The reduced yields of acetaldehyde and fusel alcohols through fermentation by Saccharomyces cerevisiae is of significance for the improvement of the flavor and health of alcoholic beverages. In this study, the ADH2 (encode alcohol dehydrogenase) and THI3 (encode decarboxylase) genes of the industrial diploid strain S. cerevisiae XF1 were deleted. Results showed that single-gene-deletion mutants by separate gene deletion of ADH2 or THI3 led to a reduced production of the acetaldehyde or fusel alcohols, respectively. In the meantime, the double-gene-deletion mutant S. cerevisiae XF1-AT was constructed by deleting the ADH2 and THI3 simultaneously. An equivalent level of the ethanol production by the S. cerevisiae XF1-AT could be achieved but with the yields of acetaldehyde, isoamyl alcohol and iso-butanol reduced by 42.09%, 15.65% and 20.16%, respectively. In addition, there was no interaction between the ADH2 deletion and THI3 deletion in reducing the production of acetaldehyde and fusel alcohols. The engineered S. cerevisiae XF1-AT provided a new strategy to alcoholic beverages brewing industry for reducing the production of acetaldehyde as well as the fusel alcohols.
Camptotheca acuminata is the major plant for the production of camptothecin (CPT), an important anticancer drug used for the treatment of various cancers throughout the world. The low accumulation of CPT in plants limited its supply in the market and led to urgent need of promoting its accumulation by means of plant metabolic engineering, which relied on deep understanding of CPT biosynthesis pathway. However, missing of most of pathway genes restricted the attempts for regulating CPT biosynthesis. To unveil the CPT biosynthesis pathway, many efforts have been done for pathway gene identification and the application of large-scale RNA-sequencing technique accelerated screening of candidate genes which could be involved in CPT biosynthesis. To identify the function of these candidate genes in planta, it needs to develop an effective approach, such as virus-induced gene silencing (VIGS) in C. acuminata. In this work, a Tobacco Rattle Virus-based VIGS method was developed and the application of this method successfully silenced the expression of four known genes involved in the early steps of CPT biosynthesis and resulted in clear decrease of CPT and 10-hydroxycamptothecin (10-HCPT) accumulation. This VIGS approach could be further applied to functional identification of candidate genes to elucidate CPT biosynthesis in C. acuminata.
Excessive fusel alcohols in red wine will bring an uncomfortable bitterness and generate an intoxicating effect, which affects the quality and attractivity of the red wine. In order to achieve better regulation of fusel alcohols in red wine, strains with LEU1 and PDC5 deletions were constructed, and seven engineered yeast strains based on THI3 and BAT2 deletions were applied to red wine fermentation to dissect the effects of four critical genes on fusel alcohols during wine fermentation. The fermentation results of these recombinant strains showed that the deletion of THI3 increased the contents of n-propanol, isobutanol, and isoamyl alcohol by 48.46%, 42.01%, and 7.84%, respectively; the deletion of BAT2 decreased isoamyl alcohol and isobutanol by 32.81% and 44.91%; the deletion of PDC5 and LEU1 decreased isoamyl alcohol by 40.21% and 68.28%, while increased isobutanol by 24.31% and 142%, respectively; the deletion of THI3 exerted a negative influence on the reduction of isoamyl alcohol caused by BAT2 or PDC5 deletion; the deletion of THI3 and PDC5 had a synergistic effect on the increase of isobutanol, while BAT2 and PDC5 deletion presented no additive property to the decrease of isoamyl alcohol. Hence, it is concluded that either BAT2, PDC5, or LEU1 deletion can effectively decrease fusel alcohols, especially isoamyl alcohol, which provides an important reference for the control of fusel alcohols in red wine.
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