Abstract
Rhizopus oryzae lipase (ROL) containing 28 C-terminal amino acids of the prosequence fused to the N-terminal mature sequence in ROL (proROL) was successfully expressed in the methylotrophic yeast Komagataella phaffii (Pichia pastoris) under the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP). Although the sequence encoding the mature lipase (rROL) was also transformed, no clones were obtained after three transformation cycles, which highlights the importance of the truncated prosequence to obtain viable transformed clones. Batch cultures of the K. phaffii strain constitutively expressing proROL scarcely influenced growth rate and exhibited a final activity and volumetric productivity more than six times higher than those obtained with proROL from K. phaffii under the methanol-inducible alcohol oxidase 1 promoter (PAOX1). The previous differences were less marked in fed-batch cultures. N-terminal analysis confirmed the presence of the 28 amino acids in proROL. In addition, immobilized proROL exhibited increased tolerance of organic solvents and an operational stability 0.25 and 3 times higher than that of immobilized rROL in biodiesel and ethyl butyrate production, respectively. Therefore, the truncated prosequence enables constitutive proROL production, boosts bioprocess performance and provides a more stable biocatalyst in two reactions in which lipases are mostly used at industrial level, esterification (ethyl butyrate) and transesterification (biodiesel).
Highlights
Biocatalysis has for some time been extensively used to obtain a variety of products including polymers, fine and bulk chemicals, flavors and pharmaceutical intermediates [1,2]
The fact that some of the positive traits of the whole prosequence have been identified with truncated sequences [21,35], led us to transform K. phaffii with proROL phosphate dehydrogenase promoter (PGAP)-plasmid, rROL PGAP-plasmid and the empty plasmid—blank—to investigate the role of the C-terminal 28 amino acids of the prosequence
The Rhizopus oryzae lipase (ROL) mature sequence must somehow hinder constitutive expression in K. phaffii. These results are consistent with those of previous work where production of the ROL mature sequence caused cell lysis in E. coli owing to its phospholipase activity but no lysis when 28 C-terminal amino acids in the prosequence were expressed together with the mature sequence, noxious effects were still observed as cell growth was inhibited [10]
Summary
Biocatalysis has for some time been extensively used to obtain a variety of products including polymers, fine and bulk chemicals, flavors and pharmaceutical intermediates [1,2]. ROL forms from a presequence of 26 amino acids that is followed by a prosequence of 97 and a mature sequence of 269 [6]. Obtaining the mature form of the lipase (rROL) entails removing the presequence and prosequence [10,11]. The native microorganism secretes a protein including the mature sequence and, attached to its N-terminal, the 28 C-terminal amino acids of the prosequence (proROL), which are subsequently proteolyzed by extracellular proteases. The 28 amino acids by themselves seemingly suffice for the previous positive traits of the whole prosequence to occur and are important with a view to developing a potentially improved ROL-based biocatalyst [7]
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