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Abstract :
[en] Komagataella phaffii, a widely recognized yeast strain utilized to produce recombinant proteins (rProt) is influenced by metabolic/operational factors. Previous reports have described the self-induced FdhKO strain, it assimilates sorbitol with high rProt-specific activity upon pAOX1 promoter induction by formate accumulation. However, its growth rate (μ) is low. SOR1, encoding sorbitol dehydrogenase (Sd), was overexpressed (SdOE) to increase the carbon source uptake. Although the μ of strain (FdhKO-SdOE) is higher, its rProt-specific activity decreases compared to the parental strain.
Given this, flask cultures with different volumes are proposed to control μ of these strains using oxy-gen availability (OA) with (coefficient oxygen transfer) KLa values: 10h-1(low), 50h-1(medium), and 100h-1(high). As expected, reducing the OA led to a decrease in the μ for both strains. The FdhKO-SdOE-eGfp strain exhibited μ of 0.10h⁻¹, 0.09h⁻¹, and 0.06h⁻¹, while the FdhKO-eGfp strain showed μ of 0.05h⁻¹, 0.04h⁻¹, and 0.03h⁻¹ for high, medium and low OA, respectively. The differences observed for μ in these strains are due to SdOE and the ability to metabolize sorbitol.
Unlike the μ, both strains' fluorescence was inversely proportional to OA. The FdhKO-eGfp strain exhibited a 12-fold lower signal at high OA than at low OA (i.e., 105267 and 8939 TFU, respectively). In the FdhKO-SdOE-eGfp strain, with a 5-fold decrease (i.e., 12184 and 2709 TFU, respectively) cor-relating with the formate accumulation, which decreased as the OA increased. 107,3-fold at low OA for FdhKO-eGfp strain (i.e., 26,5±4,8 M/gDCW) compared with the lowest value for FdhKO-SdOE-eGfp strain at high OA (i.e., 0,25±0,15 M/gDCW).
In line with these findings, rProt production is prioritized at lower μ. Thus, the SdOE increased sorbitol uptake and promoted cell growth, decreasing the rProt-specific activity. The elevated μ in the FdhKO-SdOE-eGfp strain resulted in reduced formate accumulation. Therefore, the FdhKO strain presents better specific values under evaluated conditions.
