Heterologous and efficient expression of a new alkaline pectin lyase in Pichia pastoris

Abstract

Abstract Pectin lyase (PMGL) is an industrially important enzyme with widespread applications in the food, paper, and textile industries, owing to its capacity for direct degradation of highly esterified pectin. In this study, PMGL-Ba derived from Bacillus underwent mining and heterologous expression in P. pastoris. Furthermore, diverse strategies, encompassing the optimization of expression cassette components, elevation of gene dosage, and co-expression of chaperone factors, were employed to augment PMGL-Ba production in P. pastoris. The signaling peptide OST1-pre-α-MF-pro and promoter AOX1 were finally selected as expression elements. By overexpressing the transcription factor Hac1p in conjunction with a two-copy PMGL-Ba setup, a strain yielding high PMGL-Ba production was achieved. In shake flask fermentation lasting 144 hours, the total protein concentration reached 1.81 g/L, and the enzyme activity reached 1821.36 U/mL. For further scale up production, high-density fermentation transpired in a 5 L fermenter for 72 h. Remarkably, the total protein concentration increased to 12.49 g/L, and the enzyme activity reached an impressive 12668.12 U/mL. The successful heterologous and efficient expression of PMGL-Ba not only furnishes a valuable biological enzyme for industrial applications but also contributes to cost reduction in the utilization of biological enzymes in industrial applications.