Desenvolvimento de sistema de controle de fluxo de carbono em vias metabólicas sintéticas de levedura
Carregando...
Data
2022-12-08
Autores
Lelis, Maria Cecília de Souza
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
O limoneno é um monoterpeno cíclico amplamente utilizado por seu aroma agradável na indústria de cosméticos, sendo também utilizado nas indústrias de biomateriais e biocombustíveis por ser um precursor químico nestes ramos. Assim, a produção em microrganismos surge como uma opção rápida e prática para atender a demanda comercial. Entretanto, a rota metabólica dos terpenoides compete por recursos com a maquinaria celular. Até o momento, as linhagens de Saccharomyces cerevisiae que foram construídas visando o aumento da produção de terpenos, sofrem com o acúmulo de isopentanil pirofosfato (IPP), um dos precursores da rota dos isoprenoides, que é tóxico para a célula em altas concentrações. Através da reconstrução dos genes IDI1, ERG20W e ClLS, buscamos aumentar o fluxo de carbono pela via de biossíntese de monoterpenos e reduzir a disponibilidade citoplasmática de IPP, visando direcionar o fluxo de carbono para a produção de limoneno pela canalização de substrato, que consiste na formação de complexos proteicos formados por enzimas que catalisam reações subsequentes em uma via metabólica. Assim, buscou-se clonar os genes das enzimas da via metabólica fusionados a synzips, que consistem em peptídeos sintéticos capazes de formar heterodímeros. Desta forma, sondamos três configurações para a canalização de substrato, heterodímero linear, heterotrímero linear e heterotetrâmero em anel, para comparar a capacidade de produção de limoneno entre as linhagens. Para as etapas de clonagem, utilizamos o plasmídeo pYES2, que possui origem de replicação para Escherichia coli e S. cerevisiae. As etapas consistiram em ligar ao plasmídeo os genes CILS-synzip21 e ERG20w-synzip19, para formar o heterodímero, IDI1-synzip18, ClLS-synzip21 e ERG20w-synzip19, para formar o heterotrímero, e ligar ClLS-synzip20 aos genes anteriores, para formar o heterotetrâmero.
Limonene is a cyclic monoterpene widely used for its pleasant aroma in the cosmetics industry, being also used in the biomaterials and biofuels industries for being a chemical precursor in these branches. Thus, production in microorganisms appears as a quick and practical option to meet commercial demand. However, the terpenoid metabolic pathway competes for resources with the cellular machinery. So far, the Saccharomyces cerevisiae strains that were built to increase terpene production suffer from the accumulation of isopentanyl pyrophosphate (IPP), one of the precursors of the isoprenoid pathway, which is toxic to the cell in high concentrations. Through the reconstruction of the IDI1, ERG20W and ClLS genes, we seek to increase the carbon flux through monoterpene biosynthesis and reduce the cytoplasmic availability of IPP, aiming to direct the carbon flux to the production of limonene by channeling the substrate, which consists of formation of protein complexes formed by enzymes that catalyze subsequent reactions in a metabolic pathway. Thus, we sought to clone the genes of the enzymes of the metabolic pathway fused to synzips, which consist of synthetic peptides capable of forming heterodimers. In this way, we probed three configurations for channeling substrate, linear heterodimer, linear heterotrimer and ring heterotetramer, to compare the limonene production capacity between the strains. For the cloning steps, we used the pYES2 plasmid, which has an origin of replication for Escherichia coli and S. cerevisiae. The steps consisted of linking the ClLS-synzip21 and ERG20w-synzip19 genes to the plasmid, to form the heterodimer, IDI1- synzip18, ClLS-synzip21 and ERG20w-synzip19, to form the heterotrimer, and linking ClLSsynzip20 to the previous genes, to form the heterotetramer.
Limonene is a cyclic monoterpene widely used for its pleasant aroma in the cosmetics industry, being also used in the biomaterials and biofuels industries for being a chemical precursor in these branches. Thus, production in microorganisms appears as a quick and practical option to meet commercial demand. However, the terpenoid metabolic pathway competes for resources with the cellular machinery. So far, the Saccharomyces cerevisiae strains that were built to increase terpene production suffer from the accumulation of isopentanyl pyrophosphate (IPP), one of the precursors of the isoprenoid pathway, which is toxic to the cell in high concentrations. Through the reconstruction of the IDI1, ERG20W and ClLS genes, we seek to increase the carbon flux through monoterpene biosynthesis and reduce the cytoplasmic availability of IPP, aiming to direct the carbon flux to the production of limonene by channeling the substrate, which consists of formation of protein complexes formed by enzymes that catalyze subsequent reactions in a metabolic pathway. Thus, we sought to clone the genes of the enzymes of the metabolic pathway fused to synzips, which consist of synthetic peptides capable of forming heterodimers. In this way, we probed three configurations for channeling substrate, linear heterodimer, linear heterotrimer and ring heterotetramer, to compare the limonene production capacity between the strains. For the cloning steps, we used the pYES2 plasmid, which has an origin of replication for Escherichia coli and S. cerevisiae. The steps consisted of linking the ClLS-synzip21 and ERG20w-synzip19 genes to the plasmid, to form the heterodimer, IDI1- synzip18, ClLS-synzip21 and ERG20w-synzip19, to form the heterotrimer, and linking ClLSsynzip20 to the previous genes, to form the heterotetramer.
Descrição
Palavras-chave
limoneno, canalização de substrato, Saccharomyces cerevisiae