Systematic engineering of Saccharomyces cerevisiae for efficient synthesis of Lolium perenne antifreeze protein

The antifreeze protein from Lolium perenne (LpAFP) is an efficient inhibitor of ice growth, holding significant promise for the cryopreservation of biological specimens and food preservation. Nevertheless, its productivity remains low due to incompatibility between host cells and heterologous LpAFP expression. Herein, Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) was employed to construct LpAFP-high-yield Saccharomyces cerevisiae strain. Multi-omics analysis of the mutant strains revealed that the translation initiation factor gene TIF2 and the nucleotide pyrophosphatase/phosphodiesterase gene NPP2 boost LpAFP production by increasing translational activity and ATP levels, resulting in yield improvements of 110% and 75%, respectively. Furthermore, knockout of the pyruvate decarboxylase gene PDC1 increased LpAFP production by 79.8% compared to the parental strain. By harnessing these engineering targets, the high-titer strain zYFW035 was constructed. In fed-batch fermentation using a 5 L bioreactor, the yield of LpAFP was achieved to 1.9 g/L？, the highest titer reported to date. Surprisingly, the resulting LpAFP exhibits excellent antifreeze activity, highlighting strong potential for industrial-scale manufacturing.