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Ehrlich and demethiolation pathways as two competing branches converted amino acid into alcohols. Controlling both pathways offers considerable potential for industrial applications including alcohols overproduction, flavor-quality control and developing new flavors. While how to regulate ehrlich and demethiolation pathways is still not applicable. Taking the conversion of methionine into methionol and methanethiol for example, we constructed two suppression subtractive cDNA libraries of Clonostachys rosea by using suppression subtractive hybridization (SSH) technology for screening regulators controlling the conversion. E3 ubiquitin-protein ligase gene HUWE1 screened from forward SSH library was validated to be related with the biosynthesis of end products. Overexpressing HUWE1 in C. rosea and S. cerevisiae significantly increased the biosynthesis of methanethiol and its derivatives in demethiolation pathway, while suppressed the biosynthesis of methional and methionol in ehrlich pathway. These results attained the directional regulation of both pathways by overexpressing HUWE1. Thus, HUWE1 has potential to be a key target for controlling and enhancing alcohols production by metabolic engineering.

Aroma results from the interplay of volatile organic compounds (VOCs) and the attributes of microbial-producing aromas are significantly affected by fermentation conditions. Among the VOCs, only a few of them contribute to aroma. Thus, screening and identification of the key VOCs is critical for microbial-producing aroma. The traditional method is based on gas chromatography-olfactometry (GC-O), which is time-consuming and laborious. Considering the Tuber melanosporum fermentation system as an example, a new method to screen and identify the key VOCs by combining the aroma evaluation method with principle component analysis (PCA) was developed in this work. First, an aroma sensory evaluation method was developed to screen 34 potential favorite aroma samples from 504 fermentation samples. Second, PCA was employed to screen nine common key VOCs from these 34 samples. Third, seven key VOCs were identified by the traditional method. Finally, all of the seven key VOCs identified by the traditional method were also identified, along with four others, by the new strategy. These results indicate the reliability of the new method and demonstrate it to be a viable alternative to the traditional method.

The aroma attributes of sulfurous, mushroom and earthy are the most important characteristics of the aroma of Tuber melanosporum. However, these three aroma attributes are absent in the T. melanosporum fermentation system. To improve the quality of the aroma, repeated freeze-thaw treatment (RFTT) was adopted to affect the interplay of volatile organic compounds (VOCs). Using RFTT, not only was the score on the hedonic scale of the aroma increased from the “liked slightly” to the “liked moderately” grade, but the aroma attributes of sulfurous, mushroom and earthy could also be smelled in the T. melanosporum fermentation system for the first time. A total of 29 VOCs were identified and 9 compounds were identified as the key discriminative volatiles affected by RFTT. Amino acid analysis revealed that methionine, valine, serine, phenylalanine, isoleucine and threonine were the key substrates associated with the biosynthesis of the 9 key discriminative VOCs. This study noted that amino acid metabolism played an important role in the regulation of the aroma of the T. melanosporum fermentation system.