Engineering the protein secretory pathway of Saccharomyces cerevisiae enables improved protein production

Abstract

Baker’s yeast Saccharomyces cerevisiae 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.

Multiple strategies to improve the yield of chitinase a from Bacillus licheniformis in Pichia pastoris to obtain plant growth enhancer and GlcNAc

“…Molecular chaperones were reported to be an effective method to improve the expression of heterologous protein since they can facilitate unfolded or partially folded protein to be correct folding, finally improve the It is likely that the improved activity of amylase in the ERV29 overexpressed strain was related to enhancement of the ERV29p-dependent transport. When the COG5 were overexpressed, the retrograde vesicles trafficking strengthened, which was beneficial for amylase production [31]. However, the co-expression of SEC16 and COG5 promotes the secretion of heterologous proteins for the first two days in this study, then has little effect afterwards.…”

“…It is likely that the improved activity of amylase in the ERV29 overexpressed strain was related to enhancement of the ERV29p-dependent transport. When the COG5 were overexpressed, the retrograde vesicles trafficking strengthened, which was beneficial for amylase production [ 31 ]. However, the co-expression of SEC16 and COG5 promotes the secretion of heterologous proteins for the first two days in this study, then has little effect afterwards.…”

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

“…Several gene targets, most of which are involved in the trafficking and secretory pathways, that could enhance protein production by S. cerevisiae to the gram per liter level have been identified. It was also found that intracellular retention of recombinant proteins can be considerably reduced by engineering the endosome-to-Golgi trafficking (Huang et al, 2018). The development of a synthetic biology toolkit and how those tools have been applied in the areas of drug production and screening were described in detail by Chen et al (2018).…”

RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production in Saccharomyces cerevisiae

“…With the 11C-GK3 strain integrated with ∼14 copies of the amylase gene (as estimated by qPCR, Fig. 2A), we examined the feasibility of perturbing protein producing capability via RNAi, by manipulating expression of several genes, which were reported previously to improve protein production, in particular by overexpression [protein disulfide isomerase PDI1 (17), basic leucine zipper transcription factor HAC1 (18)] or deletion [histone deacetylase HDA2 (19) and a putative oxidoreductase involved in protein transport TDA3 (20)]. Knockdowns of PDI1 and TDA3, via the complete reverse coding sequence-assisted pattern (21), significantly decreased (55.7%) and intensified (90.7%) amylase production, respectively, while down-regulations of HAC1 or HDA2 had no effect (Fig.…”