To construct small antibody-producing yeast cells, genes encoding target proteins were cloned into an expression vector containing a methanol-inducible promoter (AOX1 promoter), a zeocin- resistant gene, and a homologous sequence for single crossover recombination. The resulting vector plasmids were then linearized and electroporated into the host strain. The yielded transformants were grown under different zeocin concentrations. Because zeocin resistance of the transformants increased with an increase in the copy number of the integrated plasmid, transformants with different plasmid copy numbers were easily obtained by varying the zeocin concentration. Finally, the transformants were screened through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for target protein secretion, and the gene copy number was determined through quantitative PCR (qPCR).
To demonstrate this workflow, we parallelly evaluated the production of 14 recombinant small antibodies, including 3 nanobodies (ALX-0061 (vobarilizumab), ALX-0081 (caplacizumab), and ALX-0171 (gontivimab)), 3 camelid single-domain antibodies against the epidermal growth factor receptor (i.e., VHH; 7D12, 9G8, and EgA1)6, 6 antibody-like scaffold proteins (i.e., DARPin, adnectin, anticalin, fynomer, affibody, and KALBITOR), and 2 bispecific and bivalent antibodies (i.e., BiBian; G1 and G3)7 (Fig. 1c–1f and Supplementary Figs. 1–3). Figure 1 presents the estimated gene copy numbers and secretion levels of the target proteins. In most cases, protein secretion levels increased with an increase in the gene copy number, except for BiBian. Interestingly, the same trends were observed when the multivalent antibodies (nanobodies and BiBian) were segregated into their constituents (Fig. 2). According to our results, the gene copy number plays a vital role in the high-level secretory production of the target proteins in yeast.
To measure the scalability of the aforementioned evaluation, we conducted 5-L fed-batch fermentation of three strains producing ALX-0081, DARPin, and adnectin, respectively (Figs. 3–5). A proportional-integral-differential (PID) controller was incorporated into the jar fermenter to mitigate the large fluctuation of DO resulting from intense oxygen consumption, allowing settled DO control (Fig. 1b). pH and temperature were maintained at constant levels throughout fermentation8, 9. Glycerol and methanol concentrations in the growth and induction phases were monitored using an online biosensor. Because excess methanol is toxic to yeast, its concentration during the induction phase was gradually increased so as to allow the cells to adapt to the induction medium. This increase in methanol concentration was achieved through a stepwise increase in the flow rate of methanol feeding or through the feedback control of the methanol concentration by using the biosensor and PID controller. After the strains were grown for 40 h with glycerol, the biomass (OD600) reached approximately 350. At this point, methanol was added to induce protein production. The titers of ALX-0081, DARPin, and adnectin were 7 ± 3 g/L (N = 3), 5 ± 2 g/L (N = 2), and 3 ± 1 g/L (N = 3), respectively (Fig. 3–5, and Supplementary Figs. 4 and 5).