Heterologous Expression and Rational Design of l-asparaginase from Rhizomucor miehei to Improve Thermostability

Abstract

l-asparaginase (EC 3.5.1.1) hydrolyzes l-asparagine to produce l-aspartate and ammonia and is widely found in microorganisms, plants, and some rodent sera. l-asparaginase used for industrial production should have good thermostability. We heterologously expressed l-asparaginase from Rhizomucor miehei, selected nine loci for site-directed mutagenesis by rational design, and obtained two mutants with significantly improved thermostability. The optimal temperature of mutants S302I and S302M was 50 °C. After incubating the mutant and wild-type enzymes at 45 °C for 35 h, the residual activity of the wild-type enzyme (WT) was only about 10%. In contrast, the residual activity of S302I and S302M was more than 50%. After combination mutagenesis, Bacillus subtilis 168-pMA5-A344E/S302I was constructed using the food-safe host strain B. subtilis 168. Additionally, a 5′ untranslated region (UTR) modification strategy was adopted to enhance the expression level of R. miehei-derived l-asparaginase in B. subtilis. In a 5-L fermenter scale-up experiment, the enzyme activity of recombinant B. subtilis 168-pMA5-UTR-A344E/S302I reached 521.9 U·mL−1 by fed-batch fermentation.

Enhanced Thermostability and Molecular Insights for l-Asparaginase from Bacillus licheniformis via Structure- and Computation-Based Rational Design

“…Currently, there are two major approaches to obtain l -ASNases with high thermostability through screening new l -ASNase genes from extreme environments and reconstructing mesophilic l -ASNase by protein engineering. , Although several thermophilic l -ASNases have been extracted from hot-springs and hydrothermal vents (such as the source of Thermococcus and Pyrococcus sp), the enzymatic activities in low temperatures and substrate specificities of these l -ASNases cannot meet the demand for industrial applications and their screening processes are time-consuming and of higher cost . Instead, these limitations can be overcome by protein engineering (chemical modification, rational design, directed evolution, and semirational design).…”

“…l -Asparaginase ( l -ASNase, amidohydrolase enzyme, EC3.5.1.1) can catalyze the production of non-essential amino acid l -asparagine (L-ASN) to l -aspartic acid (L-ASP) and ammonia. , This enzyme displays great potential for a wide range of applications in the food and pharmaceutical industries, such as reducing the formation of carcinogenic acrylamide in fried foods and treating acute lymphoblastic leukemia (ALL) . Due to the short half-life, commercial l -ASNase coming from Escherichia coli (EcA) and Erwinia chrysanthemi (ErA) have a high-dose-dependent disadvantage for ALL treatment, causing severe toxic effects on patients . In addition, the low thermal stability of l -ASNase hinders acrylamide mitigation efficiency in thermally processed food, , which also increases the risk of microbial contamination and overall production costs .…”

Current state of molecular and metabolic strategies for the improvement of L-asparaginase expression in heterologous systems

“…This region is comprised of the open reading frame (ORF) and facilitates the accessibility of the Shine-Dalgarno sequences and the start codons. Thereby, this modificacion improved the efficiency of the beginning of translation (Zhang et al, 2021). Zhang et al (2021) succeeded in expressing a site-directed mutated L-ASNase from Rhizomucor miehei , using B. subtilis 168 as host microorganism, increasing the L-ASNase activity from 2.1 U/mL to 13.3 U/mL by 5' UTR modification.…”

“…Thereby, this modificacion improved the efficiency of the beginning of translation (Zhang et al, 2021). Zhang et al (2021) succeeded in expressing a site-directed mutated L-ASNase from Rhizomucor miehei , using B. subtilis 168 as host microorganism, increasing the L-ASNase activity from 2.1 U/mL to 13.3 U/mL by 5' UTR modification. This was produced in high density batch culture and reached an activity of 521.9 U/mL.…”

Production, characterization and techno-economic evaluation of Aspergillus fusant l-asparaginase

“…A common huddle with fermentative production of L-ASNases and other microbially-derived value-added products is low yield because microorganisms maintain tight control over biosynthesis of metabolites, especially extracellularly-secreted ones (Sadh et al 2018 ; Pinu et al 2018 ). A combined use of hyper-producing strains obtained naturally or through mutagenesis, protoplast fusion and genetic engineering, especially with CRISPR/Cas9 system (Zou et al 2019 ; Zhang et al 2021 ; Hu et al 2021 ), and design of experiments by response surface methodology and artificial neural network, has significantly improved yield (Farjaminezhad and Garoosi 2021 ; Asitok et al 2022a ; Kusuma et al 2022 ).…”