Abstract
BackgroundWith 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 of Homo sapiens UBIAD1 (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 in P. pastoris.ResultsFirstly, we constructed a novel synthetic pathway in P. pastoris for the production of menaquinone-4 (MK-4) via heterologous expression of HsUBIAD1. Then, the glyceraldehyde-3-phosphate dehydrogenase constitutive promoter (PGAP) appeared to be mostsuitable for the expression of HsUBIAD1 for various reasons. By optimizing the expression conditions of HsUBIAD1, 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 found HsUBIAD1 expressed in recombinant GGU-23 has the ability to catalyze the biosynthesis of MK-4 when using VK1 and VK3 as the isopentenyl acceptor. In addition, we constructed a ribosomal DNA (rDNA)-mediated multi-copy expression vector for the fusion expression of SaGGPPS and PpIDI, 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 VK3 was the isopentenyl acceptor.ConclusionsIn this study, we constructed a novel synthetic pathway in P. pastoris for the biosynthesis of the high value-added prenylated product MK-4 through heterologous expression of HsUBIAD1 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.
Highlights
With 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 of Homo sapiens UBIAD1 (HsUBIAD1)
Recent research has found that Homo sapiens UbiA prenyltransferase containing 1 (HsUBIAD1), a human homologue of Escherichia coli prenyltransferase menA and mammalian mitochondrial prenyltransferase C OQ2 catalyzes the conversion of PK to its prenylated derivative MK-4 [12,13,14]
The prediction results of ProtScale showed a GRVAY (Grand average of hydropathicity) value of 0.528, indicating that HsUBIAD1 is a hydrophobin, which was consistent with the predictions of SOSUI (Additional file 1: Figure S2)
Summary
With 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 of Homo sapiens UBIAD1 (HsUBIAD1). It is relatively difficult to screen stable high expression strains using animal cell expression systems and often requires a longer time period due to their low recombination rate. The methylotrophic yeast, Pichia pastoris has become an attractive workhorse for biotechnology, especially for the production of both secreted and intracellular heterologous proteins [18,19,20,21]. It is an important heterologous protein expressing system that has several advantages over other eukaryotic and prokaryotic expression systems. Recent studies have shown that the level of expression seen with the P GAP can be slightly higher than that obtained with the P AOX1 [29, 31, 33, 34]
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