ABSTRACT
Glycerol is a byproduct during biodiesel production. It is an important feedstock for fermentation due to its low price and high reduced status. Multiple genes of the glycerol utilization pathway were modulated in a previously engineered high β-carotene producing Escherichia coli strain CAR015 to enhance glycerol utilization capability for improving isoprenoids production. The glpR gene, encoding glycerol 3-phosphate repressor, was firstly deleted. The glpFK, glpD and tpiA genes were then modulated by three artificial regulatory parts, M1-37, M1-46 and M1-93, respectively. β-carotene titer reached 64.82 mg/L after modulating glpD with M1-46, which was 4.86 times higher than that of CAR015, and glycerol consumption rate also increased 100%. Modulating tpiA led to a little increase of β-carotene titer, whereas modulating glpFK led to a little decrease of β-carotene titer. This demonstrated that GlpD was a rate-limiting step in glycerol utilization pathway. Q-PCR of glpF, glpK, glpD and tpiA results showed that decrease the transcription level of glpF, glpK, glpD, or decrease the transcription level of tpiA could increase the cell growth and β-carotene production, probably for the decrease of methylglyoxal toxicity. Modulating glpD and tpiA genes in combination resulted in the best strain Gly003, which produced 72.45 mg/L β-carotene with a yield of 18.65 mg/g dry cell weight. The titer was 5.23 and yield 1.99 times of that of the parent strain CAR015. Our work suggested that appropriate activation of glpD and tpiA genes in glycerol utilization pathway could effectively improve β-carotene production. This strategy can be used for production of other terpenoids in E. coli.
ABSTRACT
We designed a strategy for the sequencing and bioinformatical characterization of the 1,3-propanediol operon regulator genes from the Colombian Clostridium sp. strain IBUN13A, which is taxonomically related to Clostridium butyricum. Three genes are proposed to be involved in the operon's transcriptional activity, the dhaS and dhaA genes through a two-component system and the third gene named dhaY, which encodes a putative transcriptional regulator similar to the domains of the dhaS/A system. Phylogenetic analyses indicated that the predicted proteins had a modular structure consisting of domains homologous to different signal transduction systems, but had significant differences concerning their conserved residues, pointing to the possibility that they constitute ancestral domains. In accordance with the prediction of functions, we propose a mechanism of regulation of the proteins studied of the 1,3-propanediol operon of the native strain, as a response to the presence of glycerol in the medium, which provides valuable information on the overall regulation of the glycerol metabolism in Clostridium sp.
Se diseñó una estrategia de amplificación, secuenciación y caracterización bioinformática de los genes reguladores del operón 1,3-propanediol (1,3-PD) de la cepa nativa colombiana Clostridium sp. IBUN 13A, relacionada taxonómicamente con Clostridium butyricum. Se identificaron tres genes que pueden estar involucrados en la regulación transcripcional de dicho operón: los genes dhaS y dhaA -a través de un sistema de transducción de señales de dos componentes-y un tercer gen que se denominó dhaY, que codifica para un regulador transcripcional putativo, similar a los dominios presentes en las proteínas del sistema DhaS/A. Los análisis filogenéticos indican que las proteínas predichas presentan una estructura modular con dominios homólogos a diferentes sistemas de transducción de señales, pero muestran diferencias importantes en los residuos conservados, lo que sugiere que podrían ser estos los dominios ancestrales. La predicción de funciones postula un mecanismo de regulación de las proteínas estudiadas sobre el promotor del operón 1,3-PD de la cepa nativa como respuesta a la presencia de glicerol en el medio, lo cual aporta información importante sobre la regulación global del metabolismo del glicerol en Clostridium sp.
Nesta pesquisa foi feita uma estratégia para a amplificacäo, sequenciamento e caracterizacäo bioinformática dos genes reguladores do operon 1,3 propanodiol (1,3-PD) da cepa colombiana Clostridium sp. IBUN 13A, relacionada taxonomicamente com o Clostridium butyricum. Tèm sido identificados tres genes que podem estar envolvidos na regulacáo transcricional do operon. Os genes dhaS e dhaA por meio de um sistema de dois componentes e o terceiro gene nomeado de dhaY, que codifica para um regulador transcridonal putativo, parecido com os dominios presentes nas proteínas do sistema DhaS/A. A análise filogenètica mostra que estas proteínas apresentam uma estrutura modular com dominios homólogos a diferentes sistemas de traducáo de sinais, mas pressupöem diferencas importantes nos residuos conservados, indicando provavelmente que possam constituir os dominios ancestrais. De acordo com a predicáo de funcöes, é postulado um mecanismo de regulacáo do sistema DhaS/A, DhaY sobre o promotor do operon 1,3-DP da cepa nativa, como resposta à presenca de glicerol no meio, aportando informacöes importantes da regulacáo global do metabolismo do glicerol no Clostridium sp.