Increasing proline and myo-inositol improves tolerance of Saccharomyces cerevisiae to the mixture of multiple lignocellulose-derived inhibitors.

BACKGROUND: The development of robust microbes with tolerance to the combined lignocellulose-derived inhibitors is critical for the efficient cellulosic ethanol production. However, the lack of understanding on the inhibition mechanism limited the rational engineering of tolerant strain. Here, through the metabolomic analysis of an adaptation process of Saccharomyces cerevisiae to representative inhibitors, i.e., furfural, acetic acid and phenol (FAP), we figured out the new candidates for improving inhibitor tolerance. RESULTS: After metabolomic analysis, proline and myo-inositol were identified as the potential metabolites responsible for strain tolerance to inhibitors. The deletion of genes involved in proline or myo-inositol synthesis weakened strain tolerance against FAP stress. On the contrary, the addition of proline or myo-inositol in medium exerted a protective effect on cell growth under FAP stress. Furthermore, the enhancement of proline or myo-inositol synthesis by overexpressing key gene PRO1 or INO1 conferred yeast strain significantly increased FAP tolerance. All the recombinant strains finished the fermentation within 60 h under FAP stress, while the control strain was still in the lag phase. Meanwhile, it was found that the intracellular level of reactive oxygen species (ROS) under FAP condition was decreased with the increase of proline content, suggesting the function of proline as a ROS scavenger to protect strains from inhibitor damage. CONCLUSION: Increasing proline and myo-inositol were uncovered as the new determinants for improving strain tolerance to FAP under the guidance of metabolomics. Meanwhile, this study displayed the powerful application of metabolomics to develop rational strategies to increase stress tolerance and provided valuable insights into the design of recombinant microbes for the complex traits.

Progressive regulation of sesquiterpene biosynthesis in Arabidopsis and Patchouli (Pogostemon cablin) by the miR156-targeted SPL transcription factors.

Plant metabolites vary at different stages of their life cycle. Although it is well documented that environmental factors stimulate biosynthesis of secondary metabolites, the regulation by endogenous developmental cues remains poorly understood. The microRNA156 (miR156)-targeted squamosa promoter binding protein-like (SPL) factors function as a major age cue in regulating developmental phase transition and flowering. We show here that the miR156-targeted SPL transcription factor plays an important role in the spatiotemporal regulation of sesquiterpene biosynthesis. In Arabidopsis thaliana, the miR156-SPL module regulates the formation of (E)-ß-caryophyllene in the flowering stage through modulating expression of the sesquiterpene synthase gene TPS21. We demonstrated that SPL9 directly binds to TPS21 promoter and activates its expression. In the perennial fragrant herb Pogostemon cablin, the accumulation of patchouli oil, largely composed of sesquiterpenes dominated by (-)-patchoulol, is also age-regulated, and the SPL promotes biosynthesis of sesquiterpenes in elder plants by upregulating patchoulol synthase (PatPTS) gene expression. As miR156-SPLs are highly conserved in plants, our finding not only uncovers a molecular link between developmental timing and sesquiterpene production but also suggests a new strategy to engineer plants for accelerated growth with enhanced production of terpenoids.

Metabolic engineering of the moss Physcomitrella patens to produce the sesquiterpenoids patchoulol and α/ß-santalene.

The moss Physcomitrella patens, has been genetically engineered to produce patchoulol and ß-santalene, two valuable sesquiterpenoid ingredients in the fragrance industry. The highest yield of patchoulol achieved was 1.34 mg/g dry weight. This was achieved by non-targeted transformation of the patchoulol synthase and either a yeast or P. patens HMGR gene under the control of a 35S promoter. Santalene synthase targeted to the plastids yielded 0.039 mg/g dry weight of α/ß santalene; cytosolic santalene synthase and 35S controlled HMGR afforded 0.022 mg/g dry weight. It has been observed that the final yield of the fragrance molecules is dependent on the expression of the synthase. This is the first report of heterologous production of sesquiterpenes in moss and it opens up a promising source for light-driven production of valuable fragrance ingredients.

Progressive Regulation of Sesquiterpene Biosynthesis in Arabidopsis and Patchouli (Pogostemon cablin) by the miR156-Targeted SPL Transcription Factors.

Plant metabolites vary at different stages of life cycle. Although it is well documented that environmental factors stimulate biosynthesis of secondary metabolites, the regulation by endogenous developmental cues remains poorly understood. The microRNA156 (miR156)-tageted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) factors function as a major age cue in regulating developmental phase transition and flowering. We show here that the miR156-targeted SPL transcription factor plays an important role in the spatiotemporal regulation of sesquiterpene biosynthesis. In Arabidopsis thaliana, the miR156-SPL module regulates the formation of (E)-ß-caryophyllene in flowering stage through modulating expression of the sesquiterpene synthase gene TPS21. We demonstrated that SPL9 directly binds to TPS21 promoter and activates its expression. In the perennial fragrant herb Pogostemon cablin, the accumulation of "patchouli oil", largely composed of sesquiterpenes dominated by (-)-patchoulol, is also age-regulated, and the SPL promotes biosynthesis of sesquiterpenes in elder plants by up-regulating patchoulol synthase (PatPTS) gene expression. As miR156-SPLs are highly conserved in plants, our finding not only uncovers a molecular link between developmental timing and sesquiterpene production, but also suggests a new strategy to engineer plant for accelerated growth with enhanced production of terpenoids.