Construction of a novel MK-4 biosynthetic pathway in Pichia pastoris through heterologous expression of HsUBIAD1

Abstract

AbstractBackgroundWith a variety of physiological and pharmacological functions, menaquinone is an essential prenylated product that can be endogenously converted from phylloquinone (VK1) or menadione (VK3) via the expression ofHomo sapiensUBIAD1 (HsUBIAD1). The methylotrophic yeast,Pichia pastoris, is an attractive expression system that has been successfully applied to the efficient expression of heterologous proteins. However, the menaquinone biosynthetic pathway has not been discovered inP. pastoris.ResultsFirstly, we constructed a novel synthetic pathway inP. pastorisfor the production of menaquinone-4 (MK-4) via heterologous expression ofHsUBIAD1. Then, the glyceraldehyde-3-phosphate dehydrogenase constitutive promoter (PGAP) appeared to be mostsuitable for the expression ofHsUBIAD1 for various reasons. By optimizing the expression conditions ofHsUBIAD1, its yield increased by 4.37 times after incubation at pH 7.0 and 24 °C for 36 h, when compared with that under the initial conditions. We foundHsUBIAD1 expressed in recombinant GGU-23 has the ability to catalyze the biosynthesis of MK-4 when using VK1and VK3as the isopentenyl acceptor. In addition, we constructed a ribosomal DNA (rDNA)-mediated multi-copy expression vector for the fusion expression ofSaGGPPS andPpIDI, and the recombinant GGU-GrIG afforded higher MK-4 production, so that it was selected as the high-yield strain. Finally, the yield of MK-4 was maximized at 0.24 mg/g DCW by improving the GGPP supply when VK3was the isopentenyl acceptor.ConclusionsIn this study, we constructed a novel synthetic pathway inP. pastorisfor the biosynthesis of the high value-added prenylated product MK-4 through heterologous expression ofHsUBIAD1 and strengthened accumulation of GGPP. This approach could be further developed and accomplished for the biosynthesis of other prenylated products, which has great significance for theoretical research and industrial application.