs enzyme binds ATP in an endothermic way. The dissociation constant has been determined and ATP-binding showed no significant differences between acetylated and deacetylated enzyme, although kcat was much higher for the deacetylated enzyme. However, K609 lysine mutation resulted in an increase in ATP-Acs-affinity and in a total loss of enzymatic activity, while T264 and D500 mutant proteins showed a total loss of ATP-binding ability and a decrease in catalytic activity. K609 site-specified acetylation induced a change in Acs conformation which resulted in an exothermic and more energetic ATP-binding. CONCLUSIONS: The differences in ATP-binding could explain the broadly conserved inactivation of Acs when K609 is acetylated. GENERAL SIGNIFICANCE: The results presented in this study demonstrate the importance of the selected residues in Acs ATP-binding and represent an advance in our understanding of the adenylation step of the superfamily of adenylating enzymes and of their acetylation/deacetylation regulation.

Acetilcoenzima A/química , Trifosfato de Adenosina/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Acetilcoenzima A/metabolismo , Trifosfato de Adenosina/metabolismo , Cinética , Ligação Proteica
PLoS One ; 12(12): e0189689, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29253849


Lysine acetylation has emerged as a global protein regulation system in all domains of life. Sirtuins, or Sir2-like enzymes, are a family of histone deacetylases characterized by their employing NAD+ as a co-substrate. Sirtuins can deacetylate several acetylated proteins, but a consensus substrate recognition sequence has not yet been established. Product inhibition of many eukaryotic sirtuins by nicotinamide and its analogues has been studied in vitro due to their potential role as anticancer agents. In this work, the kinetics of CobB, the main Escherichia coli deacetylase, have been characterized. To our knowledge, this is the first kinetic characterization of a sirtuin employing a fully acetylated and natively folded protein as a substrate. CobB deacetylated several acetyl-CoA synthetase acetylated lysines with a single kinetic rate. In addition, in vitro nicotinamide inhibition of CobB has been characterized, and the intracellular nicotinamide concentrations have been determined under different growth conditions. The results suggest that nicotinamide can act as a CobB regulator in vivo. A nicotinamidase deletion strain was thus phenotypically characterized, and it behaved similarly to the ΔcobB strain. The results of this work demonstrate the potential regulatory role of the nicotinamide metabolite in vivo.

Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Niacinamida/química , Sirtuínas/antagonistas & inibidores , Sirtuínas/química , Acetatos/química , Acetilcoenzima A/metabolismo , Acetilação , Deleção de Genes , Histonas/metabolismo , Cinética , Lisina/química , NAD/metabolismo , Fenótipo , Plasmídeos/metabolismo , Dobramento de Proteína , Sirtuínas/metabolismo , Especificidade por Substrato
J Biol Chem ; 290(38): 23077-93, 2015 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-26251518


Lysine acetylation is an important post-translational modification in the metabolic regulation of both prokaryotes and eukaryotes. In Escherichia coli, PatZ (formerly YfiQ) is the only known acetyltransferase protein and is responsible for acetyl-CoA synthetase acetylation. In this study, we demonstrated PatZ-positive cooperativity in response to acetyl-CoA and the regulation of acetyl-CoA synthetase activity by the acetylation level. Furthermore, functional analysis of an E809A mutant showed that the conserved glutamate residue is not relevant for the PatZ catalytic mechanism. Biophysical studies demonstrated that PatZ is a stable tetramer in solution and is transformed to its octameric form by autoacetylation. Moreover, this modification is reversed by the sirtuin CobB. Finally, an in silico PatZ tetramerization model based on hydrophobic and electrostatic interactions is proposed and validated by three-dimensional hydrodynamic analysis. These data reveal, for the first time, the structural regulation of an acetyltransferase by autoacetylation in a prokaryotic organism.

Acetiltransferases/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Modelos Moleculares , Multimerização Proteica , Acetilação , Acetiltransferases/genética , Acetiltransferases/metabolismo , Substituição de Aminoácidos , Animais , Coenzima A Ligases/química , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Camundongos , Mutação de Sentido Incorreto , Estrutura Quaternária de Proteína , Sirtuínas/química , Sirtuínas/genética , Sirtuínas/metabolismo