Enhanced β-Carotene Biosynthesis in Recombinant Escherichia Coli Harboring the Bottom Portion of Mevalonate Pathway of Enterococcus Faecium VTCC-B-935 Isolated in Vietnam

Abstract

Abbreviations: IPP, isopentenyl diphosphate; MEP, 2-C-methyl-d-erythritol-4-phosphate; MVA, mevalonate; DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; G3P, glyceraldehyde-3-phosphate Introduction β-carotene, one of the carotenoid compounds, was found mainly in plants. Because of it's highly antioxidant activity, this compound was shown to have good effects to human health. It is the precursor of vitamin A which is critically required for human.1 Due to good health effects and yellow color, β-carotene is widely used in industries including nutraceuticals, pharmaceuticals, food colorants, cosmetics, and animal feed additives. However, production of natural β-carotene is not sufficient to afford demand of the market. Currently, more than 90% of commercially available β-carotene is chemically synthesized. 2This fact leads a number of research groups to the trend of seeking for alternative sources of natural β-carotene. Natural carotenogenic microorganisms, e.g. Blakeslea trispora, Rhodotorula glutinis, and Dunaliella salina have been used for fermentation to produce β-carotene. 3-5The availability of carotenoid genes from natural carotenogenic organisms leads to another strategy of natural carotenoid biosynthesis. Carotenoid genes were cloned and introduced into non-carotenogenic organism, Escherichia coli, for production of carotenoid, including β-carotene.6-19 E. coli is considered as one of the ideal hosts because of its convenience genetic engineering system and fast growth. However, to date, the production of β-carotene is still unable to support industrial needs because of insufficient yield and stability. Therefore, attempts are required for improvement of production of carotenoid in general and β-carotene in particular. Biosynthesis of β-carotene was affected by a number of factors. In our study, we focus in i. The different expression vector backbones ii. Origin of foreign genes iii. Availability and balance of isopentenyl diphosphate (IPP) which is the building blocks of carotenoids, and iv. Effect of additional carbon sources Biosynthesis partway of β-carotene is indicated in Figure 1. DMAPP and IPP, the building blocks of carotenoid are synthesized via 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway which is autonomous in our chosen host E. coli. However, in theory the natural yield of these precursors is only sufficient for natural need of E. coli in normal growth conditions which is far less than that required once the organism is used as a microbial factory to produce β-carotene. To address this issue, addition of the exogenous mevalonate (MVA) pathway was shown as a reasonable strategy. 6,12,18,20,21 The MVA pathway is divided into two portions, the upper (from acetyl-CoA to MVA) and the lower (from MVA to DMAPP and IPP). Natural E. coli harbors MEP pathway that enable biosynthesis of FPP from G3P and pyruvate, as well as IPP isomerase catalyze the two-way conversion of IPP and DMAPP, the precursors of carotenoid (yellow part). These precursors could ...