Overexpress Gentiana Lutea IPI Gene In Metabolic Engineering Escherichia Coli To Improve Carotenoid Contents

Carotenoids have a great deal of important biological functions in human health. For instance, their pro-vitamin A activity, enhancing human immune ability and anti-cancer activity. Human body is not able to synthesis carotenoids and generally depending on food supply. Unfortunately, many potentially useful compounds are found in only trace quantities in their natural sources and are difficult or impossible to synthesize chemically. In recent years, nearly all the enzyme genes involved in carotenoid biosynthetic pathway from microorganisms and higher plants were cloned. With the cloning and characterization of these genes, it becomes possible to regulate carotenoid biosynthesis and enrich the carotenoid content in vivo via genetic engineering. This kind of research is feasible and of great importance. Although the carotenoid biosynthetic pathway was already successfully targeted by genetic engineering, the complex regulation of carotenogenesis still remains poorly understood. It is still not clear which enzymes are the key regulatory ones.The Gentiana lutea isopentenyl diphosphate isomerase (IPI) cDNA is 1146bp long without any poly A tail. It encompasses an open reading frame (ORF) with 708bp, encoding 235 amino acids whose molecular mass is 27kD and isoelectric point is 4.84. The un-translated regions of 5′end and 3′end are 197bp and 241bp in length, respectively. In this research, we over expressed Gentiana lutea IPI gene in Escherichia coli which was transformed with several genes that mediated lycopene, beta-carotene or zeaxanthin biosynthesis, in order to find out whether the enzyme encoded by this gene is a regulatory one. Moreover, we over expressed Gentiana lutea carotene beta-hydroxylase (BCH) and Zea mays 5-phosphate reductoisomerase (DXR) genes respectively, in order to find out over expressing which of these genes could reach the maximum amount of lycopene, beta-carotenoid or zeaxanthin in Escherichia coli. The results show that over expressing IPI gene in Escherichia coli with pACCRT-EBIeu produces most lycopene, which reaches 1953.75μg/gDW (4.51 times); over expressing IPI gene in Escherichia coli with pACCAR16ΔcrtX produces most beta-carotenoid, which reaches 2266.24μg/gDW (6.53 times) and over expressing IPI and BCH in Escherichia coli with pACCAR25ΔcrtX produces the maximum amount of zeaxanthin, which reaches 9341.51μg/gDW (19.95 times).