Identification of efficient key enzymes in biosynthesis pathway and optimization of the fitness between functional modules and chassis are important for improving the production of target compounds. In this study, the taxadiene biosynthesis pathway was firstly constructed in yeast by transforming ts gene and overexpressing erg20 and thmgr. Then, the catalytic capabilities of six different geranylgeranyl diphosphate synthases (GGPPS), the key enzyme in mevalonic acid (MVA) pathway catalyzing famesyl diphosphate (FPP) to geranylgeranyl diphosphate (GGPP), were predicted using enzyme-substrate docking strategy. GGPPSs from Taxus baccata x Taxus cuspidate (GGPPSbc), Erwinia herbicola (GGPPSeh), and S. cerevisiae (GGPPSsc) which ranked 1st, 4th and 6th in docking with FPP were selected for construction. The experimental results were consistent with the computer prediction that the engineered yeast with GGPPSbc exhibited the highest production. In addition, two chassis YSG50 and W303-1A were chosen, and the titer of taxadiene reached 72.8 mg/L in chassis YSG50 with GGPPSbc. Metabolomic study revealed that the contents of tricarboxylic acid cycle (TCA) intermediates and their precursor amino acids in chassis YSG50 was lower than those in W303-1A, indicating less carbon flux was divided into TCA cycle. Furthermore, the levels of TCA intermediates in the taxadiene producing yeasts were lower than those in chassis YSG50. Thus, it may result in more carbon flux in MVA pathway in chassis YSG50, which suggested that YSG50 was more suitable for engineering the taxadiene producing yeast. These results indicated that computer-aided protein modeling directed isoenzyme selection strategy and metabolomic study could guide the rational design of terpenes biosynthetic cells.

This study determined the effects of exogenous ascorbic acid (AsA) treatment on germination, membrane lipid peroxidation, activities of antioxidant enzymes and ultra-structure of embryo cells of Siberian wildrye (Elymus sibiricus L.) seeds with different moisture contents (4%, 10% and 16%) after ageing at 45°C for 48 h. Germination percentage and activities of enzymes declined, electrical conductivity value of seeds increased significantly as moisture content increased from 4% to 16%. However, ascorbic acid treatment had protective effects on alleviating the damage of ageing to seeds membrane integrity and structure of mitochondria, which was in accordance with the decrease of electrical conductivity and malondialdehyde (MDA) content, especially for seeds with 10% moisture content. Ascorbic acid treatment significantly (P<0.05) inhibited the loss of seed germination at 10% moisture content, and correspondingly electrical conductivity decreased and activities of catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) improved. In addition, ultra structure observation of embryo cells showed that serious damage occurred in seeds with 16% moisture content, including the broken cellular and nuclear membrane, pyknotic nucleolus and swollen mitochondria. Although activities of CAT, APX and GR were improved by the exogenous AsA, it had no obviously protective effect on integrity of membrane system and mitochondria. Seed moisture of Siberian wildrye played a vital role in the function of AsA, which provided an efficient treatment to deal with the antioxidant effects of storage and ageing at seed moisture of 10%.