Reprogramação do metabolismo de purina na bactéria Bacillus subtilis por tecnologia de pequenos RNAs não codificadores (sRNAs)
Carregando...
Data
2019-07-25
Autores
Lins, Milca Rachel da Costa Ribeiro
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
As técnicas mais usadas atualmente para alterar o fluxo metabólico por uma determinada via são a deleção e/ou inserção de sequências regulatórias ou genes no cromossomo bacteriano. Estes são métodos que alteram permanentemente a via metabólica em questão, perturbando o balanço metabólico durante a fase lag de crescimento, o que muitas vezes leva a um excessivo prolongamento desta fase além de diminuir a viabilidade celular. O metabolismo de purinas em Bacillus subtilis tem grande potencial para manipulação genética e produz metabólitos com valor biotecnológico. Cinco riboswitches (purE, xpt, nupG, pbuE e pbuG) controlam o metabolismo de purina em B. subtilis promovendo a terminação precoce da transcrição em ligação com guanina ou adenina. O GTP gerado nesta via é utilizado na biossíntese da vitamina B2 (riboflavina), apresentando o mesmo mecanismo de regulação com riboswitch de flavina-monofosfato (ribDG). Neste estudo, um novo modelo de controle de metabolismo de purina foi construído visando o aumento reversível do fluxo de carbono pela via em B. subtilis utilizando pequenos RNAs não-codificadores sintéticos (sRNAs). Estes foram desenhados e sintetizados para interferir na regulação da expressão gênica realizada pelos riboswitches, mantendo assim a via biossintética ativa. Os riboswitches foram caracterizados por uma nova metodologia in vitro na presença dos ligantes guanina, adenina ou FMN, e nas células de B. subtilis, sendo possível constatar o papel regulador sobre o operon da bioluminescência luxABCDE. Os sRNAs sintéticos foram desenhados no software Ribomaker para interferir na estrutura secundária dos riboswitches e, após simulações in silico da interação intermolecular riboswitch-sRNA, a funcionalidade dos sRNAs também foi ensaiada in vitro. B. subtilis reprogramado com os sRNAs desenhados contra os riboswitches purE, pbuE e ridDG foram capazes de neutralizar o efeito riborregulador comparado aos controles, após análises de bioluminescência e fluorescência em leitor de microplacas. A inserção da sequência RiboJ à extremidade 5’ UTR do sRNA(purE) proporcionou ótimas características a B. subtilis para ser utilizada como uma linhagem industrial, por exemplo, fase lag diminuída e crescimento populacional maior. Foi possível modelar exemplarmente o bem estudado metabolismo de purinas nesta bactéria amplamente utilizada pela indústria biotecnológica, e demonstrar que a tecnologia sRNA é realmente adequada para a engenharia metabólica de cepas microbianas produtoras de produtos biotecnológicos. A vantagem deste conceito inovador de controle é que a expressão do gene pode ser tanto ligada como desligada conforme necessário. O novo conceito é amplamente aplicável e pode ser um elemento inovador no portfólio da moderna biologia sintética.
The currently employed tools to redirect metabolic flux through a specific pathway are deletion and/or insertion of regulatory sequences or genes in the bacterial chromosome. These methods permanently modify the pathway perturbing the metabolic balance during the lag growth phase, which generally extends this process and also decreases cell viability. The purine metabolism in Bacillus subtilis has great potential for genetic manipulation and produces value-added biotechnological products. Five riboswitches (purE, xpt, nupG, pbuE and pbuG) control the purine metabolism in B. subtilis by promoting the premature transcription termination guided by guanine or adenine levels in the cell. The GTP generated in this pathway is used in the biosynthesis of vitamin B2 (riboflavin), which is regulated by a flavin-monophosphate riboswitch (ribDG). In this study, a new metabolism control model was developed aiming to reversibly increase the carbon flux through selected pathways employing small synthetic non-coding RNAs (sRNAs). These sRNAs were designed and synthesized to interfere with the regulation of gene expression performed by the riboswitches, thus maintaining the biosynthetic pathways active. The riboswitches were characterized by a new in vitro methodology in the presence of the ligands: guanine, adenine or FMN. The riboswitches were characterized in B. subtilis cells using the luxABCDE bioluminescence operon as reporter. Synthetic sRNAs were designed in the Ribomaker software to interfere with the secondary structure of the riboswitches and, following in silico simulations of the intermolecular riboswitch-sRNA interaction, sRNA functionality was also assayed in vitro. B. subtilis reprogrammed with the sRNAs designed against the purE, pbuE and ridDG riboswitches were able to neutralize the riboregulatory effect comparing to the controls. Insertion of the RiboJ sequence to the 5' UTR site of the sRNA(purE) provided excellent features to B. subtilis as an industrial strain, as shorter growth lag phase and increased population density. It was possible to exemplarily model the well-studied purine metabolism in this bacteria which is widely used by the biotechnology industry and to demonstrate that sRNA technology is actually suitable for the metabolic engineering of producing strains. The advantage of this novel control concept is that gene expression can be either turned ON or OFF as needed. The new concept is widely applicable and may be an innovative element in the portfolio of modern synthetic biology.
The currently employed tools to redirect metabolic flux through a specific pathway are deletion and/or insertion of regulatory sequences or genes in the bacterial chromosome. These methods permanently modify the pathway perturbing the metabolic balance during the lag growth phase, which generally extends this process and also decreases cell viability. The purine metabolism in Bacillus subtilis has great potential for genetic manipulation and produces value-added biotechnological products. Five riboswitches (purE, xpt, nupG, pbuE and pbuG) control the purine metabolism in B. subtilis by promoting the premature transcription termination guided by guanine or adenine levels in the cell. The GTP generated in this pathway is used in the biosynthesis of vitamin B2 (riboflavin), which is regulated by a flavin-monophosphate riboswitch (ribDG). In this study, a new metabolism control model was developed aiming to reversibly increase the carbon flux through selected pathways employing small synthetic non-coding RNAs (sRNAs). These sRNAs were designed and synthesized to interfere with the regulation of gene expression performed by the riboswitches, thus maintaining the biosynthetic pathways active. The riboswitches were characterized by a new in vitro methodology in the presence of the ligands: guanine, adenine or FMN. The riboswitches were characterized in B. subtilis cells using the luxABCDE bioluminescence operon as reporter. Synthetic sRNAs were designed in the Ribomaker software to interfere with the secondary structure of the riboswitches and, following in silico simulations of the intermolecular riboswitch-sRNA interaction, sRNA functionality was also assayed in vitro. B. subtilis reprogrammed with the sRNAs designed against the purE, pbuE and ridDG riboswitches were able to neutralize the riboregulatory effect comparing to the controls. Insertion of the RiboJ sequence to the 5' UTR site of the sRNA(purE) provided excellent features to B. subtilis as an industrial strain, as shorter growth lag phase and increased population density. It was possible to exemplarily model the well-studied purine metabolism in this bacteria which is widely used by the biotechnology industry and to demonstrate that sRNA technology is actually suitable for the metabolic engineering of producing strains. The advantage of this novel control concept is that gene expression can be either turned ON or OFF as needed. The new concept is widely applicable and may be an innovative element in the portfolio of modern synthetic biology.
Descrição
Palavras-chave
Bacillus subtilis, Purinas, Riboflavina, Riborregulação, Riboswitch, RNAs reguladores, RNAs sintéticos, Purines, Regulatory RNAs, Riboflavin, Riboregulation, Synthetic RNAs