Deletion of NTH1 and HSP12 increases the freeze–thaw resistance of baker’s yeast in bread dough

Abstract

Background The intracellular molecule trehalose in Saccharomyces cerevisiae may have a major protective function under extreme environmental conditions. NTH1 is one gene which expresses trehalase to degrade trehalose. Small heat shock protein 12 (HSP12 expressed) plays a role in protecting membranes and enhancing freezing stress tolerance. Results An optimized S. cerevisiae CRISPR-Cpf1 genome-editing system was constructed. Multiplex genome editing using a single crRNA array was shown to be functional. NTH1 or/and HSP12 knockout in S. cerevisiae enhanced the freezing stress tolerance and improved the leavening ability after freezing and thawing. Conclusions Deleting NTH1 in the combination with deleting HSP12 would strengthen the freezing tolerance and protect the cell viability from high rates of death in longer-term freezing. It provides valuable insights for breeding novel S. cerevisiae strains for the baking industry through a more precise, speedy, and economic genome-editing system.

An Introduced RNA-Only Approach for Plasmid Curing via the CRISPR-Cpf1 System in Saccharomyces cerevisiae

“…In a previous study, we observed longer recovery times for colony development after electroporation and higher genome editing efficiency in the CRISPR-Cpf1 system in S. cerevisiae. The efficiency of genome editing is highest at 48 h for cell recovery [27]. However, the curing efficiency did not consistently increase with cell recovery time in this study.…”

“…The S. cerevisiae wild-type strain BCRC 21447 used in this study was obtained from the Bioresource Collection and Research Center. The ∆ade2, ∆nth1, ∆hsp12, and ∆nth1/∆hsp12 strains were constructed from the previous study [26,27]. S. cerevisiae was grown at 30 • C in a YPD medium (1% yeast extract, 2% peptone, and 2% glucose)(BD, Sparks, MD, USA), and 200 mg/L G418 (Sigma-Aldrich, St. Louis, MO, USA) was added for the selection.…”

“…In the previous study, we generated the freeze-thaw resistant S. cerevisiae strains with NTH1 deleted, HSP12 deleted, and NTH1/HSP12 double deleted using the CRISPR-Cpf1 system [27]. However, the edited strains contain CRISPR plasmids, and strains containing foreign genes are difficult to apply in the food industry.…”

“…Furthermore, we also constructed the edited strains with a double-gene deletion, including the genes encoding the trehalose degradation enzyme (Nth1) and the membrane chaperone (Hsp12) by the CRISPR-Cpf1 system in S. cerevisiae. Deletion of NTH1 and HSP12 increases the freeze-thaw resistance of baker's yeast in bread dough [27]. However, the edited strains containing the CRISPR plasmids are difficult to apply in the food industry.…”

Diverse signatures of convergent evolution in cacti-associated yeasts

“…When cells return to a more favourable condition, the accumulated trehalose is hydrolysed into glucose by the trehalase Nth1. Inactivation of NTH1 by mutations, and therefore subsequent impairment of trehalose hydrolysis, can be one of the outcomes of the heat stress response in experimentally evolved strains of S. cerevisiae under high temperature stress 69,70 , suggesting that deficient trehalose hydrolysis can be beneficial under long-term thermal stress conditions. However, NTH1 is generally present in cactophilic genomes, suggesting that absence of NTH1 does not explain impairment in trehalose assimilation in these species.…”