Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 104, Issue 10, Pages 4435-4444Publisher
SPRINGER
DOI: 10.1007/s00253-020-10549-4
Keywords
Chinese rice wine; Ethyl carbamate; Saccharomyces cerevisiae; DUR3; CRISPR; Cas9
Categories
Funding
- National Natural Science Foundation of China [31701588, 31701730]
- Natural Science Foundation of Jiangsu Province, China [BK20170178]
- Fundamental Research Funds for the Central Universities [JUSRP11965]
- Program of Introducing Talents of Discipline to Universities (111 Project) [111-2-06]
- Priority Academic Program Development of Jiangsu Higher Education Institutions
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Ethyl carbamate (EC) is a potential carcinogen to humans that is mainly produced through the spontaneous reaction between urea and ethanol during Chinese rice wine brewing. We metabolically engineered a strain by over-expressing the DUR3 gene in a previously modified strain using an improved CRISPR/Cas9 system to further decrease the EC level. Homologous recombination of the DUR3 over-expression cassette was performed at the HO locus by individual transformation of the constructed plasmid CRISPR-DUR3-gBlock-HO, generating the engineered strain N85(DUR1,2/DUR3)-c. Consequently, the DUR3 expression level was significantly enhanced in the modified strain, resulting in increased utilization of urea. The brewing test showed that N85(DUR1,2/DUR3)-c reduced urea and EC concentrations by 92.0% and 58.5%, respectively, compared with those of the original N85 strain. Moreover, the engineered strain showed good genetic stability in reducing urea content during the repeated brewing experiments. Importantly, the genetic manipulation had a negligible effect on the growth and fermentation characteristics of the yeast strain. Therefore, the constructed strain is potentially suitable for application to reduce urea and EC contents during production of Chinese rice wine.
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