Constitutive Expression in Komagataella phaffii of Mature Rhizopus oryzae Lipase Jointly with Its Truncated Prosequence Improves Production and the Biocatalyst Operational Stability

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).

Enabling growth-decoupled Komagataella phaffii recombinant protein production based on the methanol-free PDH promoter

“…Besides, having detached growth and production phases represents a better choice to produce toxic proteins that have detrimental effects on growth. This is the case of the widely studied Rizhopus oryzae lipase, which cannot be constitutively expressed but has been efficiently produced in methanol-induced expressions systems ( López-Fernández et al, 2021 ).…”

Synergic kinetic and physiological control to improve the efficiency of Komagataella phaffii recombinant protein production bioprocesses

“…Expression cassette gene dosage was determined using Droplet Digital PCR (ddPCR). Further details regarding the construction and copy number determination are described elsewhere (Garrigós‐Martínez et al., 2021 ; López‐Fernández et al., 2021 ; Nieto‐Taype et al., 2020 ).…”

Producing Natural Flavours from Isoamyl Alcohol and Fusel Oil by Using Immobilised Rhizopus oryzae Lipase

“…Rhizopus oryzae lipase with 28 C-terminal amino acids of its pro-sequence fused to the N-terminal of the mature sequence (proROL) was heterologously expressed in Komagataella phaffii (Pichia pastoris) under the constitutive promoter P GAP as described elsewhere [60]. Fed-batch runs were followed by removal of biomass and concentration of the enzyme, obtaining a biocatalyst with a specific activity of 70 AU mg −1 protein [57].…”

“…Purolite ® polymethacrylate matrix support D6308 with epoxide and octadecyl groups (EO) was pre-treated and used to immobilise proROL as described elsewhere [60,62]. The resulting biocatalyst (EO-proROL) was vacuum-filtered, dried on silica gel and stored at −20 • C until use.…”

“…Enzyme operational stability was assessed identically as previously in 15 mL tubes. The relative yields of consecutive esterification cycles, as calculated by taking the final yield of the first to be 100%, were used to fit the experimental results to the following two-component first-order exponential decay equation [60,64,65]:…”