Transcriptomics-based identification of novel factors enhancing heterologous protein secretion in yeasts

Abstract

Efficient production of heterologous proteins with yeasts and other eukaryotic hosts is often hampered by inefficient secretion of the product. Limitation of protein secretion has been attributed to a low folding rate, and a rational solution is the overexpression of proteins supporting folding, like protein disulfide isomerase (Pdi), or the unfolded protein response transcription factor Hac1. Assuming that other protein factors which are not directly involved in protein folding may also support secretion of heterologous proteins, we set out to analyze the differential transcriptome of a Pichia pastoris strain overexpressing human trypsinogen versus that of a nonexpressing strain. Five hundred twenty-four genes were identified to be significantly regulated. Excluding those genes with totally divergent functions (like, e.g., core metabolism), we reduced this number to 13 genes which were upregulated in the expression strain having potential function in the secretion machinery and in stress regulation. The respective Saccharomyces cerevisiae homologs of these genes, including the previously characterized secretion helpers PDI1, ERO1, SSO2, KAR2/BiP, and HAC1 as positive controls, were cloned and overexpressed in a P. pastoris strain expressing a human antibody Fab fragment. All genes except one showed a positive effect on Fab fragment secretion, as did the controls. Six out of these novel secretion helper factors, more precisely Bfr2 and Bmh2 (involved in protein transport), the chaperones Ssa4 and Sse1, the vacuolar ATPase subunit Cup5, and Kin2 (a protein kinase connected to exocytosis), proved their benefits for practical application in laboratory-scale production processes by increasing both specific production rates and the volumetric productivity of an antibody fragment up to 2.5-fold in fed-batch fermentations of P. pastoris.

FIG. 1.

Kinetics of growth-related parameters of the secretion-engineered P. pastoris and the control strain in fed-batch cultures. Development of biomass concentration (g liter⁻¹ yeast dry mass [YDM]) (A) and specific growth rates (μ) (B) are shown for the P. pastoris expression strains (SMD1168 secreting 2F5 Fab under the control of the GAP promoter) coexpressing the novel secretion helper factors and the control strain expressing only the antibody fragment.

FIG. 2.

Increased production of secreted antibody fragments in P. pastoris coexpressing novel secretion helper factors in fed-batch cultures. The development of product titer (rectangles with solid black lines) and Q_P (triangles with trend lines) are shown for each of the secretion-engineered strains and their parental strain (control) individually.

FIG. 3.

Improvements in antibody fragment secretion yields by overexpression of novel helper factors in yeast. The increases (n-fold) of q_P (striped bars), maximum Q_P (Q_Pmax) (gray bars), the product yield coefficient Y_P/X (dotted bars), and the final antibody fragment titer (black bars) during fed-batch cultivations are displayed for the secretion-optimized P. pastoris strains and their parental strain.