Truncated Prosequence of Rhizopus oryzae Lipase: Key Factor for Production Improvement and Biocatalyst Stability

Abstract

Recombinant Rhizopus oryzae lipase (mature sequence, rROL) was modified by adding to its N-terminal 28 additional amino acids from the C-terminal of the prosequence (proROL) to obtain a biocatalyst more suitable for the biodiesel industry. Both enzymes were expressed in Pichia pastoris and compared in terms of production bioprocess parameters, biochemical properties, and stability. Growth kinetics, production, and yields were better for proROL harboring strain than rROL one in batch cultures. When different fed-batch strategies were applied, lipase production and volumetric productivity of proROL-strain were always higher (5.4 and 4.4-fold, respectively) in the best case. rROL and proROL enzymatic activity was dependent on ionic strength and peaked in 200 mM Tris-HCl buffer. The optimum temperature and pH for rROL were influenced by ionic strength, but those for proROL were not. The presence of these amino acids altered lipase substrate specificity and increased proROL stability when different temperature, pH, and methanol/ethanol concentrations were employed. The 28 amino acids were found to be preferably removed by proteases, leading to the transformation of proROL into rROL. Nevertheless, the truncated prosequence enhanced Rhizopus oryzae lipase heterologous production and stability, making it more appropriate as industrial biocatalyst.

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

“…Key production and growth parameters were compared with reported values for proROL PAOX1-strain and rROL PAOX1-strain grown in methanol [7] (Table 1). The greatest differences between proROL PAOX1-strain and proROL PGAP-strain were those in final activity and volumetric productivity, which were 6.5 times higher in the latter.…”

“…Thus, ROL forms from a presequence of 26 amino acids that is followed by a prosequence of 97 and a mature sequence of 269 [6]. Whereas the presequence is known to act as a signal peptide, the specific functions of the prosequence are incompletely known but thought to include increasing protein stability, reducing stress during recombinant protein expression, and minimizing unfolded protein response (UPR) in rROL production-which has an adverse impact on microbial growth [7][8][9]. Obtaining the mature form of the lipase (rROL) entails removing the presequence and prosequence [10,11].…”

“…However, 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 (pro-ROL), 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 thus important with a view to developing a potentially improved ROLbased biocatalyst [7].…”

“…Although the expression of ROL has been already reported under this constitutive promoter, w-proROL-gene, which encodes both the prosequence and the mature sequence, is needed to alleviate the adverse effects of producing the mature sequence of ROL under PGAP [32], as this lipase form is harmful to the host cell [10]. In fact, rROL-gene expression has only been accomplished under the inducible PAOX1 [7,33]-inducible promoters are less troublesome than constitutive promoters in this respect [34].…”

A Valuable Product of Microbial Cell Factories: Microbial Lipase

“…The 1, 3-specific lipases catalyze the release of fatty acids from the 1 and 3 carbon sites of the of triglyceride substrate; they are not capable of performing hydrolysis at the middle, or second ester bond. The 1, 3-specific lipases promote hydrolysis of triglycerides into monoglycerides at a more rapid rate than performed by non-specific lipases ( Kavadia et al, 2018 ; López-Fernández et al, 2019 ; Gao et al, 2020 ). By contrast, fatty-acid-specific lipases demonstrate substrate selectivity; these enzymes target long-chain fatty acids with cis -double bonds between C-9 and C-10 ( Kapoor and Gupta, 2012 ).…”