RESUMO
The Ntr regulon in Escherichia coli has previously been engineered to control the expression of a heterologous metabolic pathway. In this study, we reengineered the same system for protein production. In the absence of NRII (glnL gene product), we showed that glnAp2 can be an effective promoter for protein production that is inducible by exogenous acetate, but both the induction ratio and the range of modulation are low. To deal with this issue, we inactivated phosphotransacetylase (pta gene product), which disrupts the acetate pathway and denies the cell the ability to synthesize acetate. With this additional modification, gene expression from glnAp2 can be controlled by directly adding acetate into the growth medium. Using a lacZ reporter fusion, we found that glnAp2 induction was modulatable over a range of potassium acetate concentrations, and the induction/noninduction ratio increased to 77 in the absence of pta. The extracellular acetate required for maximal induction is lower than the concentration that causes toxicity, and thus growth inhibition by acetate addition was not a matter of concern. Furthermore, compared to the P(tac) promoter, overexpression of a model protein using the modified glnAp2 promoter system did not cause significant growth inhibition, although a higher level of protein expression was achieved.
Assuntos
Ácido Acético/farmacologia , Escherichia coli/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Biossíntese de Proteínas , Biotecnologia/métodos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Fixação de Nitrogênio/genética , Proteínas/genética , Regulação para CimaRESUMO
One issue that must be addressed in the rational design of metabolic pathways is the elimination of potential bottlenecks in the upstream pathways. We have reconstructed the isoprenoid pathway to overproduce the carotenoid lycopene in Escherichia coli. Here we show that the distribution between pyruvate and glyceraldehyde 3-phosphate (G3P), the originating precursors of the isoprenoid pathway, is a major factor that can limit isoprenoid production yields in E. coli. In particular, alterations in the central metabolism that redirect flux from pyruvate back to G3P enhance lycopene production, while alterations that channel carbon flux away from the G3P pool have the opposite effect. These results suggest that G3P may be limiting in the biosynthesis of lycopene, and modifications that achieve a more equitable distribution between the two precursors are able to increase the lycopene yield in metabolically engineered E. coli.
Assuntos
Carotenoides/biossíntese , Escherichia coli/metabolismo , Gluconeogênese , Glicólise , Cinética , LicopenoRESUMO
Metabolic engineering has achieved encouraging success in producing foreign metabolites in a variety of hosts. However, common strategies for engineering metabolic pathways focus on amplifying the desired enzymes and deregulating cellular controls. As a result, uncontrolled or deregulated metabolic pathways lead to metabolic imbalance and suboptimal productivity. Here we have demonstrated the second stage of metabolic engineering effort by designing and engineering a regulatory circuit to control gene expression in response to intracellular metabolic states. Specifically, we recruited and altered one of the global regulatory systems in Escherichia coli, the Ntr regulon, to control the engineered lycopene biosynthesis pathway. The artificially engineered regulon, stimulated by excess glycolytic flux through sensing of an intracellular metabolite, acetyl phosphate, controls the expression of two key enzymes in lycopene synthesis in response to flux dynamics. This intracellular control loop significantly enhanced lycopene production while reducing the negative impact caused by metabolic imbalance. Although we demonstrated this strategy for metabolite production, it can be extended into other fields where gene expression must be closely controlled by intracellular physiology, such as gene therapy.
Assuntos
Proteínas de Bactérias , Carotenoides/biossíntese , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Engenharia Genética/métodos , Transativadores , Fatores de Transcrição , 3-Desoxi-7-Fosfo-Heptulonato Sintase/biossíntese , 3-Desoxi-7-Fosfo-Heptulonato Sintase/genética , Anticarcinógenos/metabolismo , Antioxidantes/metabolismo , Isomerases de Ligação Dupla Carbono-Carbono/biossíntese , Isomerases de Ligação Dupla Carbono-Carbono/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/metabolismo , Retroalimentação , Dosagem de Genes , Glicólise , Hemiterpenos , Licopeno , Metabolismo/genética , Nitrogênio/deficiência , Organofosfatos/metabolismo , Proteínas PII Reguladoras de Nitrogênio , Fosfoproteínas Fosfatases/genética , Fosfotransferases (Aceptores Pareados)/biossíntese , Fosfotransferases (Aceptores Pareados)/genética , Proteínas Quinases/genética , RegulonRESUMO
Acetate excretion by Escherichia coli during aerobic growth on glucose is a major obstacle to enhanced recombinant protein production. We report here that the fraction of carbon flux through the anaplerotic pathways is one of the factors influencing acetate excretion. Flux analysis of E. coli central metabolic pathways predicts that increasing the fraction of carbon flux through the phosphoenolpyruvate carboxylase (PPC) pathway and the glyoxylate bypass reduces acetate production. We tested this prediction by overexpressing PPC and deregulating the glyoxylate bypass by using a fadR strain. Results show that the acetate yield by the fadR strain with PPC overexpression is decreased more than fourfold compared to the control, while the biomass yield is relatively unaffected. Apparently, the fraction of carbon flux through the anaplerotic pathways is one of the factors that influence acetate excretion. These results confirm the prediction of our flux analysis and further suggest that E. coli is not fully optimized for efficient utilization of glucose.