초록
<P><B>Significance</B></P><P>Recombinant proteins production by cell factories for medical and industrial use is one of the most prominent achievements in the modern biotech industry. It is constantly necessary to explore the underlying mechanisms of protein secretion to identify new targets for design and construction of improved cell factories. Here, we identified several gene targets, most of which are involved in the trafficking and secretory pathways, that could improve protein production by <I>Saccharomyces cerevisiae</I> to the gram per liter level. We also found that intracellular retention of recombinant proteins can be significantly reduced by engineering the endosome-to-Golgi trafficking. Genes and pathways identified here may provide general guidelines for engineering other cell factories for efficient protein secretion.</P><P>Baker’s yeast <I>Saccharomyces cerevisiae</I> is one of the most important and widely used cell factories for recombinant protein production. Many strategies have been applied to engineer this yeast for improving its protein production capacity, but productivity is still relatively low, and with increasing market demand, it is important to identify new gene targets, especially targets that have synergistic effects with previously identified targets. Despite improved protein production, previous studies rarely focused on processes associated with intracellular protein retention. Here we identified genetic modifications involved in the secretory and trafficking pathways, the histone deacetylase complex, and carbohydrate metabolic processes as targets for improving protein secretion in yeast. Especially modifications on the endosome-to-Golgi trafficking was found to effectively reduce protein retention besides increasing protein secretion. Through combinatorial genetic manipulations of several of the newly identified gene targets, we enhanced the protein production capacity of yeast by more than fivefold, and the best engineered strains could produce 2.5 g/L of a fungal α-amylase with less than 10% of the recombinant protein retained within the cells, using fed-batch cultivation.</P>