Reconstruction of the Biosynthetic Pathway of Santalols under Control of the GAL Regulatory System in Yeast.

Sandalwood oil has been widely used in perfumery industries and aromatherapy. Santalols are its major components. Herein, we attempted to construct santalol-producing yeasts. To alter flux from predominant triterpenoid/steroid biosynthesis to sesquiterpenoid production, expression of ERG9 (encoding yeast squalene synthase) was depressed by replacing its innate promotor with PHXT1 and fermenting the resulting strains in galactose-rich media. And the genes related to santalol biosynthesis were overexpressed under control of GAL promotors, which linked santalol biosynthesis to GAL regulatory system. GAL4 (a transcriptional activator of GAL promotors) and PGM2 (a yeast phosphoglucomutase) were overexpressed to overall promote this artificial santalol biosynthetic pathway and enhance galactose uptake. 1.3 g/L santalols and 1.2 g/L Z-α-santalol were achieved in the strain WL17 expressing SaSS (α-santalene synthase from Santalum album) and WL19 expressing SanSyn (α-santalene synthase from Clausena lansium) by fed-batch fermentation, respectively. This study constructed the microbial santalol-producing platform for the first time.

Characterization of santalene synthases using an inorganic pyrophosphatase coupled colorimetric assay.

We developed a colorimetric assay using yeast inorganic pyrophosphatase (IPP1) as a coupling enzyme to measure the activities of terpene synthases. IPP1 hydrolyzes pyrophosphate, the byproduct of terpene synthase catalyzed reactions, into orthophosphate, which can then be quantitated by reacting with molybdic acid to form a blue color compound. As a proof of concept, this method was used to quantitatively characterize three santalene synthases, SaSSy and SspiSSy involved in sandalwood oil biosynthesis, and a phylogenetically distant SanSyn from Clausena lansium. Our study provided the kinetic parameters of all three santalene synthases and demonstrated the validity of the enzyme couple colorimetric assay by the comparison of this assay with the existing GC-MS (Gas Chromatography-Mass Spectrometry) method.

Production of farnesene and santalene by Saccharomyces cerevisiae using fed-batch cultivations with RQ-controlled feed.

Terpenes have various applications as fragrances, cosmetics and fuels. One of the most prominent examples is the sesquiterpene farnesene, which can be used as diesel substitute in its hydrogenated form farnesane. Recent metabolic engineering efforts have enabled efficient production of several terpenes in Saccharomyces cerevisiae and Escherichia coli. Plant terpene synthases take on an essential function for sesquiterpene production as they catalyze the specific conversion of the universal precursor farnesyl diphosphate (FPP) to the sesquiterpene of interest and thereby impose limitations on the overall productivity. Using farnesene as a case study, we chose three terpene synthases with distinct plant origins and compared their applicability for farnesene production in the yeast S. cerevisiae. Differences regarding the efficiency of these enzymes were observed in shake flask cultivation with maximal final titers of 4 mg/L using α-farnesene synthase from Malus domestica. By employing two existing platform strains optimized for sesquiterpene production, final titers could be raised up 170 mg/L in fed-batch fermentations with RQ-controlled exponential feeding. Based on these experiments, the difference between the selected synthases was not significant. Lastly, the same fermentation setup was used to compare these results to production of the fragrance sesquiterpene santalene, and almost equivalent titers were obtained with 163 mg/L, using the highest producing strain expressing a santalene synthase from Clausena lansium. However, a reduction of the product yield on biomass by 50% could indicate a higher catalytic efficiency of the farnesene synthase.

Response of Chinese wampee axes and maize embryos to dehydration at different rates.

Survival of wampee (Clausena lansiumSkeels) axes and maize (Zea mays L.) embryos decreased with rapid and slow dehydration. Damage of wampee axes by rapid dehydration was much less than by slow dehydration, and that was contrary to maize embryos. The malondialdehyde contents of wampee axes and maize embryos rapidly increased with dehydration, those of wampee axes were lower during rapid dehydration than during slow dehydration, and those of maize embryos were higher during rapid dehydration than during slow dehydration. Activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) of wampee axes markedly increased during the early phase of dehydration, and then rapidly decreased, and those of rapidly dehydrated axes were higher than those of slow dehydrated axes when they were dehydrated to low water contents. Activities of SOD and APX of maize embryos notable decreased with dehydration. There were higher SOD activities and lower APX activities of slowly dehydrated maize embryos compared with rapidly dehydrated maize embryos. CAT activities of maize embryos markedly increased during the early phase of dehydration, and then decreased, and those of slowly dehydrated embryos were higher than those of rapidly dehydrated embryos during the late phase of dehydration.