CRISPR-Cas9 based Metabolic Engineering of Industrial Saccharomyces cerevisiae for Acrylamide Reduction

Abstract

Acrylamide, known as a toxic substance and potential carcinogen, is present in large amounts in foods including potato chips and breads. Acrylamide is produced from asparagine in raw food materials through the Maillard reaction during the cooking process. Therefore, reduction of asparagine is the key of the acrylamide reduction. The objective of this thesis is to reduce acrylamide in foods by degrading asparagine by metabolically engineered S. cerevisiae. A recently developed CRISPR-Cas9 system was employed to engineer industrial S. cerevisiae since the CRISPR-Cas9 genome editing technology would ensure safety for food applications. First, to isolate industrial S. cerevisiae to be used for acrylamide reduction, 7 stains of S. cerevisiae were isolated from nuruk which is a Korean traditional microbial resource. Among 7 strains of S. cerevisiae, S. cerevisiae N1 was selected as the host strain for further studies because S. cerevisiae N1 showed the best glucose fermentation ability. Also S. cerevisiae N1 was expected to be used efficiently under acidic pH conditions or osmotic stress conditions as compared with the other isolated S. cerevisiae strains. Next, metabolic engineering was performed for reducing acrylamide using the CRISPR-Cas9 gene editing technology. To prove the rationality of metabolic engineering strategies for acrylamide reduction, a laboratory strain S. cerevisiae D452-2 was engineered prior to industrial S. cerevisiae N1. S.cerevisiae D452-2 was engineered by overexpression of the general amino acid permease (GAP1), overexpression of asparaginases (ASP1, ASP3), regulation of nitrogen catabolite repression (NCR). Engineered strains were grown in flasks containing asparagine rich YPD media to test the ability of asparagine degradation. For the laboratory S. cerevisiae D_PASP3 strain overexpressing ASP3 (cell-wall asparaginase) through a promoter replacement, the asparagine consumption rate was enhanced by 9.9 times relative to the D452-2 WT strain. Therefore, the ASP3 overexpression strategy was applied to the industrial N1 strain. Since APS3 did not exist in N1, an ASP3 expression cassette was constructed and inserted into either the URE2 site or the GZF3 site of the N1 chromosome to construct N1_ΔURE2::ASP3 and N1_ΔGZF3::ASP3. To determine the asparagine consumption ability of the two strains, engineered strains were grown in flasks containing asparagine rich YPD media. For the S. cerevisiae N1_ΔURE2::ASP3, the asparagine consumption rate increased by 20.6 times, compared with the N1 WT strain. For the S. cerevisiae N1_ΔGZF3::ASP3, the asparagine consumption rate increased by 11.4 times, compared with the N1 WT strain. When they were applied to food doughs, asparagine was consumed fast to result in 95% less acrylamide than the control strain N1 WT. In this study, metabolically engineered industrial S. cerevisiae strains were constructed to reduce acrylamide in foods by degrading asparagine. The engineered strains reduced acrylamide in foods by 95%, and it is believe that these engineered strains could be used effectively for acrylamide reduction in food industry.