Simultaneous Modulation Of Multiple Genes Expression For Improved β-carotene Yield

The key of metabolic or biosynthetic pathway optimization lies in expression regulation of genes in this pathway, especially in the ways to regulating multiple genes simultaneously. In this study, for β-carotene production, the upstream and downstream pathways genes of β-carotene biosynthesis are regulated simultaneously.For the regulation of the upstream synthesis pathway of β-carotene, The five genes mvaE、mvaS、mvaK1、mvaD、mvaK2 of MVA pathway, originating from E.faecalis and S.Pneumoniae, was introduced into CAR005 of β-carotene production bacteria which constructed in library before. The plasmid library constituted of pathways genes with different pattern of regulation was obtained by regulating the five genes by using RBS Library and with golden gate assembly method, through a golden gate enzyme digestion ligation reaction. The plasmid library obtained by specially designed method, was transferred to CAR005. Through the early screening by β-carotene color and second screening by spectrophotometric method for the determination of β-carotene yield, representative strains with different β-carotene yield were obtained from the library and were proved to have different expression pattern of mvaE、mvaS、mvaK1、mvaD、mvaK2 by sequencing, and different β-carotene yield. The highest yield achieved was increased by 129.2% compared to the control strain. The RBS clung to genes of MVA was processed by the software to calculate the theoretical strength, of which the combination condition was also analyzed.Similarly, as to the regulation of the downstream synthesis pathway of β-carotene, through the Golden Gate DNA assembly method to construct the plasmid, the expression of β-carotene synthetic pathway genes crtE、crtY、crtI、crtB from P. agglomerans were simultaneously modulated with promoters of various strength to optimized the efficiency of this pathway. Through the screening by color and the determination of β-carotene yield, A series strains with different β-carotene yield were obtained, in which the highest yield achieved was increased by 65.2% compared to the control strain. The expression pattern of crtE、crtY、crtI、crtB was obtained by sequencing and analyzed.This study proved the concept of the simultaneous regulation of multiple genes in the metabolic pathway by constructing library with Golden Gate DNA assembling method by RBS library and promoters of various strength to improve target production, while it further proves the necessity of simultaneous regulation of multiple genes.