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  Promoter choice is an important step in recombinant protein production, which directly determines the expression manner as constitutive or inducible and the expression level of the recombinant protein. This study aims to investigate the applicability of heterologous yeast promoters ( Kluyveromyces marxianus TPI , Hansenula polymorpha PMA , Candida tropicalis ICL , and Saccharomyces cerevisiae CUP ) in Pichia pastoris . The regulation mode of the CtICL and ScCUP promoters in P. pastoris was found to be inducible and that of the KmTPI and HpPMA was constitutive. The carbon sources in which the promoters exhibited the highest activity were determined as glycerol for PMA and TPI , glucose for CUP , and ethanol for ICL . The DNA region showing the highest activity was determined as 1000 bp for all promoters by promoter deletion analysis. Results from the study demonstrate the potential of inducible and constitutive heterologous promoters allowing expression under different conditions in the P. pastoris expression system and offers alternatives to frequently used promoters. Key points • Heterologous promoters exhibited similar expression pattern in P. pastoris with its native host . • HpPMA has the highest promoter activity among the heterologous promoters tested . • Reporter gene expression with ScCUP is responsive to elevating Cu 2+ in P. pastoris .

Pichia pastoris is a successful expression system that is frequently preferred in the secretion of proteins for both basic research and industrial purposes. In this study, recombinant Rhizomucor miehei (RmASNase) L-asparaginase was produced in Pichia pastoris. The impact of gene copy number on increasing protein production was examined with six clones harboring various gene copy numbers (1–5 and 5 +). The results demonstrated that the clone with three copies of the expression cassette integrated had the highest production level. Also, biochemical characterization of the enzyme was performed. It was determined that the optimum pH and temperature values of the purified enzyme were pH 7.0 and 50 °C, respectively. Stability analyses of the enzyme showed that it maintains its activity of 80% in the pH range of 5–9 and 67% in the temperature range of 20–50 °C. Ca+2 and Mn+2 ions increased the enzyme activity to 121% and 138%, respectively. In future studies, it is also possible to improve the activity and stability values of the enzyme with advanced molecular techniques and to increase production efficiency by producing at fermenter scale and under optimum conditions.

OBJECTIVES: To characterize the genes responsible for ethanol utilization in Pichia pastoris. RESULTS: ADH3 (XM_002491337) and ADH (FN392323) genes were disrupted in P. pastoris. The ADH3 mutant strain, MK115 (Δadh3), lost its ability to grow on minimal ethanol media but produced ethanol in minimal glucose medium. ADH3p was responsible for 92 % of total Adh enzyme activity in glucose media. The double knockout strain MK117 (Δadh3Δadh) also produced ethanol. The Adh activities of X33 and MK116 (Δadh) strains were not different. Thus, the ADH gene does not play a role in ethanol metabolism. CONCLUSION: The PpADH3 is the only gene responsible for consumption of ethanol in P. pastoris.