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The high cost of exogenous glucoamylase and bacterial contamination risk are major bottlenecks in industrial starch-based ethanol fermentation. Multilocus genome engineering offers a solution but requires compact, efficient editing tools. Compared with SpCas9, the FIRE TnpB platform achieved locus-specific integration efficiencies of 13.5–84.4% across genomic loci in industrial Saccharomyces cerevisiae, comparable to SpCas9 (33.1–81.3%). Integrating 30 signal peptide fusions with FIRE TnpB identified α-factor as optimal for protein secretion. Among three fungal glucoamylases, Saccharomycopsis fibuligera-derived enzyme showed the highest fermentative activity. Co-integration of a phage endolysin conferred antibacterial activity against lactic acid bacteria. In 5-L corn mash fermentations at 30 °C, engineered strains produced 20.0–20.1% (v/v) ethanol without exogenous glucoamylase, and the dual-functional strain reduced the glucoamylase requirement from 5.5 to 1.375 U/mL (75% saving), with no significant difference between TnpB and SpCas9. Thus, compact TnpB matches SpCas9 editing efficiency in industrial yeast, providing a size-advantaged alternative for consolidated bioprocessing.
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Zhang et al. (2026) studied this question.
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