Biosynthesis Of A Ginsenoside Precursor By Assembly Of Two Key Enzymes In Pichia Pastoris

Ginsenosides are bioactive triterpenoids found in the popular oriental medicinal plant, ginseng. Production of ginsenosides by extraction from ginseng roots is a time-consuming and labor-intensive process. Biotechnological strategies including tissue culture, transgenic plants, and engineered yeast cells, have been used for more rapid and efficient production of ginseng biomass and ginsenosides. Yeast is one of the best candidates for natural products production through synthetic biology methods. Dammarenediol-Ⅱ, as a ginsenoside precursor, has been successfully produced in Saccharomyces cerevisiae. However, heterologous expression of dammarenediol-II synthase (PgDDS) alone is insufficient to achieve a satisfactory yield of dammarenediol-II. In this study, strategies for assembling squalene epoxidase (ERG1) and PgDDS, both of which are key enzymes in the ginsenoside biosynthesis pathway, were developed in Pichia pastoris to enhance the biosynthesis of dammarenediol-Ⅱ. Moreover, optimization of the culture conditions was carried out for further improvement of dammarenediol-II yield.PgDDS gene and ERG1 gene were integrated into the genome of P. pastoris GS115 to construct the dammarenediol-II biosynthetic pathway in P. pastoris. PgDDS and ERG1 were co-localized through three different strategies:co-expression, fusion-expression and self-assemby method via interactional protein domain and its ligand. There is no significant difference in growth rate of yeast cells among different strains, while the self-assembly strategy was more efficient for the biosynthesis of dammarenediol-II, resulting in a 2.1-fold enhancement in dammarenediol-II yields.A mCherry-based bimolecular fluorescence complementation (BiFC) was performed to examine the assembly dynamics of ERG1 and PgDDS in yeast cells. Chimeric proteins were constructed by fusing the mN159 fragment and mC160 fragment of mCherry to ERG1 and PgDDS. As indicated by fluorescence complementation experiments with KMM and KMPMP, localization of PgDDS was originally observed to be close to that of ERG1 in lipid particles. The specific interaction between PDZ and its ligand brought PgDDS and ERG1 closer in space resulting in higher fluorescence intensity.To improve dammarenediol-Ⅱ production in KDPEP strain, several factors such as temperature, pH, methanol concentration and medium composition were examined and the influence of these factors on growth rate and production was measured.Optimal conditions for dammarenediol-II production were BMGY/BMMY medium,28℃, pH=6.0 and 1% methanol/24 h. Dammarenediol-II production was increased by 51% under this optimal condition.The assembly strategy in this study could also be applied to the biosynthesis of other high value-added natural products, which presents a potential method in synthetic biology.