Type IIs restriction based combinatory modulation technique for metabolic pathway optimization

Abstract

BackgroundOne of the most important research subjects of metabolic engineering is pursuing a balanced metabolic pathway, which is the basis of an efficient cell factory. In this work, we dedicated to develop a simple and efficient technique to modulate expression of multiple genes simultaneously, and select for the optimal regulation pattern.ResultsA Type IIs restriction based combinatory modulation (TRCM) technique was designed and established in the research. With this technique, a plasmid library containing variably regulated mvaE, mvaS, mvaK1, mvaD and mvaK2 of the mevalonate (MVA) pathway were obtained and transformed into E. coli DXS37-IDI46 to obtain a β-carotene producer library. The ratio of successfully assembled plasmids was determined to be 35%, which was increased to 100% when color based pre-screening was applied. Representative strains were sequenced to contain diverse RBSs as designed to regulate expression of MVA pathway genes. A relatively balanced MVA pathway was achieved in E. coli cell factory to increase the β-carotene yield by two fold. Furthermore, the approximate regulation pattern of this optimal MVA pathway was illustrated.ConclusionsA TRCM technique for metabolic pathway optimization was designed and established in this research, which can be applied to various applications in terms of metabolic pathway regulation and optimization.