中国生物工程杂志

Objective: The natural synthesis pathway of isopentenyl pyrophosphate, the precursor of isoprene compounds in microorganisms, is subject to strict metabolic regulation, which limits the efficient biosynthesis of isoprene compounds, and the use of the artificial isopentenol utilization pathway (IUP) is independent of the biological endogenous metabolic pathways. By introducing IUP into microorganisms, a large amount of isopentenol pyrophosphate can be synthesized, thereby promoting the large amount of synthesis of isoprene compounds. Methods: Introducing the artificial IUP into the oleaginous yeast Yarrowia lipolytica to strengthen the isopentenyl pyrophosphate biosynthesis and promote the efficient accumulation of β-carotene. Results: The tertiary structures of two key proteins involved in IUP, ScCK (Choline kinase from Saccharomyces cerevisiae) and AtIPK (isopentenyl phosphate kinase from Arabidopsis thaliana), are predicted to be acidic hydrophilic proteins without transmembrane domain and signal peptide. Both proteins have loose and unstable structural features and contain a variety of post-translational features. Modification sites and multiple interacting proteins are involved in phosphorylation. The β-carotene synthesis pathway was constructed by introducing carRP and carB, and strengthening the expression of thmgR and ggs1 in Yarrowia lipolytica by using homologous recombination technique, and 2.68 mg/L β-carotene was accumulated in the engineered strain. The ura on the genome were removed by the Cre-loxP system, and then IUP was integrated into the genome of this engineered strain. The yield of β-carotene was achieved at the levels of 410.2 mg/L in the engineered strain, which was nearly 200 folds greater than that from the original engineered strain. The fermentation conditions were as follows: cells were incubated for 96 h in the medium containing 20 mmol/L isoprenol and the ratio of carbon to nitrogen was set as 4/3 in the medium. Conclusion: IUP can promote the efficient accumulation of β-carotene in Y. lipolytica. This study provides a new strategy for the efficient biosynthesis of β-carotene and other isoprene compounds by using IUP.