Thermostability improvement of Aspergillus awamori glucoamylase via directed evolution of its gene located on episomal expression vector in Pichia pastoris cells

Abstract

Novel thermostable variants of glucoamylase (GA) from filamentous fungus Aspergillus awamori X100 were constructed using the directed evolution approach based on random mutagenesis by error-prone PCR of the catalytic domain region of glucoamylase gene located on a new episomal expression vector pPEHα in Pichia pastoris cells. Out of 3000 yeast transformants screened, six new thermostable GA variants with amino acid substitutions Val301Asp, Thr390Ala, Thr390Ala/Ser436Pro, Leu7Met/His391Tyr, Asn9His/Ile82Phe and Ser8Arg/Gln338Leu were identified and studied. To estimate the effect of each substitution in the double mutants, we have constructed the relevant single mutants of GA by site-directed mutagenesis and analyzed their thermal properties. Results of the analysis showed that only Ile82Phe and Ser8Arg substitutions by themselves increased enzyme thermostability. While the substitutions Leu7Met, Asn9His and Gln338Leu decreased the thermal stability of GA, the synergistic effect of double mutant variants Leu7Met/His391Tyr, Asn9His/Ile82Phe and Ser8Arg/Gln338Leu resulted in significant thermostability improvement as compared to the wild type GA. Thr390Ala and Thr390Ala/Ser436Pro mutant variants revealed the highest thermostability with free activation energy changes ΔΔG of 2.99 and 3.1 kJ/mol at 80°C, respectively.

Improving the Heterologous Production of Fungal Peroxygenases through an Episomal Pichia pastoris Promoter and Signal Peptide Shuffling System

“…Despite being a heterologous host of outstanding interest, the use of P. pastoris for high throughput enzyme engineering endeavors remains rather limited to few examples. − This is likely due to a limited number of suitable autonomously replicating sequences (ARS) for P. pastoris , which confer episomal stability. For the model yeast organism S. cerevisiae , a multitude of directed evolution examples exist utilizing well-known episomal systems. ,, …”

Development of synthetic biology tools to engineer Pichia pastoris as a chassis for the production of natural products

“… Zeocin r Episomal [ 46 , 47 ] pPEHα a PARS1 N.A. HIS4 Episomal [ 48 ] Note. a These plasmids contain a signal peptide for protein secretion.…”

Improving Thermostability of Chimeric Enzymes Generated by Domain Shuffling Between Two Different Original Glucoamylases

“…Because the saccharification processes are usually followed by a liquefaction process of starch and are performed at 60°C for 48–72 h, the glucoamylases required in starch industrials have to possess good thermostability and catalytic activities ( Lim and Oslan, 2021 ; Tong et al, 2021 ). So, searching for a new source of glucoamylase with potentially applicable properties encompassing elevated temperature, extreme pH, high salinity, organic solvents, surfactants, and specificities (substrate and product) is still of considerable importance ( Schmidt et al, 2019 ). Although some novel glucoamylases have been found and characterized for industrial applications ( Guo et al, 2019 ; Karim et al, 2019 ; Lincoln et al, 2019 ; Zhang et al, 2019 ; Wang et al, 2020 ; Lago et al, 2021 ; Wayllace et al, 2021 ), they did not achieve industrially desirable traits.…”

“…In addition to exploring novel enzymes with desirable properties in nature, attempts are being made to improve the properties of the existing enzymes by protein engineering techniques to make them suitable for industrial applications ( Parashar and Satyanarayana, 2016 ; Sharma et al, 2019 ). These techniques mainly include rational design, semi-rational design, directed evolution (error prone PCR and DNA shuffling), and fusion ( Schmidt et al, 2019 ; Sharma et al, 2019 ; Lim and Oslan, 2021 ; Tong et al, 2021 ). However, the design of chimeric enzymes by fusing different domains from native enzymes is considered to be a straightforward method for generating a novel enzyme with improved catalytic properties ( Parashar and Satyanarayana, 2016 ; Ali et al, 2020 ).…”